Giovanni's house

Formins: from plants to eukaryotic cells. Phylogenetic evolution of a protein family.

2012-01-08T20:31:00.000+01:00

FORMINS

All eukaryotic cell types have morphologies that are uniquely tailored to their physiological functions. The great variation in cell shape depends crucially on an underlying network of dynamic, iterconnected network of actin filaments and microtrubule polymers. The dynamic turnover of these filamentous meshwork is used to direct cell polarity and to facilitate membrane and organelle traffic, cell adhesion, chromosome segregation, cell migration, cell proliferation and differentiation. Cytoskeletal polymers are organised to form an high order force-generating structures.

Two kind of proteins are present on cell cytoplasm in order to organize actin and tubulin monomers:

- inhibiting factors such as profilin and thymosin ( for actin ) and stathmin ( for tubulin)

- stimulating factors such as formin family proteins ( for actin polymerization and for tubulin stabilization), WAVE-ARP2/3 ( for actin polymerization), Spire, Cordon-Bleu (COBL), Leiomodin (LMOD), junction-mediating and regulating protein (JMY)

However, formins might be unique in their ability to directly regulate both actin filaments and microtubules.

ormins are large ( 120-220 kDa) multidomain proteins that interact with many binding partners to performs their functions. Fungal species have 3 formin genes, whereas mammals have 15 formin genes and plants species have more than 20 formin genes.

Human 15 genes have been subdivided into seven subfamilies:

DAAM : DAAM 1 and 2Dishevelled-associated activator of morphogenesis
Delphilin: Neuronal expression near Glutamate receptor
Dia: Diaphanous (lamellopodial, filopodial stress fibers ubiquitarious)
FHOD: FH1/FH2 domain containing protein (ubiquitarious)
FMN: Formin 1 and 2 ( developing nervous system, kidney and oocytes)
FMNL: Formin-like 1, 2 and 3 (phagocytosis, ubiquitarious)
INF: Inverted Formin 1 and 2 ( role in microtubule stabilization and organization of endoplasmic reticulum)

No three dimensional structure of intact formin has been reported, but these proteins are depicted such as dimers, each subunit formed by three major functional regions:

the amino terminal regulatory region formed by Rho Binding Domain (RBD), Diaphanous Inhibitory Domain (DID), Dimerization Domain (DD)
Formin Homology Domain 1 (FH1) , Formin Homology Domain 2 (FH2)
The carboxy terminal domain Dia Autoregulatory Domain (DAD)

RhoGTPases have a central role in activating formins. Rho-associated protein kinases (ROCKs) must be activated by extracellular signals such as pherormones, growth factors, chemoattractant proteins. When activated Rho proteins dind to the RBD region of formins and help to disrupt adjacent autoinhibitory DID-DAD interactions. Moreover Rho proteins play a central role in recruiting formins to specific locations such as at inner layer of plasmamembrane. For this reason formin activation must be configured to specific locations, typically at the cell cortex; so that Rho interactions regulate formin cortical localization and activation.Mounting evidences suggested that Rho proteins are not sufficient to full formin activation process.

Farnesylation is a postranslational process in which three isoprene units are linked usually at CAAX motif present in a given protein. Usually Ras GTP ases are farnesylated at C terminal region promoting their insertion into lipid bilayer.

The same seems to be true for Inverted Formin-2 (INF2) those farnesylation might help INF2 to target to endoplasmic reticulum membranes.

Profilin interact with FH1 where formin shows a rope-like structure ( probably due toloss of a true secondary structure) in middle region of its molecule. Profilin is an ubiquitarious actin monomer-binding protein with separate binding sites for monomeric actin (G-actin) and polyproline tracts. Profilin is associated with most acin monomers in cells, and therefore, profilin actin-complexes are predominant substrate for actin assembly in vivo.

The formin adjacent FH2 domain forms a head-to-tail doughnut-shaped dimer that encircles the “barbed end” of the actin filament and it links directly to microtubules and microtubules-tip associated proteins such as EB1, Adenomatous Polyposis Coli (APC), Citoplasmic Linker Protein 170 (CLIP170).

Interestingly all known plant formin proteins lacks RBD, DID and DD domains, whereas they contain a transmembrane domain or PTEN domain.

All formins govern two distinct phases of actin assembly : nucleation and elongation, requiring only 5-200 nM formin for a robust nucleation process or an elongation activity in vitro. Interestingly some formins have additional effects on actin beyond eleongation and nucleation.

Myosins and motor proteins

Generally speaking three classes of molecular motor proteins are present in human cells: dyneins, kinesins, and myosins; these proteins have evolved to transport organelles and other cargoes inside cells.

Humans have 38 myosin genes; some of them are adapted to transport vescicles and other cell products inside the cytoplasm, whereas others function as load-bearing tension sensors.

Myosin molecular structure has been extensively elucidated, so that any given myosin molecule is composed by three domains: the first is the motor domain or head, usually located at amino terminal regionable to bind ATP and actin. This domain is mechanically linked to a second domain consisting of an extended alfa helical “neck” containing variable number of IQ motifs, able to bind to proteins of calmodulin family.This alfa helical domain is sometime referred such as “lever arm”, because it moves as a rigid body to generate “the power stroke” in response to small ATP dependent changes in the conformation of N terminal domain. The C terminal domain is called “tail” domain containing most subclass specific variations , including a coiled-coil motifs, intervening in the dimerization process, as well's as membrane binding motifs or cargo receptor linking motifs. A clear example of myosin membrane linked is those of MYO1E normally engulfed into the plasma membrane , whereas cytoskeletal filaments of glomerular podocytes foot processes contain nonmuscle myosins IIa and IIb. Nonmuscle myosin activity generates tension, and the interaction among actin, myosins and alfa actinin-4 probably allows the foot processes to generate the contractile forces that help glomerular capillaries to resist to high intraluminal hydrostatic pressure and to change their morphologic structure actively, modifying the permeability of the glomerular filtration barrier.

Formin domain structure

It seems quite clear from the data available that Inverted Formins play a unique role in cytoskeletal pathway organization. INF1 and INF 2 seems to be very unique proteins for human cellular cytoskeletal organization, allowing some speculations on a possible role in microtubular assembly at reticulo endothelial membrane level. INF2 ia a unique member of the formin family . Although it has a domain structure similar to the diaphanous formins (DID, FH1, FH2, DAD), it is capale of not only accelerate acting polymerization, but also accelerating actin depolymerization. INF2’s depolymerizing activity relies on the combination of its FH2 domain and C terminous, including a DAD that also serves as an actin monomer-binding WH2 domain (Wasp homology domain 2). INF2’s depolymerization activity is regulated via an autoinhibitory interaction of its DID and DAD; this DID/DAD interaction could be relieved by CDC42-GTP bound to the DID domain of INF2. Recently GTP-bound CDC42 and Rac2 have been shown to bind to the DID domain of INF-2 and to mudulate INF2’s function in transcytosis.

The integral membrane MAL2 protein has been characterized as an essential component of the vescicle machinery for apical transcytosis. INF2 has been demonstrated to interact at its C terminal region with MAL2 in a region not conserved in other formins. MAL2 fulfils its trancytotic function by shuttling in tubulovescicular structures between the apical zone, which constitute the main site of residence of MAL2, and the cell periphery, where it encounters internalized cargo that is subsequently introduced into MAL2 positive transcytotic carriers destined to the apical membranes. Interesting INF2 silencing produced cargo retention in endosomal structures distant from the apical membrane. These results showed a cooperation beween the actin and microtubule cytoskeletons in apical transcytosis. In addition to its localization at the apical cell zone, INF2 is predominantly localized in the endoplasmic reticulum raising the possibility of an involvement of endoplasmic reticulum in apical transcytosis or to ther transport processes. It has been proposed that assembly and disassembly of short actin filaments , as we see in actin stress fibers, by INF2 provide the force for propelling MAL2 positive vescicle movement to encounter internalized cargo.

Other formins, such as mDia3 have been demonstrated to be involved in the control of microtubules during mitotic process promoting the attachment of microtubules to kinetotochores.

Brown Elizabeth J recently demonstrated that mutations in INF2 cause a form of autosomal dominant focal and segmental glomerulosclerosis (FSGS). INF2 is highly expressed in glomerular podocytes, terminally differenatiated cells whose characteristics are higly cytoskleleton dependnet. These characteristics include a branched structure that terminates in filopodia-like foot processes, and the maintenance of a specialized cell to cell junction known as the glomerular slit diaphragm. The importance of actin cytoskeleton in maintaining the glomerular filtration barrier is supported by the experiment demonstrating that mutation in alfa actinin 4 and actin cross-linking protein, cause a similar form of autosomal doinant FSGS.

FSGS associated mutant form of INF2 induce distinct patterns of actin polymerization in cultured podocytes compared with wild type of INF2 podocytes. The mechanism underlying these differences is largely unknown.

From phylogenetic point of view, lower species developed actin filaments based motor pathways, so that they don’t require motor protein dimerization. On the contrary human cells developed two kind of motor pathways: actin based submembrane pathways and reticulo-endothelial microtubular based pathway. If transcytotic carriers can involve actin filaments nucleation, polymerization and depolymerization though interactions between formins family proteins like INF2 and other components of plasmamembrane such as MAL2, this process can probably involves only structures moving under inner layers of plasmamembrane on different side of yet polarized cells.

References

Brown EJ, Schlondofrff JS, Becker DJ et al. Mutations in the formin gene INF2 cause Focal Segmental Glomerulosclerosis. Nat Genet 2010;42:72-6.

Bryant DM, Mostov KE. From cells to organs: building the polarized tissue. Nat Rev Mol Cell Biol 2008;9:887-901.

Sun H, Schlondorff JS, Brown EJ et al. Rho activation of mDia formins is modulaed by an interaction with Inverted Fomin 2 (INF2). Proc Natl Acad Sci USA 2011;108:2933-8.

Boyer O, Nevo F, Plaisier E et al. INF2 mutations in Charcot-Marie-Tooth disease with glomerulopathy. N Engl J Med 2011;365:2377-88.

Lupski JR, Reid JG, Gonzaga-Jauregui C et al. Whole genome sequencing ina patient with Charcot-Marie-Tooth Neuropathy. N Engl J Med 2010;362:1181-91.

Mele C, Iatropoulos P, Donadelli R et al. MYO1E mutations and childhood familial focal segmental glomerulosclerosis. N Engl J Med 2011;365:295-306.

Ingelfinger JR. MYO1E Focal segmental glomerulosclerosis and the cytoskeleton. N Engl J Med 2011;365:368-9.

Tryggvason K, Patrakka J, Wartiovaara J. Hereditary proteinuria syndromes and mechanisms of proteinuria. N Engl J Med 2006;354:1387-401.

D’Agati VD, Kastel FJ, Falk RJ. Focal segmental glomerulosclerosis. N Engl J Med 2011;365:2398-411.

Hammer JA III, Seller JR. Walking to work: roles for class V myosins as cargo transporters. Nat Rev Mol Cell Biol 2012;13:13-26.

Chesarone MA, DuPage AG, Goode BL. Unleasing formins to remodel the actin and microtubule cytoskeletons. Nat Rev Mol Cell Biol 2010;11:62-74.

As always many thanks to IVY for her patience and encouragement during my scientific studies.

Increasing iodine intake: a non dangerous solutions for thyroid diseases.

2011-12-20T11:16:00.001+01:00

Thyroid hormones and iodine metabolism

Iodine can come only from external sources; mostly food but also water. It is not widely distributed in nature, so that in the past iodine deficiency was common among people on every continent.

A minimum of approximately 70 μg of iodine is therefore needed to produce two hormones triiodothyronine and tetraiodothyronine in the thyroid gland each day. However more than that is required, because iodine when ingested, released from thyroid when the iodotyrosine precursors of the hormones are deiodinated in extrathyroid tissues, is rapidly excreted in the urine. Pregnant or lactating women need more iodine, because their tyroxine production rate is relatively high.

Worldwide iodine nutrition based on measurement of median Urinary Iodine excretion.

The World Health Organization (WHO) has recommended that children 5 years of age or younger ingest 90 μg of iodine daily; children 6 to 12 years of age , 120 μg of iodine daily; adults, 150 μg of iodine daily; pregnant and lactating women, 200 μg of iodine daily. The standard measure of iodine nutrition in a community or country is the median urinary iodine excretion, expressed in micrograms per liter. The values correspond to 70 to 80 percent of daily iodine intake, which often varies widely among people in the same community or country. Iodine deficiency is defined as urinary iodine excretion of less than 100 μg per liter.

International efforts have been conducted by major national and international efforts to increase iodine intake, primarily through the voluntary or mandatory iodination of salt.

However, iodine deficiency persists in many countries such as Australia, Russia and some African and European countries. However the salt iodination is only the first step in amelioration of iodine deficiency. The increase in amount of iodinated salt must be sustained. In normal adults, the daily production rate of two biologically active thyroid compounds tiroxine and triiodothyronine is approximately 100 μg and 30 μg respectively. Only about 20% of triiodothyronine is produced by the thyroid gland; the remainder of triiodothyronine is produced through the extrathyroidal deiodination of thyroxine.

Epidemiologic studies indicated that in United States from 1971-74 to 2001-2002 urine iodine excretion changed from 320 μg per liter to 168 μg per liter.

Among pregnant women, the frequency of moderate iodine deficiency (50 μg per liter ) is also more sustained from 1 % in 1971-74 to 7% in 2001-2002.

An important issue is if some level of chronic iodine intake is harmful. Indeed, fear of iodine-induced thyroid dysfunction has delayed or limited the implementation of iodine supplementation in regions with iodine deficiency.

It seems quite clear in view of molecular studies and genetic data that we have to study more than world regions with low iodine availability, people presenting susceptibility to a diffuse low iodine concentration present everywhere in the world. In particular mutations in thyrotropin receptor present on thyroid cells can account for most portion of this “susceptibility” to iodine intake variations.

The Thyrotropin Receptor

Constitutively activating mutations and inactivating mutations of the thyreotropin receptor gene are shown, as in the location of somatic mutations found in thyroid carcinomas. All gain-of-function mutations are in exon 10, except Sr281Asn/The that is in exon 9.

Circles denotes hyperthyroid adenomas, squares hyperthyroidism, diamonds congenital hyperthyroidism, octagons thyroid carcinomas.

Loss-of-function mutations are denoted by triangles.

Iodine deficiency

The consequences of iodine deficiency are broad. In mother and fetuses we can have pregnancy loss or cretinism, with irreversible mental retardation, neurologic disfunction, and growth retardation.

The consequences of mild iodine deficiency are easlily understood, tinking that production of thyroid hormones is reduced, and the thyroid gland enlarges to compnsate for the hormonal reduction.

In adults, mild iodine deficiency is also associated with nontoxic nodular goiter and also with toxic nodular goiter, due to the constitutive (thyrotropin independent) growth and functional increase in some thyroid cells clones.

Peter Kopp on 1995 reported a report of congenital hyperthyroidism caused by a mutation in the thyrotropin receptor gene. So that both constitutive activation of G-protein coupled receptors and germline mutations on the thyrotropin receptor gene can be ascribed to hyperfunctioning thyroid adenomas and autosomal dominant hyperthyroidism. Almost five independent mutations have been found to be responsible for constitutive activation of the thyroid receptor. These mutations can disrupt the structure of a domain than normally inhibit the coupling of thyroid receptor with G protein, leading to constitutive activation thyrotropin independent of the receptor itself.

These are:

Valine to Alanine substitution at position 509
Phenylalanine to Leucine substitution at position 631
Cysteine to Tyrosine substitution at position 672
Alanine to Isoleucine substitution at position 623
Aspart acid to Glycine substitution at position 619

Mutations in other locations in the transmembrane and intracytoplasmatic domains of thyrotropin receptor can cause autonomous thyroid function.

However, certain receptor locations may be at “hot spot” mutational risk possibly because DNA sequence at these sites is more susceptible to mutagenesis (exon 10) or , more likely, because mutations at these sites can lead to clonal espansion leading to phenotype of hyperthyroidism.

Iodine excess

Thyroid tissue can maintain normal thyroid function also in the presence of iodine excess.

In 1948 it was demonstrated that large doses of iodide, when given acutely in the rat, induce an inhibition of thyroid-hormone synthesis. This phenomenon , termed Wolff-Chaikoff effect, was later shown to also in man.

The acute Wolf-Chaikoff effect results from the increased intrathyroidal, rather than plasma, iodide concentration leading to inhibition of thyroid peroxidase activity. The net effect is the thyroid hormone synthesis decrease perduring for 24 to 48 hours.

The “escape” from acute Wolff-Chaikoff effect is due to a decrease in the expression of the sodium-iodide symporter in plasmamembrane of thyrocytes, leading to stable resumption of normal thyroid function.

Iodide mixedema

In patients with thyroid diseases such as Hashimoto’s thyroiditis or some pre-existing functional defects, the exposure iodine excess can lead to the so that so called “iodide mixedema”, in particular when large dose of iodide are given for prolonghed periods to these patients. A sort of prolonghed and stable Wolff-Chaikoff effect with a net reduction in the production of thyroid hormones and clinical hypothyroidism.

Jod-Basedow

In euthyroid patients with a nodular goiter or an autonomous thyroid nodule, so where Wolff-Chaikoff effect is not present at all, hyperthyroidism may develop, configuring the so called “Jod-Basedow” phenomenon. That’s why in region where dietary iodine was insufficient and in euthyroid patients with Graves’ disease treated with antithyroid drugs, iodine-induced hyperthyroidism is more common.

Diagnosis of iodine induced hyperthyroidism is based on 24 hours thyroid uptake of iodine-123 those normal values would be of 15 to 30% and in these patients would be reduced.

Therapy for Jod-Basedow phenomenon is based on usual therapy with anthyroid drugs ( methymazole better than propyltiouracil ) and on inhibition of the thyroid sodium-iodide symporter with oral sodium or potassium perchlorate given at 200 mg every 8 hours.

Teng W on 2006 reported data from chineese patients in whom iodine intake was mildly deficient, more than adequate, or excessive for five years. The investigators found that more than adequate or exccessive iodine intake was associated with a slightly increased cumulative five-year incidence of subclinical hypothyroidism and autoimmune thyroiditis, but not over hypothyroidism or hyperthyroidism.

Moreover, in many people the first two disorders where manifested with a non sustained and uncertain clinical importance.

Thyroid effects on cardiovascular system

It’s clear from invasive and noninvasive measurements in patients with thyroid disease that cardiac functions are closely related to thyroid status. In addition to the well-recognized action of thtroid hormones to increased peripheral oxygen consumption and substrate requirements, the hormone also increases cardiac contractility directly.

Triiodothyronine decreases system vascular resistance by dilating the resistance arterioles of the peripheral circulation. The vasodilation is due to a direct effect of triiodothyronine on vascular smooth-muscle cells that promotes relaxation.

The renin-angiotensin-aldosterone system is activated and renin release is increased as the result of the decrease in systemic vascular resistance. Consequently the rate of sodium reabsorption at kidney level results increased.

Interestingly thyroid hormones also stimulate the release of erytropoietin by renal tissue.

Thyroid hormones show many effects on heart and vascular system. Many of the clinical manifestations of hyperthyroidism are due to the ability of thyroid hormone to alter cardiovascular hemodynamics.

The effects of triiodothyroine, the active cellular form of thyroid hormone, on cardiovascular physiology are the following:

1. Increased tissue thermogenesis

2. Decreased systemic vascular resistance

3. Increased cardiac inotropy and chronotropy

Dedreased thermogenesis per se can induces low levels of vascular resistances; the last phenomenon leads to decreased affective arterial filling volume, increased renal sodium reabsorption and increased blood volume.

Increased blood volume per se can contribute to increased contranction energy and increased heart rate with subsequent increased cardiac output.

To understand the alterations of cardiac functions in thyroid diseases, we need to understand the mechanisms by which thyroid hormone acts on cardiac myocytes and on vascular smooth-muscle cells.

Triiodothyronine is the biologically relevant thyroid hormone molecule in cardiac myocytes, as in other cells. And there is evidence that cell membrane contains specific transport proteins for triiodothyronine.

Conversion of thyroxine to triiodothyronine by enzyme deiodinase does not occur in cardiac myocytes. Once in myocytes , triiodothyronine enters the nucleus and it binds to nuclear receptors that then can bound to thyroid hormone responsive elements in target genes.

Different subtypes of thyroid receptors exist into myocytes : Tyroid Receptor α1, α2 and β1, so that they can regulate the expression of different genes and encode both structural and regulatory proteins.

The thyroid hormone receptors binds to DNA as monomers or homodimers, or as heterodimers composed of a triiodothyronine nuclear receptor and another receptor from steroid hormone receptor family.

Occupancy of receptors by triiodothyronine in combination with the recruitment of coactivators leads to optimal transcriptional activation.

Interestingly in the absence of triiodothyronine, the receptors repress genes that are positively regulated by thyroid hormone.

Genes positively regulated by thyroid hormones are:

α Myosin heavy chain
β 1 Adrenergic receptor
Voltaged gated potassium channels (Kv1.5, Kv4.2, Kv4.3)
Na/K –ATPase
Sarcoplasmic reticulum Ca-ATPase
Guanine nucleotide regulatory proteins

Genes negatively regulated by thyroid hormones are:

Β Myosin heavy chain
Triiodothyronine nulear receptor alfa 1
Adenyl Cyclase type V and type VI
Na/ Ca exchanger
Phospholamban

The two Myosin heavy chains isoforms ( alfa and beta ) are myofibrillar proteins that make up the thick filament of the contractile apparatus of cardiac myocytes. In humans beta isoform predominates in cardiac myocytes; however the derangement in contractile function that is markedly impaired in thyroid disease is likely to be influenced by other factors beside the selective transcription of myosin heavy chains.

Increased heart rate , widened pulse pressure, and increased cardiac output are characteristics of hyperthyroid patients, strictly resembling a state of increased adrenergic activity. In hyperthyroid state despite normal plasma levels of catecholamines, the sensitivity of the heart to adrenergic stimulation is increased or normal. It has been demonstrated that different components of adrenergic receptor complex at plasma membrane level are under strict control of thyroid hormones in particular beta adrenergic receptor itself, guanine nucleotide regulatory proteins, and adenyl cyclase type V and VI.

Plasma membrane ion channels including Na/K ATP ase, Na / Ca exchanger, and voltage-gated potassium channel (Kv1.5, Kv4.2, Kv4.3) coordinated the electrochemical activity of depolarization rate in myocardial sinus node and transmission of depolarization signals through atrial tissue in atrio-ventricular node. That’s why in patients affected by increased thyroid activity freqquently we can found a fibrillar atrial pulse activity.

In short term, triiodothyronine changes the performance characteristics of various sodium, potassium and calcium channels in the heart, altering the inotropic and chronotropic activity of specialized cardiac myocytes.

In most patients with hyperthyroidism, cardiac output is elevated, and the response to exercise is subnormal; these characteristics of hyperthyroid myocardial disease are the result of an inability to increased heart rate maximally or to lower vascular resistance further, as normally occurs during exercise.

These findings support the observation that elderly patients with few symptoms of hyperthyroidism may present cardiac-rhythm disturbances or unexplained tachycardia.

So that serum thyrotropin should be measured in all elderly patients with systolic hypertension, a widened pulse pressure, recent-onset angina, atrial fibrillation , or exacerbation of ischemic heart disease.

In summary, patients with overt heart failure also have low serum triiodothyronine concentration, and the decrease is proportional to the degreee of heart failure. In children undergoing bypass surgery for correction of complex congenital heart disease serum triiodothyronine concentration fall by more tha 60 percent and remain low for up to 8 days after surgery. Triiodothyronine repletion could be accomplished safetly and with a resulting improvement in postoperative cardiac function in children surgically treated for congenital heart disease.

References

Wolff J, Chaikoff IL. Plasma inorganic iodide as homeostatic regulator of thyroid function. J Biol Chem 1948;174:555-64.

Wolff J, Chaikoff IL, Goldberg RC et al. The temporary nature of the inhibitory action of excess iodide on organic iodine synthesis in the normal thyroid. Endocrinology 1949;45:504-13.

Braverman LE, Woeber KA, Ingbar SH. Induction of myxedema by iodide in patients euthyroid after radioiodine or surgical treatment of diffuse toxic goiter. N Engl J Med 1969;281:816-21.

Vagenakis AG, Wang CA, Burger A et al. Iodide-induced thyrotoxicosis in Boston. N Engl J Med 1972;287:523-7.

Kopp P, van Sande J, Parma J et al. Congenital hyperthyroidism caused by a mutation in the thyrotropin receptor gene. N Engl J Med 1995;332:150-4.

Sunthornthepvarakul T, Gottschalk ME, Hayashi Y et al. Resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 1995;332:153-60.

Utiger RD. Thyrotropin receptor mutations and thyroid dysfunction. N Engl J Med 1995;332:183-5.

Paschke R, Ludgate M. The thyrotropin receptor in thyroid disease. N Engl J Med 1997;337:1675-81.

Teng W, Shan Z, Teng X et al. Effect of iodine intake on thyroid disease in China. N Engl J Med 2006;354:2783-93.

Utiger RD. Iodine nutrition – More is better. N Engl J Med 2006;354:2819-21.

Klein I, Ojamaa K. Thyroid hormone and the cardiovascular system. N Engl J Med 2001;344:501-9.

Pramyothin P, Leung AM, Pearce EN et al. A hidden solution. N Engl J Med 2011;365:2123-7.

Thanks to Ivy for hearing my “silly” talks during dinner times, always too late in nightime, and to her Ukraine family for their support and encouragement in follow my studies efforts.

Prostate cancer : the result of an inflammatory disease.

2011-11-06T15:26:00.000+01:00

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Tensegrity model: the biochemical basis of structural network in the skin.

2011-10-27T12:49:00.001+02:00

HUMAN NANOSTRUCTURES AS RESPONSIBLE OF COMMON SKIN DISEASES.

Simpson CL, Patel DM, Green KJ. Deconstructing the skin: cytoarchitectural determinants of epidermal morphogenesis. Nature Rev. Mol Cell Biol 2011;12:565-80.

Irvine AD, McLean I, Leung DYM. Filaggrin mutations associated with skin and allergic diseases. N Engl J Med 2011;365:1315-27.

Bieber T. Atopic dermatitis. N Engl J Med 2008;358:1483-94.

Holick MF. Clinical efficacy of 1,25 dihydroxyvatamin D3 and its analogues in the treatment of psoriasis. Retinoids 1998;14:12-7.

Lindhurst MJ, Sapp JC, Teer JK et al. A mosaic activating mutation in AKT1 associated with the Proteus Syndrome. N Engl J Med 2011;365:611-9.

Kim BYS, Rutka JT, Chan WCW. Nanomedicine. N Engl J Med 2010;363:2434-43.

Treating the hypertension and end-organ damage diseases: a cause-answer problem ?

2011-10-17T15:06:00.000+02:00

Waiting for new Guidelines….

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Chobanian Aram V. Isolated systolic hypertension in the elderly. N Engl J Med 2007;357:789-96.

The death of Vitamin K inhibitors.

2011-10-01T16:14:00.000+02:00

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Lassen MR, Gallus A, Raskob GE et al. Apixaban versus enoxaparin for thromboprophylaxis after hip replacement. N Engl J Med 2010;363:2487-98.

The EINSTEIN Investigators. Oral rivaroxaban for symptomatic venous thrombosis. N Engl J Med 2010;363:2499-510.

Hylek EM. Therapeutic potential of Oral Factor Xa inhibitors. N Engl J Med 2010;363:2559-61.

Patel MR, Mahaffey KW, Garg J et al. Rivaroxaban verus Warfarin in nonvalvular atrial fibrillation. N Engl J Med 2011;365:883-91.

del Zoppo GJ, Eliasziw M. New options in anticoagulation for Atrial Fibrillation. N Engl J Med 2011;365:952-3.

Granger CB, Alexander JH, McMurray JJV et al. Apixaban versus Warfarin in patients with Atrial Fibrillation. N Engl J Med 2011;365:981-92.

Mega JL. A new era for anticoagulation in Atrial Fibrillation. N Engl J Med 2011;365:1052-4.

VITAMIN D: how a vitamin becomes a true hormone.

2011-08-26T15:57:00.000+02:00

Calcium intake is well know to represent a very important factor affecting bone mass, bone biomechanical alteration and consequntly bone fracture incidence. Low calcium intake in particular with consequent decrease in intestinal calcium absorption is the gatekeeper in bone disease induction. It has been demonstrated from basic studies on osteoporotic patients that low intestinal calcium absorption is striclty related to loss or decrease activity of steroid hormonal compounds such as estrogens and activated form of Vitamin D3 (1,25 dihydrixyVitamin D3) at epithelial intestinal level. With aging in particular there is a decline in calcium absorption efficiency, related to intestinal loss of Vitamin D3 receptors or resistance/lower actictivity of these receptors to the action of 1,25 dihydroxyVitamin D3. Also seasonal variations in sunlight exposure profundly affect the level of Vitamin D3 synthetized in our skin by irradiation with ultraviolet wave length of our skin tissue. Pre Vitamin D2 and Pre Vitamin D3 better called ergocalciferol and colecalciferol, introduced with the diet respectively from vegetable and from animals foods, are efectively transformed on skin tissue in true Vitamin D2 and Vitamin D3 by automaintained non enzymatic reaction due only to exposure of our skin tissue to sunlight ultraviolet irradiation.

CYP27A1 hydroxylase

After sunlight activation Vitamin D3 is transported into our body linked to protein carriers on the first time to the liver, where it is hydroxylated by a cytocrome P450 dependent enzyme called 25 hydroxylase CYP27A1. At this level a first selection is performed by hydroxylating electively the choleclaciferol derivatives (i.e. Vitamin D3) more than ergocalciferol derivatives (i.e. Vitamin D2). From this first evidence is not effective the therapeutical administration of vegetable form of vitamin D, leading on the contrary to adecrease in levels of active hydroxylated forms of Vitamin D3. Measurement of plasma levels of 25-hydroxy Vitamin D3 is universally considered the main index of individual body reserve of Vitamin D3. However, many controversies exist, and recently published Guidelines outlined this topic, about optimal plasmatic levels achievable in human in order to prevent Vitamin D3 deficiency. Accordingly, a seasonal fluctuation in hormonal vitamin D3 are present in humans related to sunlight exposure with greater levels reached during spring and summer seasons, and lower levels during fall and winter seasons. Again, interindividual variations between individuals are present according to proportion of time spent in indoor and outdoor activities. Moreover, also variations according to latitute degree of countries where people live are present if we consider that sunlight irradiation is most importantat near tropical area of the world compared to northern countries.

CYP27B1 hydroxylase

After liver activation 25 hydroxyVitamin D3 is transported to the kidney proxymal convoluted tubules in order to be fully activated by 1-alfa hydroxylase enzyme cytocrome P450 CYP27B1. This hydroxylative enzyme has been profoundly studied thanks to the more evident regulation by hormonal axis devoted to calcium-phosphate homeostasis. However, as we can see later, 1 alfa hydroxylase activity is not so relevant to know the individual needs of Vitamin D3 supplementation with fortified foods. On the contrary, it is important for the synthesis of active hormonal Vitamin D3 hydroxylated in 25 and 1 alfa carbons allowing a global interaction with zinc fingher sites of intranuclear Vitamin D receptors present in target tissues. In particular , it has been largely demonstrated both in vitro and in vivo that 1 alfa hydroxylase is sensitive to stimulating action of parathyroid hormone, estrogens and probably androgenic steroids hormones, Growth Hormone/Insulin growth factor 1.

CYP24A1 hydroxylase

Responsible for catabolism of Vitamin D3 into liver secreted “ calcitroic acid “ 24 hydroxylase enzyme CYP24A1 has been only recently subjected to an accurated studies. In particular the genetic demonstrations of genetic polymorphisms leading to inactive mutated forms of CYP24A1 hydroxylase has been recently confirmed also in clinical background as responsible of Vitamin D3 toxicity. In particular the Idiopathic Infantile Hypercalcemia ( milder form ) also called Lightwood type (OMIM 143880) has been demonstrated in 3 affected patients linked to mutations in CYP24A1 gene expression. It is possible that clinically noted interindividual variations in Vitamin D3 need by fortified foods in humans could be due to inactivating mutations in CYP24A1 gene. Interestingly a new hormonal axis has been invoved into regulation of CYP24A1 expression: called FGF23/KLOTHO axis. Klotho is a protein with glycosidase activity able to modify the FGF Receptor present at kidney level for FGF23. FGF23 as bee demonstrated to be a potent stimulator of CYP24A1 and a potent inhibitor of CYP27B1 suggesting in this way a more complex picture compared to the past, involving two homeostatic system for phospho-calcium metabolism:

PTH/ Vitamin D3 axis: CYP27B1

FGF23/ KLOTHO axis: CYP27B1 and CYP24A1

References

Koro-o M, Matsumura Y, Aizawa H et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45-51.

Urakawa I, Yamazaki Y, Shimada T et al. Klotho converts canonial FGF receptor into a specific receptor for FGF23. Nature 2006;444:770-4.

Makishima M, Lu TT, Xie W et al. Vitamin D erceptor as an intestinal bile acid sensor. Science 2002;296:1313-6.

Kurosu H, Yamamoto M, Clark JD et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309:1829-33.

Chang Q, Hoefs S, van der Kemp AW et al. The beta glucosidase Klotho hydrolyzes and activates the TRPV5 Channel. Science 2005;310:490-3.

Imura A, Tsuji Y, Murata M et al. Alfa Klotho as a regulator of calcium homeostasis. Science 2007;316:1615-6.

Cai Q, Hodgsan SF, Kao PC et al. Inhibition of renal phosphate transport by a tumor product in a patients with a oncogenic osteomalacia. N Engl J Med 1994;330:1645-9.

Econs MJ, Drezner MK. Tumor induced osteomalacia – unveiling a new homone. N Engl J Med 1994;330:1679-81.

Kronenberg HM. NPT2a – The key to phosphate homeostasis. N Engl J Med 2002;347:1022-4.

Priè D, Huart V, Bakouh N et al. Nephrolithiasis and osteoporosis associated with hypophosohatemia caused by mutations in the type 2a sodium-phosphate cotransporter. N Engl J Med 2002;347:983-91.

Johnsson KB, Zahradnik R, Larsson T et al. Fibroblast growth factor 23 in oncogenic osteomalacia and X linked hypophosphatemia. N Engl J Med 2003;348:1656-63.

Karim Z, Gérard B, Bakouh N et al. NHERF1 mutations and responsiveness of renal parathyroid hormone. N Engl J Med 2008;359:584-92.

Priè D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:362:2399-2409.

Schlingmann KP, Kaufmann M, Weber S et al. Mutations in CYP24A1 and Idiopathic Infantile Hypercalcemia. N Engl J Med 2011;365:410-21.

Selenoproteomes : the selenium metabolism as key players against oxidative injury.

2011-06-06T23:43:00.000+02:00

Selenoproteins

Selenium (se) is an essential nutritional trace element that is critical to the normal physiology of a wide range of species, including humans. While Selenium deficiency in humans is rare, there is evidence that less overt changes in Selenium status may affect aspects of human health such as immune responses, neurodegerative diseases, cardiovascular diseases, cancer and thyroid metabolism. It is likely that selenium supplementation benefits certain populations only, such as Selenium deficient or genetically predisposed groups; moreover selenium supplementation is particularly effective at lowering the risk of some health disorders. Dissecting the mechanisms by which dietary selenium levels affects different aspects of human health is essential in order to understanding how altering selenium levels we can affect human health.

In humans 25 selenoproteins coding genes have been identified; members of this family exhibit different tissue distribution patterns and different subcellular localizations ( transmembrane, cytoplasmic, endothelial reticulum associated, plasmatic selenoproteins ).

The mechanism by which selenoproteins are synthetized included an evolutionary selection for an increased efficiency of cathalitic activity of selenoproteins due to physicochemical reactivity due to a lower pKa for cysteine containing reactive sites (5.2 pKa) compared to selenocysteine (Sec) containing cathalitic site (8.3 pKa). Higher pKa value means higher cathalitic reactivity and efficiency counterbalanced by lower availability of selenium in environment compared to sulfur. It has been demonstrated that selenoprotein translation relies in a special feature in recoding the UGA codon, normally readed such as termination signal with the release of nascent polypeptide from the ribosome complex, into a Selenocysteine insertion codon. Most selenoproteins mRNAs, with the exception of selenoprotein P, that is mostly secreted protein, contain a single Selenocysteine residue located at aminoacid position 21, within their active sites

During translational process of selenoproteins, cis and trans acting factors work in concert to redirect the translational machinery to insert a selenocysteine at UGA codon during polypeptide synthesis.

The “factors” include:

- Selenocysteine insertion (SECIS) elements

- A specific transfert RNA unique for Serine/Selenocyteine

- An enzyme that systhetizes this specific transfert RNA: Sec synthase

- A Phosphoseryl-tRNA kinase (Pstk)

- A translating protein SecP43 methylating Sec-tRNA and shuttling the SecS-Sec-tRNA(Ser/Sec) between nucleus and cytoplasm

- A SBP2 RNA binding protein

- A specialized eleongation factor EFsec

- A ribosomal protein L30

- SECIS interacting nucleolin

- Sec-tRNA gene transcription activating factor (STAF)

As we can understand the growing lists of specific translational ribosomal interacting factors is directed to the synthesis of a specific tRNA in the presence of selenium and incorporation of this trace elements into a growing polypeptide chain. Interestingly transgenic mouse model involving deletion of the gene encoding tRNA serine/ selenocysteine results in embryonic lethality.

Under low selenium states, mRNA is deraded through nonsense-mediated decay (NMD), representing a pathway that targets mRNAs conatining premature termination codons for degradation. The presence of both a UGA codon or an intron downstream of the UGA was shown to be required for delenium dependent regulation of mRNA turnover. Interestingly, degradation selenoprotein mRNA under condition of low Selenium is nor uniform. Probably the position of the UGA codon relative to the actual stop codon can play an important role in modulating the NMD process.

Selenoproteins are collectively essential for life; reduced selenoprotein actrivity is counterbalanced by a transcriptional system involved into maintainement of cellular redox homeostasis and viability, including the induction of a Nrf2 nuclear transcription factor.

Several selenoproteins have been characterized by an enzymatic antioxidating activity, serving to mitigate the damage caused by reacive oxigen species (ROS).

ROS are produced mainly by:

- Mitochondrial oxidative phosphorylation

- NADH/NADPH oxidase activity

- P-450 monooxygenase

- Lipoxygenase

- Cyclooxygenase

- Xantine oxidase

Through antioxidant selenoenzymes such as:

- Glutathione peroxidase: detoxification od peroxides.

- Thioredoxin reductase: regeneration of reduced thioredoxin (NADPH)

- Methionine sulfoxide reductase: reduction of oxidized methionine residues

Cellular damage caused by reactive oxigen species is mitigated. However the emerging concept of ROS as secondary messengers of cell signaling requires a closer examination of potential roles for selenoproteins as modulators of redox-modulating signal transduction pathways. Not all slenoproteins are enzymes involved in regulating ROS and the list of selenoproteins have yet to be determined, but much progress has been made recently in this rapidly evolving field.

Gluthatione Peroxidases

The first identified selenoproteins was gluthatione peroxidase 1 today called GPx1. In humans GPx1 through 4 and GPx6 are Selenocysteine containing enzymes. All members of this group use gluthatione (GSH) to catalyze the reduction of hydrogen peroxide and/or phospholipid peroxides collectively providing a wide spectrum of antioxidant protection. While GPx1 through GPx3 are 22-25 kDa proteins acting as homotetrameric enzymes, GPx4 is a 20 kDa protein that acts in a monomeric form.

GPx1 is one of the most abundant and ubiquitously expressed selenoproteins, and also most highly sensitive to changes in selenium levels. In vivo conditions of oxidative stress like Asthma have been studied for effects on GPx1 expression; so that some studies have demonstrated that GPx1 levels increase in lung during asthma.

GPx1 plays an important role in protecting against neurodegenerative diseases such as Parkinson’s disease and Dementia with Lewy bodies, due to higher content of GPx1 in microglia compared to neurons in normal people.

GPx1 activity against cancer has been studied intensively; recently some studies have demonstrated a relationship between GPx1 genetic polymorphisms and cancer.

Recently, GPx1 overexpressing mice were described showing spontaneous development of hyperglycaemia, hyperinsulinasemia, insulin resistance and obesity. The effect of GPx1 overexpression on diabetes involves upregulated pancreatic duodenal homeobox 1 (PDX1) and downregulated uncoupling protein 2 (UCP2) in pancreatic islet cells, and emphatizes the requirement for some degreee of ROS for maintaining proper cell function. Supporting the notion of a central role of oxidative stress in the pathogenesis of diabetic complications 4 biochemical changes induced by hyperglycaemia are all activated by a common mechanism: i.e. the overproduction of superoxide radicals:

- Increased flux through the polyol pathway: reducing the level of both NADPH and glutathione

- Increased formation of advanced glycation end products (AGEs)

- Activation of protein kinase C

- Incresed shunting of excess glucose through the hexosamine pathway: mediating increased transcription of genes for inflammatory cytokines

GPx3 is the only family member to be secreted and constitutes probably 20% of selenium found in plasma. The main source of GPx3 is kidney, produced by cells of proximal tubule and in the parietal cells of Bowman’s capsule and later released into the blood.

GPx3 are also high in the thyroid gland where it likely serves to reduces oxidative stress. In vitro studies have shown that GPx3 may use glutathione to reduce hydrogen peroxide or tert-butylhydroxide.

Another imprtant role for GPx3 was identified in experiments showing a role in regulating the bioavailability of nitric oxide (NO) produced from platelets and vascular cells. Decreased GPx3 activity led to platelet hypereactivity and to an increased risk of thrombosis.

Thioredoxin Reductases

Thioredoxin reductases (Txnrd) enzymes are oxidoreductases that use NADPH to cathalize the reduction of oxidized thioredoxin (Trx). Txnrx in turn is used by several cellular enzymes as cofactor in dithiol-disulfide exchange reactions and this is a major mechanism by which a reduced environment is maintained within cells, particularly serving to maintain reduced cysteine groups.

Mammalian thioredoxins reductases contain a conserved N-terminal disulfide motif (Cys-Val-Asn-Val-Gly-Cys) and a C-terminal active site sequence (Gly-Cys-Ser-Gly). Higher form of life, such as mammals in the active site at C-terminal segment, contain Cysteine-Selenocysteine sequence, instead of Cys-Cys as we can see in Drosophila melanogaster ( fruit fly mosquitos) and in Caernorhabditis Elegans (worm). The presence of selenocysteine confers a mechanistic advantages to the mammalian enzymes including the ability to work at acidic PH environment.

Thioredoxin is not the only substrate for this selenocysteine peptides including:

- NK-Lysin ( effector peptide in T lymphocytes )

- Alloxan

- Lipoic acid

- Selenite

These enzymes are housekeeping proteins expressed in cells under non-stressed conditions and given the ubiquitarious nature of of the Thioredoxin/ Thioredoxin reductase complex, it is not suprising the involvment of this system in numerous cellular processes, including actin polymerization related to cell membrane restructuring or as hydrogen donors for mammalian S-phase ribonucleotide reductase.

Whereas Txnrd type 3 is specific for testes, Txnrd type 1 is also called cytoplasmic and type 2 is mitochondrial but are ubiquitarious.

With the emergence of ROS role as secondary messengers of cell signaling, there also has emerged a role of the Trx/Txnrd system as modulators of cell signaling, for example in activation of small G protein Ras in myocardiocytes through oxidation of thiol groups on Ras protein.

Evidence is emerging regarding a specific mechanism by which Txnrd1 may affect tumor growth and progression. This system has been implicated as a crucial electron donor system during DNA replication that occurs during the S-phase of cell cycle in all cell type and in particular in active replicating cells such as cancer cells. Aggressive tumors such as melanomas, breast, prostate, colorectal and thyroid cancers significalntly overexpressed both Trx1 and Txnrd1 demonstrating the direct relation existing between cell replication rate and thioredoxin system activity.

Thyroid hormones metabolism and deiodinases

Thyroid hormone synthesis is dependent on a normally developed thyroid gland, an adequate nutritional intake of iodide, and a series of biochemical steps to organify iodide.

Iodide is scarce in most environments. Despite important progress in eradicating iodine deficiency disorders, which lead to the development of endemic goiter and are associated with various degree of mental retardation, iodine deficiency remains a major public health issue.

The first step in thyroid hormone biosynthesis consists of iodide uptake at the basolateral membrane by the sodium-iodide symporter. Iodide is then released into the follicular lumen, presumably in part by the anion transporter “pendrin”, and oxidized by the membrane-bound enzyme thyroperoxidase.

This oxidation requires the presence of hydrogen peroxide, which is generated by dual oxidase 2, an enzyme that requires a specific maturation factor ( dual oxidase 2A ).

The subsequent iodination of selected tyrosyl residues within thyroglobulin, the matrix for thyroid hormone synthesis, leads to the synthesis of monoiodotyrosine and diiodotyrosine. After this organification step, iodinated donor and acceptor iodotyrosines are fused in the coupling reaction to form either triiodothyronine (T3) and Thyroxine (T4). Only a small fraction of iodotyrosines are used in this process.

To release T3 and T4, thyroglobulin is engulfed by the thyrocytes through pinocytosis, digestion in lysosomes, and then secreted into the bloodstream ( approximately 80% as T4 and 20% as T3).

In contrast, monoiodotyrosine and diiodotyrosine are found only in minute amounts in the bloodstream.

In 1952 Roche charaterized a thyroidal iodothyrosine dehalogenase, which was later found to be a flavin mononucleotide (FMN) dependent enzyme. This enzyme was found to efficiently deiodinate monoiodotyrosine and diiodotyrosine but not T4, and the investigators founded that the released iodide was reused for hormone synthesis.

At the same time, McGirr and Hutchinson described a large, inbred family of tinkers, itinerants from Scotland and Ireland, that had 12 goitrous members with hypothyroidism and mental retardation in its pedigree. The authors speculate that these family members might have an autosomal recessive defect in iodotyrosine deiodination bacause they rapidly lost a large fraction of a radiolabeled compound that didi not correspond to either T4 or T3.

The iodothyrosine deiodinase is a family of selenoproteins formed by three enzymes which are membrane anchored enzymes of a mean molecular weight ranging from 29 to 33 kDa, that share substantial sequence homologies and catalytic properties. These selenoproteins are called:

- D1

- D2

- D3

Thyroid hormone action is initiated by the activation of Tetraiodotyronine (T4) prohormone to Triodotironine (T3). This conversion is carried out by the enzymes deiodinases type 1 and type 2, able to cathalizing the monodeiodination reaction of the outer ring of T4.

Moreover T4 and T3 are subjected to monodeiodination reaction at the site of inner ring, that is an irreversible reaction leading to inactivation of thyroid hormones to T2, reaction cathalized by D3 ( and partly by D1).

In this way we can understand that thyroid metabolism is strictly dependent from the combined actions of the three deiodinases and it is regulaed mainly through D2 stability in response to changes in iodine supply, to cold exposure, and to changes in thyroid gland functions.

All three deiodinases are expressed in a number of fetal and adult tissues. Their tissue and developmental expression patterns suggest that deiodinases may control the concentration of active thyroid hormone available to specific tissues or cell types at certain stages of development.

Generally speaking it is believed that D2 generates T3 from T4 for local use in specific tissues including pituitary, brown fat and brain, whereas D1 generates T3 from T4 in the thyroid and peripheral tissues primarly for export to plasma.

These physiological insights have been confirmed by studies on KO mice for D1 and D2 as well’s on double KO mice for D1 and D2. In particular it has been suggested that D1 and D2 activity are not essential for the maintenance of the serum T3 levels, but they serve as important enzymes in thyroid hormone homeostasis with D2 playing an important role in local T3 production whereas D1 vontributing to iodine conservation by serving s scavenger enzyme in peripheral tissues and in the thyroid too.

Interestingly, the thyroid gland has an exceptionally high number of selenoproteins expressed ( at least 11 ), several of which may be involved in the protection of the gland against the high amounts of hydrogen peroxide produced during thyroid hormone biosynthesis.

Dietary levels od selenium may affect human thyroid hormone metabolism, but co-factors such as iodine deficiency and thiocyanate overload appear to be required for manifestation of disease such as:

- Kashin-Beck disease

- Endemic myxedematous cretinism

Explanation is that optimal function of the deiodinases requires very little selenium intake. However, mutations that result in defective expression or function of the key selenoproteins synthesis factor, such as SBP2, manifest in thyroid abnormalities associated with decreased D2 activity. This data suggests that this selenoenzyme play the most important role in the stability of selenoproteins in condition of low Selenium conditions.

Selenium and iodine are also linked geographycally, most notably in Asia. Severe deficiency of both iodine and selenium occurs in a region extending from northeastern China and adjacent regions of Siberia and Korea to southwestern China, including Tibet.

Severe deficiency of both elements occurs also in Central Africa, in the Democratic Republic of Congo. There, as in China, iodine deficiency is more widespread.

Three disorders have been associated with selenium deficiency:

- Keshan disease

- Kashin-Beck disease

- Hypothyroid cretinism

Keshan disease is a cardiomyopathy affecting mostly young and middle aged women. It is characterized by multifocal myocardial necrosis and fibrosis and leads to cardiogenic shock and congestive heart failure. The name derives from Keshan country in northeastern China, the site of a large outbreak of the disease in the 1930s. Keshan disease can be prevented, though not treated, by selenium supplementation, and widespread supplementation and more varied diets have led to its near-disappearance in China.

The second disorder, Kashin-Beck disease, is named for two investigators who studied affected patients in Siberia in the 19^th century. It is an osteoarthropathy of the hands and fingers. Elbows, knee and ankles in children and adolescents. It is charcterized by the necrosis of growth plate and epiphyseal chondrocytes, as well’s proliferation of the surrounding chondrocytes, and it leads to short stature. Kashin-beck disease occurs in the same regions of China as Keshan disease, but it is more common and its prevention requires more selenium. The frequency of this disease in China is decreasing probably because of changes in diet; this trend has made it difficult to document the benefit from selenium supplementation alone.

Given the fact that selenium deficiency and iodine deficiency occur in the same regions of China snd tha goiter can occur both in people wit Keshan disease and in those with Kashin-Beck disease, a question arise spontly.

Can Iodine deficiency exacerbate or even cause some of the disorders attributed to selenium deficiency ?

Moreno-reyes in 1998 described the results of a study of iodine and selenium metabolism in children and adolescent in 11 villages in Tibet in which many of the subjects had Kashin-Beck disease and 1 village in which none had the disease. Severe selenium and iodine deficiency was present in all the villages. Among the subjects with Kashin Beck disease, the investigators found more severe iodine deficiency and moe hypothyroidism ( with some cretinism) than among unaffected people of the same villages and those from village in which there was no Kashin Beck disease. Interestingly the severity of selenium deficiency was similar in all three groups; suggesting that Kashin-Beck disease results from deficiency of both iodine and selenium. From this study Kashin-Beck disease is more a clinical consequence of iodine deficeincy than one of selenium deficiency.

The third disease is hypothyroid cretinism. Neurologic cretinism, consenquence of iodine deficiency during early pregnancy, is characterized from motor rigidity, a shuffling gait, deaf-mutism, and mental retardation, but not by hypothyroidism. Hypothyroid cretinism, consequence of iodine deficiency during infancy, is characterized by hypothyroidism, mental retardation, and growth retardation. It has been postulated that a deficiency of glutathione peroxidase could results in oxidative damage to the thyroid by hydrogen peroxid, which is produced in increased amounts in the thyroid gland of iodine-deficient subjects as a result of stimulation by thyrotropin.

Graves’ disease

Flaiani-Basedow-Graves’ disease affects approximately 0.5% of the population and is underlying cause of 50 to 80% of cases of hyperthyroidism. The hyperthyroidism of Graves’ disease is the result of circulating IgG antibodies that bind to and activate the G-protein-coupled thyrotropin receptor. This activation stimulates follicular hyperthrophy and hyperplasia, causing thyroid enlargement, as well as increases in thyroid hormone production and the fraction of triiodothyronine (T3) relative to thyroxine (T4) in thyroid secretion, from approximately 20% to as high as 30%.

Thyroid function testing in Graves’ disease typically reveals a suppressed serum thyrotropin level and elevated levels of T4 and T3, whereas a suppressed serum thyrotropin level with normal serum levels of T4 and T3 is referred as subclinical hyperthyroidism.

The female to male ratio among patients with Graves’ disease is between 5:1 and 10:1. The peak incidence is between 40 and 60 years of age, although the disease can occur at any age. The concordance rate for Graves’ disease among monozygotic twins is 35%. Triggers of Graves’ disease in people with genetic susceptibility to the disease include stressful life events, infections, and recent childbirth.

A family history of thyroid disease and several associated genetic loci have been identified , conferring susceptibility to Graves’ disease alone or to both Hashimoto’s thyroiditis and Graves’ disease.

Graves’ orbitopathy or ophalmopathy is clinically apparent in approximately 30 to 50% of patients affected by Graves’ disease, but it is detected in more than 80% of patients undergoing assessment by means of orbital imaging. As other manifestations of Graves’ disease it is believed that ocular manifestations of Graves’ disease are of autoimmune origin.

Graves’ ophtalmopathy is probably initiated by autoreactive T lymphocytes reacting with one or more antigens shred by the thyroid and orbit; after reaching the orbit and recognizing the shared antigens T lymphocytes trigger a cascade of events, including a secretion of cytokines. These cytokines stimulate the proliferation of orbital fibroblasts, expansion of adipose tissue and secretion of hydrophilic glycosaminoglycans from fibroblasts. Genetic determinants of Graves’ ophalmopathy remains poorly defined. Environmental factors appear to play a major role in the development and progression of Graves ophthalmopathy.

Concerning treatment option all patients with Graves ophtamopathy should discontinued smoke , should correct eventually present thyroid disfunctions.

Restoration of euthyroid state by antihtyroid drugs is associated with ophtalmopathy amelioration; whereas radioiodine therapy for Graves’ hyperthyroidism caused progression of ophtalmopathy in about 15% of patients. Risk factors for ophtalmopathy progression after radioiodine therapy are cigarette smoking, severe hyperthyroidism (T3), high levels of thyrotropin receptor antibodies, and uncontrolled hypothyroidism after radioiodine therapy.

Prophylactic treatment with glucocorticoid agents may be appropriate for many patients with Graves’ ophtalmopathy whose hyperthyroidism is treated with radioiodine therapy.

It is not clear at present if hyperthyroidism treatment with antithyroid drugs, thyroidectomy or radioiodine should have better outcomes in patients affected by Graves’ ophtalmopathy.

Specific treatment of Graves’ ophtamopathy varied depending from the severity of the disease.

Mild eyes disease usually does not require any treatment except local measures such as lubricans, ointments, dark lenses, prisms to reduce diplopia and now selenium administration has been demonstrated to be useful.

Moderate to severe eye disease should be treated with methylprednisolone intravenously at dose regimen of 1 gr a day for 3 consecutive days in particular if optic neuropathy is present.

If there is little or no improvement after 1 to 2 weeks, patients should be treated with surgical decompression, eye-muscle surgery and eyelid surgery to correct eyelid retraction.

Orbital irradaition may be an useful addition to therapy, particularly when eye motility is impaired. Orbital irradiation should be avoided in patients youngher than 35 years of ageand in patients with diabetic retinopathy or severe hypertension due to possible additional retinal damage.

Moreover in Moderate to severe and active ophtalmopathy intravenous or oral methylprednisolone treatment at lower doses should be used.

Combination of orbital irradiation and oral glucocorticoids therapy is more effective than either treatment alone.

Each treatment options is associated with a significant risk of relapse, particularly those receiving radioactive iodine and anti-thyroid drugs.

Recently small-molecule ligands of TSHReceptor are emerging sd promising targets. A research group led by Marvin C. Gershengorn at the National Institutte of Diabetes and Digestive and Kidney diseases in Bethesda, Maryland, USA have analyzed tens of thousands compounds, in orderto identify a family of small-molecule TSHR ligands, mostly agonists, and someones antagonists.

TSH and TSAbs bind to the large N-terminal domain of the receptor outside the cell, whereas the small molecule antagonists they identified likely bind to a transmembrane pocket.

These small molecule allosteric antagonists inhibit TSHR by preventing it from undergoing the conformational changes necessary for activation, rather than by interfering with TSH or TSAb binding. Seeking to optimizing the potency and solubility of the most promising small molecule anti-thyroid drug candidate of those screened, the researchers sythetized and tested six analogs of the TSHR “inverse agonist”, so termed because it inhibits basal, agonist independent TSHR signaling. Analog-1 has been demonstrated to be a better inverse agonist nad more efficicient TSHR antagonist than its parent. Micromolar concentrations of analog 1 inhibited TSHR basal signaling and TSH stimulated signaling in HEK-EM 293 cells engineered to stably express human TSHR. In this cells culture and in huan thyrocyte cultures, analog-1 also interfred with the TSAbs’ ability to induce TSHR signaling in sera from 30 different patients with Graves’ disease.

These are preliminary in vitro studies and much work should be done to investigate the response of analog-1 administration in vivo. However oral administration of a structurally similar molecule to mice showed enhanced thyroid hormone secretion and radioiodine uptake.

Another possible clinical application of analog-1 might be alleviating the symptoms of related ophtalmopathy, thought to be associated with reactivity to TSHR antibodies.

References

Gluthatione peroxidases

Flohe L, Gunzler WA, Schock HH. Glutathione peroxidase: a selenoenzyme. FEBS Lett 1973;32:132-34.

Chambers I, Frampton J, Goldfarb P et al. The structure of the mouse glutathione peroxidase gene:the selenocysteine in the active ite is encoded by the “termination” codon, TGA. EMBO J 1986;5:1221-7.

Rahman I, MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J 2000;16:534-54.

Rahman I, Mulier B, Gilmour PS et al. Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factor-kappaB. Biochem Pharmacol 2001;62:787-94.

Hoffmann PR. Selenium and asthma: a complex relationship. Allergy 2008;63:854-6.

Arsova-Sarafinovska Z, Matevska N et al. Glutathione peroxidase 1 (GPx1) genetic polymorphism, erythrocyte GPX activity, and prostate cancer risk. Int urol Nephrol 2008;41:63-70.

Ceriello A, Testa R. Antioxidant anti-inflammatory treatment in type 2 diabetes. Diabetes Care 2009;32:s232-6.

Ceriello A. The glucose triad and its role in comprehensive glycaemic control: current status, future management. Int J Clin Pract 2010;64:1705-11.

Ceriello A, Ihnat MA, Thorpe JE. The “Metabolic Memory”: is more than just tight glucose control necessary to prevent diabetic complications? J Clin Endocrinol Metab 2009;94:410-5.

Thioredoxin reductases

Dammeyer P, Damdimopoulos AE, Nordman T et al. Induction of cell membrane protrusions by the N-terminal glutaredoxin domain of a rare splice variant of human thioredoxin reductase I. J Biol Chem 2008;283:2814-21.

Kuster GM, Siwik DA, Pimentel DR et al. Role of reversible, thioredoxin-sensitive oxidative protein modifications in cardiac myocytes. Antioxid Redox Signal 2006;8:2153-9.

Deiodinases

Hutchinson JH, McGirr EM. Hypothyroidism as an inborn error of metabolism. J Clin Endocrinol 1954;14:869-86.

Hutchinson JH, McGirr EM. Sporadic non-endemic goitrous cretinism. Hereditary transmission. Lancet 1956;270:1035-7.

Murray P, Thomson JA, McGirr EM et al. Absent and defective iodotyrosine deiodination in a family some of whose members of goitrous cretins. Lancets 1965;1:183-5.

Bianco AC, Salvatore D, Gereben B et al. Biochemistry, cellular and molecular biology, and physiological roles of the iodothyronine selenodeiodinases. Endocr Rev 2002;23:38-89.

Gereben B, Goncalves C, Harvey JW et al. Selective proteolysis of human type 2 deiodinase: a novel ubiquitin proteasomal mediated mechanism for regulation of hormone activation. Mol Endocrinol 2000;14:1697-1708.

Steinsapir J, Harney J, Larsen PR. Type 2 iodotyronine deiodinase in rat pituitary tumor cells is inactivated in proteasomes. J Clin Invest 1998;102:1895-99.

Steinsapir J, Bianco AC, Buettner C et al. Substrate-induced down regulation of human type 2 deiodinase (hD2) is mediated through proteasomal degradation and requires interaction with the enzyme’s active center. Endocrinology 2000;141:1127-35.

Schneider MJ, Fiering SN, Pallud SE et al. Targeted disruption of the type 2 selenodeiodinase gene (DIO2) results in a phenotype of pituitary resistance to T4. Mol Endocrinol 2001;15:2137-2148.

Schneider MJ, Fiering SN, Thai B et al. Targeted disruption of the type I selenodeiodinase gene (DIO1) results in marke changes in thyroid hormone economy in mice. Endocrinology 2006;147:580-9.

De Jesus LA, Carvalho SD, Ribeiro MO et al. The type 2 iodothyronine deiodinase is essential for adaptive thermogenesis in brown adipose tissue. J Clin Invest 2001;108:1379-85.

Bartalena L, Marcocci C, Bogazzi F et al. Use of corticosteroids to prevent progression of Graves’ ophtalmopathy after radioiodine therapy for hyperthyroidism. N Engl J Med 1989;321:1349-52.

Bartalena L, Marcocci C, Bogazzi F et al. Relation between therapy for hyperthyroidism and the course of Graves’ ophtalmopathy. N Engl J Med 1998;338:73-8.

Wiersinga WM. Preventing Graves’ ophtalmopathy. N Engl J Med 1998;338:121-2.

Moreno-Reyes R, Suetens C, Mathieu F et al. Kashin-Beck osteoarthropathy in rural tibet in relation to selenium and iodine status. N Engl J Med 1998;339:1112-20.

Moreno JC, Bikker H, Kempers MJ et al. Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 2002;347:95-102.

Moreno JC, Klootwijk W, van Toor H et al. Mutations in iodotyrosine deiodinase gene and hypothyroidism. N Engl J Med 2008;358:1811-8.

Brent GA. Graves’ disease. N Engl J Med 2008;358:2594-605.

Bartalena L, Tanda ML. Graves’ Ophthalmopathy. N Engl J Med 2009;360:994-1001.

Marcoccci C, Kahaly GJ, Krassas GE et al. Selenium and the course of mild Graves’ orbitopathy. N Engl J Med 2011;364:1920-31.

Woeber KA. Triiodothyronine production in Graves’ hyperthyroidism. Thyroid 2006;16:687-90.

Toft AD. Subclinical hyperthyroidism. N Engl J Med 2001;345:512-6.

Neumann S, Huang W, Titus S et al. Small-molecule agonists for the thyrotropin receptor stimulate thyroid fuction in human thyrocytes and mice. Proc Natl Acad Sci USA 2009;106:12471-6.

Neumann S, Huang W, Eliseeva E et al. A small molecule inverse agonist for the human thyroid stimulating hormone receptor. Endocrinol 2010;151:3454-9.

Lawrence-Moon Biedl-Bardet Syndrome and ciliopathies

2011-05-15T19:51:00.000+02:00

Zimmermann is reported to be the first person to observe cilia in 1898. Immotile cilia have been considered unuseful organelles; however in the last 10 years many progresses have been made in understanding the role of immotile ”primary” cilia in normal mammalian tissues. These progresses have been possible thanks to the positional cloning of genes encoding structural components of primary cilia, experiments on imbred mouse and rat models of polycystic kidney disease (PKD), phylogenetic analysis of known ciliary genes, and charcterization of ciliary proteomes. In particular mutations in ciliary genes give rise to a multitude of human monogenic disorders that are now collectively called “Ciliopathies”, recently reviewed in a Review Article on the issue n. 16 of New England Journal of Medicine. Primary cilia are now acknowledged as vital cellular components, with specific functions as chemosensors or mechanosensors of the extracellular environment, so tha the function of ciliary proteins has been included into chapters of molecular pathology and cell biology. Ciliopathies have a broad range of phenotypes encompassing a number of different autosomal recessive or dominant syndromes of previously unknown aetiology. Ciliated cells are usually higly specialised, post-mitotic cell type, and they are therefore non-proliferating, differentiated cells. Almost all epithelial cells are ciliated, and they commonly exist as a sheet of polarized cells forming a tube or tubule with the cilia projecting into the lumen. In this way the cilia are exposed to the contents of the lumen, where they can provide a sensory role, mediating a specific signalling cascade. The results are the release of soluble factors in the external environment, a secretory role in which soluble proteins are released to have an effect downstream of the fluid flow. The cell biology of the ciliopathies can be considered in terms of functional loss or complete absence of a particular protein. The most common example of this functional loss of specialized proteins by ciliary epithelial cells is represented by outer segment of retinal rod cells, where the altered primary cilia with a compromised protein transport across the photoreceptors-connecting cilium causes the retinitis pigmentosa or retinal dystrophy. Ciliopathies can be classified according to the presence of an intact epithelium with aberrant functions or the complete absence of a mature ciliated epithelial cells. To the first group we can include the aetiology of anosmia; this symptom can be explained as an occasional manifestation of ciliopathies, because odour receptors of the olfactory epithelium are positioned on the cilia of olfactory neurons.

One obvious characteristic of the ciliopathies is the extensive heterogeneity for conditions such as Lawrence-Moon Biedl-Bardet syndrome (BBS). This is a an autosomal recessive pleiotropic condition with multiorgan involvment and variable severity. It is characterized by obesity, retinal degeneration, polydactyly, renal and gonadal malformation and behavioural and developmental problems. Twelve BBS associated genes have been identified to date called progressively BBS1 to BBS12; at least seven of the enchoded proteins (BBS1,3,4,5,6,7, and 8) are known to be associated with either centriole/basal body or the axoneme of primary cilia. The BBS proteins have now been shown to interact in a multisubunit complex know such as “ BBS-some “; where BBS4 forms a complex with BBS1,2,5,7,8, and 9 , with BBS9 acting as a possible organizing subunit. This observation provides a mechanicistic explanation for both the potential epistatic interactions of BBS genes, and the wide variability in the severity of the phenotype.

BBS7 and BBS8 have been demonstrated to be essential for Intraflagellar Transport Cargo proteins (IFT) in normal cilial along axoneme of primary cilia. A possible role of BBS proteins in ciliary assembly and disassembly is suggested from observation that BBS proteins are present at centriolar satellites in non-ciliated cells, followed by translocation during cilia formation to primary cilia.

Interestingly BBS1 is associated with the vescicular trafficking protein Rabbin-8, the loss of which leads to depleted ciliation and disruption of BBS4 localization in pre-ciliated cells. The normal function of Rabin-8 at the basal body appears to be the activation of protein that migrate to the cilia and that are involved in cilia elongation.

Once inside the cilium, it is possible that the BBS-some may act as an IFT cohesion factor responsible for linking IFT anterograde with IFT retrograde membrane raft vescicles transport.

In summary, we can affirm that the carefull study of a syndromic pleiomorphic complex such as BBS leds many insights to understanding normal physiopathology of previously underecognized structures such as immotile cilia. That these structures are almost ubiquitarious in epithelial cells, accounting for chemosensory and mechanosensory functions, through mediation of interaction between intracellular and extracellular environment, including chemical and physical extracellular alterations.

The study of this syndromic complex, I have the chance to follow in a patient, during my Medical Studies courses at Turin University, at Institute of Internal Medicine, with Professor GianMichele Molinatti, has been object of intensive research also during early late ‘80 for his peculiar clinical features, suggesting to professors working at this time an interesting area of research.

References

Tayeh MK, Yen HJ, Beck HS et al. Genetic interaction between Bardet-Biedel syndrome genes and implications for limb patterning. Hum Mol Genet 2008;17:1956-67.

Zaghloul NA, Liu Y, Gerdes JM et al. Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl Syndrome. Proc Natl Acad Sci USA 2010;107:10602-7.

Ansley SJ, Badanao JL, Blacque OE et al. Basal body dysfunction is a likely cause of pleiotropic Bardet_Biedl syndrome. Nature 2003;425:628-33.

Hildebrandt F, Benzing T, Katsanis N. Ciliopathies. N Engl J Med 2011;364:1533-43.

Are vertebral fractures to be diagnosed and treated irrespective from Bone Mineral Density values ?

2011-05-02T10:22:00.000+02:00

Vertebral fractures

Accurate detection of vertebral fracture is essential for risk assessment of individual patients in clinical practice, for determining the drug efficacy in clinical trials and for evaluating the prevalence and incidence of osteoporotic disease in a given population.

A prevalent vertebral fracture is the strongest predictor of subsequent vertebral fracture as well’s of any subsequent osteoporotic fracture.

A vertebral fracture results in a 4.4 fold increase risk of future vertebral fracture in people with a prevalent fracture.

Although bone mass is an important component of the risk of fracture, other abnormalities occur in the skeleton that contribute to fragility fractures.

In addition, a variety of nonskeletal factors, such as the liability to fall and force of impact, contribute to fracture risk. In this view an accurate assessment of fracture risk should ideally take into account other readly measured indices of fracture risk, including in particular those adding more informations to that provided by Bone Mineral Density measurement.

It has been suggested that the ability of Bone Mineral Density to predict a fracture is comparable to the use of blood pressure measurements to predict stroke and better than serum cholesterol levels to predict myocardial infarction.

At the age of 50 years, the proportion of women with osteoporosis who will fracture their hip, spine, forearm or proximal humerus in the next 10 years is about 45%. However the detection rate of these fractures (i.e. sensitivity) is low, and 90% of such fractures would occur in women without osteoporosis.

Low sensitivity is one of the reasons why widespread population-based screening is not widely recommended in women at the time of menopause.

Vertebral fractures assessment

The problem is besides all the definition of vertebral fracture, at present there’s no Consensus Giudelines on osteoporotic vertebral fracture definition.

Identification of vertebral fracture can be very difficult, because the shape of normal vertebral bodies varies widely between individuals. Vertebral bodies can be present abnormal features because non-osteoporotic deformities and errors in radiological projection can induce a misdiagnosis of fractured body.

We have to remember that about 50% of vertebral fractures are asymptomatic and therefore are only casually identified. They are not the source of pain !Even when chest radiographs or vertebral images are correctly obtained only 35 to 50% of all radiographic vertebral fractures are correctly reported. It has been estimated that only 19% of these fractures reach clinical attention and can be correctly treated with a antiosteoporotic treatment. In view of high radiation exposure routine chest and lumbar radiographs are not recommended, but the availability of vertebral imaging using DEXA take the advantage of utilize an image of near radiographic quality available with a low fraction of the radiation dose.

Imaging vertebral fractures using DXA is called Vertebral Fracture Assessment (VFA).

The disadvantage of VFA is poor image resolution compared to conventional radiography, CT or MRI and the increased difficulties in imaging the thoracic spine, expecially above T7. Between 5 to 15% of thoracic vertebrae can be visualized only by conventioonal radiography.

The sensitivity and specicificty of this approach compared with conventional radiography varies with the kind of approach used to definy a vertebral fracture:

- Morphometric

- Semiquantitative (SQ)

- Visual identification

Irrespective of the approach, sensitivity is only moderated for for mild fractures corresponding to Genant grade 1 vertebral fracture showing a sensitivity of 54%, due to low imge resolution of this technique.

The sensitivity for identifying moderate to severe vertebral fractures (corresponding to Genant grade 2 and 3 fractures ) is substantial higher ranging from 90 to 94%.

Specificity is high, with a result between 94 to 99%, compared to conventional radiography.

Morphometric and visual identification using DXA images in children can be especially problematic because currently available software cannot detect the vertebrae in most children. That’s why we use total body DXA evaluation in children.

One advantage of DXA imaging is that the scan are not subjected to the same degree of projection distortion as conventional radiography because the X-ray beam is always orthogonal to the spine. Reducing the X ray diffraction effect.

Moreover DXA reduces the frequency that soft tissue obscure the endplates compared to single energy mode.

Side-by-side viewing facilitates the identification of incidental vertebral fractures.

Morphometrical analysis

It uses the measurement of vertebral height to define vertebral fractures. A normative daatabase is established against which the vertebrae are compared, There are a number of different morphometric approaches that vary with the criteria by which they define a vertebral fracture and in the reference data used, The most widely used approaches to identify prevalent and incidental vertebral fractures are the two different algorithms proposed by McCloskey et al (3) and Eastell R et al (4). Morphometric analysis has a high sensitivity and moderate high specificity in discriminating between normal vertebrae and fractured vertebrae. Moreover, all the morphometric approaches for defining prevalent and incident vertebral fractures are correlated with clinical risk factors for vertebral fractures.

The approach used can ahve a significant impact on the prevalence of vertebral fractures identified, varying from 3% to 90%.

A loss of vertebral height of 20 to 25 % is usually used to define an incident vertebral fracture; using this definition comparable ability to identify any vertebral frcture is present irrespective of aproach used to define a baseline fracture.

As mentioned above VFA is more effective in identifying moderate to severe deformities with a sensitivity of 81.6% for grade 2 deformities, whereas mild grade 1 deformities identifiation has a sensitivity as low as 22%.

Finally , the precision error is small if compared with the reduction in vertebral height of 20 to 25% threshold used to define vertebral fractures and it is less using conventional radiology than using VFA.

Semiquantitative analysis

SQ analysis combines measurements of vertebral height with subsequent evaluation of all vertebrae with a short vertebral height by an expert reader. This combined approach enables the identification of non-osteoporotic fracture vertebral deformities, which are not identified using morphometric analysis alone. As a consequence, SQ analysis is able to reduces false positive results.

The most widely used SQ analysis is that of Genant HK (6). Baseline or prevalent vertebral fractures are graded from “0” equal to normal to “3” equal to severe fracture, and incident fractures are defined as an increase of more than or equal than 1 grade on follow-up radiographs.

Genant grade 1 corresponds to an 20 to 25% reduction in anterior, middle or posterior height

Genant grade 2 corresponds to a 25 to 40% reduction in any height

Genant grade 3 corresponds to more than 40% reduction in any vertebral height

Mild grade 1 SQ vertebral deformities are frequently not associated with low BMD values.

The interobserver agreement for conventional radiographs or DXA images is similar with a K score of 0.53 and 0.51 respectively.

This approach is currently those recommended by International Society of Clinical Densitometry for diagnosing vertebral fractures with VFA.

Visual Identification by an expert reader (Algorithm Based Qualitative Approach)

ABQ approach differs from SQ analysis because the last one is based only on variations of vertebral height; not considering variations on endplates cracks or breaks as the primary event with a subsequent evaluation of vertebral height. ABQ focus more attention on the vertebral endplate alterations rather than on short vertebral height. Using ABQ we have a greater association with low BMD and interobserver agreement for radiography and DXA images of 0.74 and 0.65 respectively. So that mild vertebral fractures identified with ABQ are more strngly associated with osteoporosis than when this mild fractures are identified with SQ method.

The definition of vertebral fractures includes the presence of breaks in the cortex of vertebral body; these breaks always occurs in the center or either the superior or inferior endplates that are the weakest area of endplate because it is more distant from the strong outer vertebral ring. As a consequence, the endplate buckles or collapses under pressure because of interventebral disc and it results in a concave appearance to the superior and/or inferior endplate. If the concavity extend beyond the inner border of the vertebral ring , it is unlikely to represent an osteoporotic fracture. A vertebral fracture initially involves a crack of the superior or inferior endplate with or without the simultaneous loss of vertebral height. As severity of the fracture progresses, the vertebral ring fractures resulting in loss of height and buckling of the anterior, lateral and occasionally posterior cortex. It is important to outlined these aspects because there is considerable variation in vertebral shape resulting in osteoporotic and non osteoporotic deformities that can result in considerable intraobserver error even among expert readers.

Commonly we can see wedge deformity fracture associated with endplate fracture where is present a fracture of the anterior cortex of vertebral body.

A true compression fracture associated with endplate fracture is an osteoporotic compression fracture of superior endplate associated with fracture of anterior and posterior cortex of vertebral body.

It is also important to identify the different characteristics of high trauma burst fractures. There is usually an history of high trauma injury (such as a car accident, fall from a significant height) immediately resulting in acute, severe, localized back pain with localized tenderness.

Also the presence of intravertebral edema during MRI, used in the studies under discussion is not universally accepted target of vertebral osteoporotic fracture. Such as CT scan, also MRI, are usually only required in the presence of localized pain, focal neurological signs, or symptoms suggesting cord compression or a radiculopathy, or the clinical suspicion of primary or metastatic lesions, but not in osteoporotic patients.

Osteoporotic fractures are rare above T4 and in this settings, it is very important to consider metastatic lesions and , if appropriate to investigate for a primary neoplastic lesion.

Variations in shape or size of the vertebrae or of the vertebral endplate can be caused by degenerative diseases (osteoarthrosis), congenital deformities and metastatic lesions.

In addition, the aging skeleton, particularly in women, may develop slight wedging because of remodelling without depression or break in the endplate or cortex.

Scheuermann’s disease is frequently associated with a short anterior vertebral height in combination with irregularity of the whole superioe and/or inferior endplates. It appears to be isolated in a single vertebral body or involving adjacent vertebrae.

Schmorl’s nodes consist of a rounded flash-like break in the superior or inferior endplatein either the anteroposterior or lateral view, which rarely affects more than 25% of the endplate. They are found in about 35-75% of population and are formed by extrusion or erniation of the nuclear material from the interventebral disk into the vertebral body.

Degenerative osteomalacic changes are present in a vertebral body involved by an uniform or symmetrical concavity of the superior and inferior endplates. It is associated with a generalized thinning and reduced density of all vertebral bodies.

Clinical recommendations for screening for vertebral fractures

The current recommendations for using fracture assessment through DXA imaging (VFA) by the International Society of Clinical Densitometry are:

When the results may influence clinical management
If BMD is indicated then consider performing VTA if clinically indicated in:

- Documented height loss greater than 2 cm

- Historical height loss greater than 4 cm since young adult

- History of fracture after 50 years old

- Commitment to long term oral or parental glucocorticoid therapy

- History or findings suggestive of vertebral fracture not documented by previous radiographic imaging

Althoough risk factors can provide guidance to identify which patients require screening for osteoporosis, very few subjects would be prepared to initiate long-term therapy to prevent a fracture without confirmation of a diagnosis of osteoporosis using DXA scan.

If a patient has osteopenia and a fragility fracture at any site, the majority of physicians would intervene with therapy.

Therefore, it is reasonable to screen all patients with osteopenia using VFA, if it will alter the management of the patient. In a study at Mayo Clinic 16% of patients 60 to 69 years old and 45% of those older than 70 years had a previously undiagnosed vertebral fracture on VFA.

From Kanis JA ( Professor Emeritus at Sheffield University, UK )

Fracture Risk Quantification

Concurrent considerations of risk factors that operate independently of BMD improuve evaluation of fracture risk.

The best example is age. The same T-score (i.e. the number of Standard Deviations from BMD found on people 35 years old) has different significance at different ages. For any BMD value, fracture risk is much higher in the elderly than in the young people. This is because age contributes to fracture risk indepently of BMD.

In general, risk factor scores show relative poor specificity and sensitivity in predicting either BMD or fracture risk. However, some risk factors vary in importance according to age. For example risk factors for falling, such as reduced mobility, sedatives use, visual impairment are more strongly predictive of fracture in the elderly than in younger individuals.

A series of meta-analyses has been undertaken to identify clinical risk factors that could be used in case finding strategies with or without the use of BMD measurement:

Low body mass index (BMI): A low BMI is a significant risk factors for hip fracture. 20 kg/m2 vs 25 kg/m2 shows a RR of 1.27, whereas 30 Kg/m2 vs 25 Kg/m2 shows a RR of 0.89.
Fragility fracture after 50 years of age : RR 1.86. In other words the presence of prior vertebral fracture approximately doubles the risk of having another fracture.
Parental history of hip fracture : RR 1.54 an increase in risk independent from BMD value.
Smoking is a RR 1.29
Ever use of corticosteroids: RR 1.65
Alcohol intake shows an increase in fracture risk that is dose dependent. Where the alcohol intake is on average 2 Units or less daily,there is no increase in risk. Intakes of 3 or more Units daily are associated with a dose-dependent increase in risk. RR 1.38
Rheumatoid Arthritis RR 1.56. In contrast to many causes of secodary forms of osteoporosis rheumatoid arthritis causes an increase in fracture risk indenpendently of BMD and the use of glucocorticoids.

The multiplicity of these risk factors poses problems in the units of risk to be used. Also if the Relative Risk can be used, the best suited for clinician is the absolute risk ( or probability ) of fracture. The absolute risk dependes on age and life expenctancy, as well’s from the current relative risk. In general lifetime risk of fracture decreases with age, in particular after 70 years of age, because the risk of death with age outweight the increasing incidence of fracture. Estimates of lifetime risk are less relevant in assessing individual clinical risk of fracture in order to choose a therapeutic intervention. So that it has been recommended the use of a short term absolute risk ( i.e. a probability over 10-year interval), 10 years interval covers the likely duration of treatment and average life expectancy in elderly 60 year or older.

What about heart failure ?

Recently Heart failure has been identified such a risk factor for fragility fractures in two large studies by Ezekowitz JA on 2008 and by Carbone L on 2010.

Heart Failure is a leading cause of hospitalization and mortality in Europe and North America. Successfully enhanced treatment rates of hypertension and survival after myocardial infarction have produced a delay in the incidence in heart failure. So that the median age of heart failure patients in clinical trials and large epidemiological studies ranges between 65 and 75 years of age. Such patients are notably at risk for other co-morbid conditions causally related or not such as bone fracture.

Osteoporosis is one of such co-morbidity affecting 1 in 4 women and 1 in 8 men over 50 years old and it is known to be clinically evident with fragility fractures.

A central unanswered question is now evident: does heart failure lead to osteoporosis and fragility fractures or is it a passive participant in a population at risk of both diseases ?

A first answer comes on 1997 in a description of 101 patients with endstage heart failure awaiting cardiac transplantation, low bone mass was common, as vitamin D deficiency and Hyperparathyroidism.

What are the possible links between osteoporosis, fragility fractures, and heart failure ? Many scientists point to shared risk factors for both diseases such as older age, smoking, diabetes, renal dysfunction, inactivity, and poor nutrition.

An interesting role in linking bone loss to heart failure should be hyperaldosteronism always present in heart failure.

Elevated aldosterone levels have been associated with urinary magnesium and calcium wasting, causing secondary hyperparathyroidism.

Heart failure patients treated with spironolactone, a known aldosterone antagonist, showed few fractures compared to matching heart failure affected patients. It is not established if the consequent reduction in hyperparathyroidism is the cause of increased bone repair, increased mineralization, or mineral retentions in particular calcium and magnesium.

Future researches are needed in order to examine biomarkers, imaging, and clinical outcomes related to bone health after carefully clinically phenotyping patients with heart failure.

References

Cameron JR, Sorenson J. Measurement of bone mineral in vivo: an improved method. Science 1963;142:230-2.

Smith R, Walker R. Femoral expansion in aging women: implications for osteoporosis and fractures. Science 1964;217:945-8.

Tinetti ME. Clinical Practice. Preventing falls in elderly persons. N Engl J Med 2003;348:42-9.

Ahlborg HG, Johnell O, Turner CH et al. Bone loss and bone size after menopause. N Engl J Med 2003;349:327-334.

Khosla S, Melton LJ III. Osteopenia. N Engl J Med 2007;356:2293-300.

Abendschein W, Hyatt GW. Ultrasonic and selected physical properties of bone. Clin Orthop 1970;69:294-301.

Ahlborg HG, Nguyen ND, Nguyen TV et al. Contribution of hip strength indeces to hip fracture risk in elderly men and women. J Bone Min Res 2005;20:1820-27.

Bolotin HH, Sievanen H. Inaccuracies inherent in dual-energy X-ray absorptiometry in vivo bone mineral density can seriously mislead diagnostic/prognostic interpretations of patients-specific bone fragility. J Bone Miner Res 2001;16:799-805.

Looker AC, Beck TJ, Orwoll ES. Does body size account for gender differences in femur bone density and geometry? J Bone Miner Res 2001;16:1291-9.

Richards JB, Leslie WD, Joseph L et al. Changes to osteoporosis prevalence according to method of risk assessment. J Bone Miner Res 2007;22:228-34.

Lewis CE, Ewing SK, Taylor BC et al. Predictors of non-spine fracture in elderly men: the MrOS study. J Bone Miner Res 2007;22:211-9.

Mussolino ME, Looker AC, Madans JH et al. Risk fators for hip fracture in white men: the NHANES I epidemiologic follow up study. J Bone Miner Res 1998;13:918-24.

Melton LJ III, Atkinson EJ, O’Connor MK et al. Bone density and fracture risk in men. J Bone Miner Res 1998;13:1915-23.

Amin S, Zhang Y, Felson DT et al. Estradiol, testosterone, and the risk for hip fractures in elderly men from the Framingham study. Am J Med 2006;119:426-33.

McCloskey EV, Spector TD, Eyres KS et al. The assessment of vertebral deformity: A method for use in population studies and clinical trials. Osteoporosis Int 1993;3:138-47.

Eastell R, Cedel SL, Wahner HW et al. Classification of vertebral fractures. J Bone Miner Res 1991;6:207-15.

Genant HK, Jergas M, Palermo L et al. Comparison of semiquantitative visual and quantitative morphometric assessment of prevalent and incident vertebral fractures in osteopororsis. The Study of Osteoporotic Fractures Research Group. J Bone Miner Res 1996;11:984-96.

Rea JA, Li J, Blake GM et al. Visual assessment of vertebral deformity by X-ray absorptiometry: A highly predictive method to exclude vertebral deformity. Osteoporosis Int 2000;11:660-8.

Siminoski K, Jiang G, Adachi JD et al. Accuracy of height loss during prospective monitoring for detection of incident vertebral fractures. Osteoporosis Int 2005;16:403-10.

Siminoski K, Warshawski RS, Jen H et al. The acuracy of historical height loss for the detection of vertebral fractures in postmenopausal women. Osteoporosis Int 2006;17:290-6.

Schousboe JT, Ensrud KE, Nyman JA et al. Cost-effectiveness of vertebral fracture assessment to detect prevalent vertebral deformity and select postmenopausal women with a femoral neck T-score lower than 2.5 SD for alendronate therapy: A modeling Study. J Clin Densitom 2006;9:133-43.

Hiu SL, Slemenda CW, Johnston CC. Age and bone mass sd predictor of fracture in a prospective study. J Clin Invest 1988;81:1804-9.

Kanis JA, McCloskey EV. Evaluation of the risk of hip fracture. Bone 1996;18:127-32.

Kanis JA, Johnell O, Oden A et al. Ten-year risk of osteoporotic fracture and the effect of risk factors on screening strategies. Bone 2001;30:251-8.

Kanis JA. Diagnosis of osteoporosis and assessment of fracture risk. Lancet 2002;359:1929-36.

Shane E, Mancini D, Aaronson K et al. Bone mass, vitamin D deficiency, and hyperparathyroidism in congestive heart failure. Am J Med 1997;103:197-207.

Jankowska EA, Jakubaszko J, Cwynar A et al. Bone mineral status and bone loss over time in men with chronic systolic heart failure and their clinical and hormonal determinats. Eur J Heart Fail 2009;11:28-38.

Van Diepen S, Majumdar SR, Bakal JA et al. Heart failure is a risk factor for orthopedic fracture: a population-based analysis of 16.294 patients. Circulation 2008;118:1946-52.

Carbone L, Buzkova P, Fink HE et al. Hip fractures and heart failure: findings from the Cardiovascular Health Study. Europ Heart J 2010;31:77-84.

Carbone LD, Cross JD, Raza SH et al. Fracture risk in men with congestive heart failure risk reduction with spironolactone. J Am Coll Cardiol 2008;52:135-8.

Aknowledgements: I would thanks Professor JA Kanis for his unvaluable studies on osteoporosis and bone diseases prevention, I’ve meet first time on Jerusalem, Israel; attending to The XXI European Symposium on Calcified Tissues hold on March 12-16 1989 ( at this time working on Paget’s disease of bone ! ) and later on many International Congresses worldwide. A man plenty of entusiasm and love for bone diseases and human health problems worldwide.

Proteoglycans as mechanotransducers: syndecans and dystroglycans.

2011-03-31T09:50:00.000+02:00

Mechanotransduction in cell physiology

A common denominator of many mechanobiology diseases is the disruption of the intricate force transmission between the extracellular matrix, the cytoskeleton and the interior of the nucleus.

Cellular mechanosensing is based on force-induced conformational changes in mechanosensitive proteins that are subjected to molecular forces, leading to opening of membrane channels, altered affinities to binding partners, thereby activating signalling pathways.

Any changes in normal intracellular force transmission through changes in cellular ( or extracellular ) structure and organization can lead to altered molecular forces acting on these proteins, resulting in changes in mechanosensitive signals.

All cells and organisms across the evolutionary spectrum, from the most primitive to the most complex, are mechanosensitive. This universal property allows cells to relay mechanical stimuli from their physical surroundings or from within the organism to electrochemical or biochemical signals, which the regulate a wide repertoire of physiological responses.

This mechanotransduction is essential to developmental pathways and to normal tissue homeostasis, primarly in the maintenance of tissues in which cellular adaptive responses are crucial to counteract substantial variations from normal conditions. This is the case for sensing externally applied forces such as “touch sensation” or in regulating forces and tension in the body such as in muscle tension and blood pressure regulation. In the first example muscle tissue responds to exercise with hyperthrophic growth ( i.e. an increase in cell size ), whereas in the second example vascular system can maintain a constant blood pressure despite changes in cardiac output by vasoconstriction and vasodilatation ( i.e. contraction or relaxation of smooth muscle cells that surrounds blood vessels ).

Generally speaking skeletal and cardiac muscles can respond to increased load such as intensive resistance exercise, with hypertrophic growth, whereas immobilization lead to muscle atrophy over time.

Bone tissue paradigm

Another example for the role of mechanotransduction in tissue maintenance is bone.

Compact bone is formed by concentric layers of bone matrix, in which small cavities known as Lacunae are interspersed at regular intervals. These lacunae harbour osteocytes so that they are connected through canaliculi, forming a true network of interconnecting canals.

Gravity and compressive forces that are generated by muscle contractions during locomotion result in small deformations of the poro-elastic bone, resulting in a pressure gradient that drive interstitial fluid flow through the lacunae-canalicular network.

This load induced fluid flow is thought to stimulate localized bone remodelling and optimize the physical performance of the bone through mechanotransduction signalling.

Likewise chondrocytes adapt to widely varying stresses by secreting glycosaminoglycan-ric ECM that gives cartilage its dynamic mechanical properties.

The ability to cells to respond to changes in their physical environments is crucial in the development and maintenance of tissue integrity, when it has been exposed to varying mechanical stresses as we have for muscle and bone tissue.

On the cellular level, mechanotransduction can modulate different functions such as protein synthesis, secretion, adhesion, migration, proliferation, apoptosis. Consequently, defects in cellular mechanotransduction can result in, or contribute to various human diseases.

Alternatively, changes in cellular physical environment can elicit pathological consequences, even when mehanotranduction function properly. For skeletal tissue we can think to effects of microgravity on bone mass loss due to increased bone remodelling.

Many human diseases are caused by defects in cells-extracellular matrix coordination so that they are potentially correctable by altering tissue environment or cells structure. For example, progressive tissue remodelling in chronic atherosclerosis, wound repair, bone and neuronal degenerative diseases ultimately are caused by a loss of tissue integrity. The pathogenesis of inflammatory diseases, thrombotic disease and cancer too is ultimately related to altered tissue adhesion, aberrant cell aggregation and pathologic cell migration. Interestingly, the incidence of many of these conditions is growing in parallel with increasing longevity in the population.

Cell surface receptors that mediate cell-extracellular matrix interactions are primarily members of two gene families: the integrins and the syndecans.

Nearly all ECM molecules contain binding sites for both types of receptor and there is substantial evidence that a full cell response requires engagement of both types of receptor. Integrins and syndecans are required for generating a physical link to the cytoskeleton, for force transduction, for spatial control of assembly of the adhesion signalling complex and for regulation of cytoskeletal dynamics.

The syndecans are a family of membrane-linked proteoglycans, each with a core protein with covalently attached Heparan Sulphate or Chondroitin Sulphate glycosaminoglycan sugar. There are four members of the syndecan family in mammals, of which three have a restricted distribution, whereas syndecan 4 is ubiquitously expressed. The syndecans act as receptors for ECM proteins such as fibronectin, for growth factors such as FGF2 or VEGF, engaging ligands with the large flexible glycosaminoglycan chains that make them ideal receptors for ligands that are diluted or distant from the membrane.

The cytoplasmic domain of syndecans comprise a pair of conserved regions, present in all isoforms, and a variable region that is unique to each syndecan. The variable region of syndecans-4 is the binding site for protein kinse C alfa, whereas conserved regions binds respectively Src/Fyn Tyrosine Kinases and PDZ-domain containing proteins.

The most intriguing cooperation demonstrated between two kind of receptors is the cooperation between alfa2beta1 integrin and alfa6beta4 integrin with syndecans during adhesion to laminin.

The molecular connection between adhesion receptor occupancy and cytoplasmic signalling concerning integrin receptors has only recently been clarified by the demonstration of a complex between cytoplasmic tail of beta3 integrin and the head domain of the cytoskeletal protein “talin”.

On the contrary cytoplasmic signalling downstream of synecan-4 is much easier to explain, owing to the presence of well characterized PKC alfa and PDZ domain-binding sites in he cytoplasmic domain of syndecan-4. Moreover putative interactions wit cytoplasmic portion of syndecan-4 with tyrosine kinase complexes and membrane binding proteins shoould also play a very important role.

Synthenin is a PDZ scaffold protein involved into syndecan-4 mediated endosome formation and cell spreading. So that communication between integrin and syndecan is responsible for the delicate balance in integrin cycling that is necessary for rapid cell migration.

References

Burger EH, Klein-Nulend J. Mechanotransduction in bone – role of the lacuno-canalicular network. FASEB J 1999;13:S101-S112.

Kein-Nulend J, Bacabac RG, Veldhuijzen JP. Microgravity and bone cell mechanosensitivity. Adv Space Res 2003;32:1551-9.

Trautman MS, Kimelman J, Bernfield M. Developmental expression of syndecan, an integral membrane proteoglycan, correlates with cell differentiation. Development 1991;111:213-20.

Hozumi K, Suzuki N, Nielsen PK et al. Laminin alfa 1 chain LG4 module promotes cell attachment through syndecans and cell spreading through integrin alfa2beta1. J Biol Chem 2006;281:32929-40.

Ogawa T, subota Y, Hashimoto J et al The short arm of laminin gamma 2 chain of laminin 5 ( laminin-332) bids to syndecan-1 and regulates cellular adhesion and migration by suppressing phosphorylation of integrin beta4 chain. Mol Biol cell 2007;18:1621-33.

Takadoro S et al. Talin binding to integrin beta tails: a final common step inintegrin activation. Science 2003;301:103-6.

Davies KE, Norwak KJ. Molecular mechanism of muscular dystrophies: old and new players. Mature Rev Mol cell Biol 2006;7:762-72.

Morgan MR, Humphries MJ, Bass MD. Synergistic control of cell adhesion by integrind and syndecans. Nature Rev Mol Cell Biol 2007;8:957-69.

Jaalouk DE, Lammerding J. Mechanotransduction gone awry. Nature rev Mol Cell Biol 2009;10:63-73.

Note: all scientific material here writed must be view as “non original” proper scientific deductive material. However it is originated from scientific reading during mine studies of “senior-lecturer” and so reported from scientific articles cited in the References and report only points of scientific literature jet clarified or anyway postulated by scientists cited in the articles reported in References.

I’m sorry; I’m always late about autoinflammatory diseases, you know …..

2011-02-26T12:39:00.001+01:00

URIC ACID

The sharp increase in hypertension, obesity, diabetes and kidney diseases in the last century has also been associated with an increase in serum uric acid levels in men from 3.5 mg/dl in 1920 to 6.5 mg/dl in 1970, in women from 2.5 in 1920 to 5.5 in 1970 probably thanks to an uricosuric effect of estrogens.

It has been hypothetized a physioogical role for uric acid such as antioxidative agent, so that elevation of uric acid levels can be due to reduced glomerular filtration rate, hyperinsulinemia, diuretic use, alcohol use, oxidative stress.

It is worth noting that humans and apes have higher uric acid levels, since they lack the hepatic enzyme uricase, which degrades uric acid to allantoin. Rats for example have an uricase enzyme so that they can degrade plasma uric acid.

Hypertension linked to uric acid production seems to be related to reduction in endothelial nitric oxide levels due to an uric acid mediated renal vasoconstriction, with activation of renin-angiotensin system.

In cultured vascular smooth muscle cells uric acid induces cellular proliferation, inflammation, oxidative stress and activation of renal-angiotensin system. Interestingly hypertension in rats model treated with uricase inhibitor, also after the uricase inhibitor remotion so with stimulation of nitric oxide production, and blockage of renin-angiotensin system, hypertension is maintained. Only a diet low in salts can lower blood pressure in hypertensive rats.

Over the past 200 years there has been a wide increase in fructose intake in developed world temporally linked to the increase in hypertension and obesity. We know that fructose causes a rapid decrease in ATP levels, increasing the generation of uric acid and its release into plasmatic fluids.Ingestion of other foods such as purine rich fatty-meats or drinks such as beer or exposure to toxins such as lead may alter the uric acid metabolism leading to an hyperuricemic hypertension.

Genetic polymorphism of genes coding for renal transporters or metabolism of uric acid can account for presence of hyperuricemia and hypertension in particular in young people.Solute Carrier Family 2, Member 9 (SLC2A9) a newly identified transporter for fructose and uric acid at kidney level presented various genetic polymorphisms that are associted with an increase risk of gout. Nevethless these polymorphisms have not been associated with hypertension.Other important geens involved into uric acid transport and founded to be associated with gout are SLC17A3, SLC22A12 and ABCG2.

Hystorically hyperuricemia has been observed in the metabolic syndrome and it was attributed to hyperinsulineamia because insulin reduces renal excretion of uric acid. Strong evidences suggest a role of uric acid in the development of metabolic syndrome; two mechanisms have been suggested to explaine how hyperuricemia can induces the metabolic syndrome. The first mechanism is related to the fact that muscle cells glucose uptake is partly dependent from increased blood flow mediated by the release of nitric oxide. Experimental evidences on hyperuricemic rats seem support such mechanism.The second point concernes the inflammatory and oxidative changes that uric acid induces in adipocytes. In addition xantine oxidoreductase, the enzyme that generate uric acid from xantine, is expressed into adypocytes and it is critical to the adipogenetic process.Hyperuricemia is trongly associated with vascular dementia, carotid artery stenosis, ictus and minor strokes as well’as preeclampsia. Interestingly some of the cardiovascular actiond attributed to Losartan therapy evidenced during LIFE Study (Losartan Intervention for Endpoints Reduction in Hypertension)and for atorvastatin in the Greek Atorvastatin and Coronary Hearth Disease evaluation (GREACE Study) have been attributed also to the activity of these drugs to lower uric acid levels. Concerning the molecular mechanisms that could explaine the action of uric acid at cellular level ; two main points have to be consider: first it seems to be involved into free radical formations, acting sometimes as antioxidant or pro-oxidant. This last action could be due to generation of free radicals during its degradation or by stimulating NAPH oxidase. Second uric acid can also stimulate innate immunity through the effect of microcrystalline uric salts on the function of dendritic cells and T lymphocytes.

INFLAMMATION AND URIC ACID

THE INFLAMMASOME

The notion of autoinflammatory diseases delineates a heterogeneous group of pathologies characterized by spontaneous periodic inflammation and fever in the absence of infection or autoimmune causes.

Hereditary Periodic fevers, Systemic Onset Juvenile Idiopathic Arthritis, Still’s disease, Behcet disease, and the metabolic disorders gout and pseudogout are examples of such inflammatory maladies. Increased production of the inflammatory IL-1beta was recently identified as the cause of several autoinflammatory diseases, providing clear evidence for a pivotal role of this cytokine in triggering autoinflammation.

Il-1 beta also known such as endogenous pyrogen, is a highly inflammatory cytokine whose production is tightly controlled at gene transcription and postranslational levels. The final activation of IL-1 beta involves the caspase1-activating complex, the best characterized being the inflammasome.

Inflammasome is formed by a member of NALP protein family, such as NALP1, NALP2 and NALP3 and the adaptor protein ASC that connects the NALPs with Caspase-1. Signals and mechanisms leading to activation of inflammasome are still poorly understood. Products of bacterial cell wall peptidoglycan (muramyl peptide) and Lipopolysaccaride (LPS) are known to be potent activators probably through the mediation of Toll-like receptors. However, the inflammasome is also efficient in sensing stress or endogenous danger signals, such as ATP depletion or hypotonic, hypoxic stress.

Monosodium urate crystals (MSU) have been identified as danger signals formed after the release of uric acid from dying cells. This observation and the well known rol of uric acid crystals in gouty arthritis, prompted to investigate whether MSU crystals could activate the inflammasome.Interestingly, calcium pyrophosphate dihydrate (CPPD) crystals another type of pathogenic crystal involved in pseudogout disease, has been demonstrated to be active such as MSU crystals. Interestingly crystals such as MSU and CPPD were able to activate caspase 1 processing of pro-IL1 beta.The human genome harbours a repertoire of 14 NALPs. It is currently not clear how many of them form inflammasomes.In addition to IL1 beta , MSU and CPPD crystals are known to induce the release of other cytokines such as TNF alfa, suggesting that inflammasome-independent activity of these crystals in release of TNF. Experments demonstrated that TNF production was relatively slow and was anyway preceded by IL 1 beta release. It was therefore possible that TNF secretion was initiated by the release of mature IL 1 beta in both macrophages and monocytes.

References

Wollaston HW. On gouty and urinary concretions. Phil Trans 1797;87:386-400.

Shi Y, Evans JE, Rock KL. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 2003;425:516-21.

Martinon F, Petrilli V, Mayon A et al. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 2006;440:237-41.

Vitart V, Rudan I, Hayward C et al. SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout. Nat Genet 2008;40:437-42.

Doring A, Gieger C, Mehta D et al. SLC2SA9 influences uric acid concentration with pronounced sex specific effects. Nat Genet 2008;40:430-6.

Taniguchi A, Urano W, Yamanaka M et al. A common mutation in an organic anion transporter gene, SLC22A12 is a suppressing factor for the development of gout. Arthritis Rheum 2005;52:2576-7.

Feig DI, Kang D-H, Johnson RJ. Uric acid and cardiovascular risk. N Engl J Med 2008;358:2811-21.

Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and proessing of pro-IL1beta. Mol Cell 2002;10:417-26.

Kisspeptins: how neuropeptides could “kiss” human brain and regulate reproductive activity.

2011-02-09T21:05:00.000+01:00

KISSPEPTINS

Nearly 40 years after the discovery of Gonadotropic Releasing Hormone (GnRH), the identification of neuronal pathways through which internal hormonal signals, such as gonadal steroids, stress hormones, and nutrient signals as well’s external environmental cues, such as day length and social relationship, regulate GnRH release is still a major unresolved issue in the field of neuroendocrinology.

Most of recent studies on GnRH secretion have been directed on discovery of KISSPEPTIN-neurons actions, studying the brain areas of Pre-Optic Area (POA) and Hypothalamic neurons.

Originally discovered as a metastasis suppressor gene in 1996, KISS1 was named for its role as a suppressor sequence “ SS “; whereas the letters “KI” were appended to the prefix SS to form KISS in homage to the location of its discovery , HERSHEY, PENNSYLVANIA, USA, home of the famous “ Hershey Chocolate Kiss “.

In 2001, four independent groups identified kisspeptin as a high-affinity RF amide (Arg – Phe – NH2) peptide ligand for a then orphan G protein coupled membrane receptor, GPR54.

In 2003, kisspeptin-GPR54 signaling pathway has been the object of reproductive physiologists when two independent groups nearly simultaneously reported that mutations in KISS1 Receptor (GPR54) were associated with the idiopathic hypogonadotropic hypogonadism and impaired pubertal maturation found in their patients.

It has been demonstrated in experimental studies that mutations in kisspeptin receptor can cause Hypogonadotropic Hypogonadism in humans and animal models. Compelling evidences now suggests that Kisspeptin plays a key role in conveying the feedback effects of gonadal steroid hormones on GnRH neurosecretory activity during puberty, the estrous cycle, and seasonal reproductive transitions.

The preovulatory GnRH/LH surge occurs at the end of the follicular phase and it is induced by the positive feedback actions of high Estradiol concentrations from preovulatory follicle(s). There is growing evidence suggesting an involvment of kisspeptin-neurons as mediators for ovarian steroids feedback. From these studies it has been suggested that Estradiol inhibits GnRH/LH pulse amplitude by suppressing Kisspeptin release from neurons of Arcuate Nucleus.

Biochemistry

Receptor

G protein coupled receptor transduce a variety of inputs to activate signaling pathways involved in different cellular functions such as growth, migration, proliferation. G protein coupled receptors superfamily can be classified into three groups: rhodopsin-like, secretin-like and metabotropic glutamate receptors –like. KISS1 Receptor is typical rhodopsin family receptor containing seven transmembrane domains, with three glycosylation sites at N-terminal side. Putative ligands screening identified several neuropeptides of RF amide family, RW amine family. The binding of Kisspeptin to GPR54 leads to the activation of a G protein activated phospholipase (PLC beta), suggesting a signal mediated by G alfa q. As usually PLC activated an intracellular second messenger, the Inositol triphopshate (IP3) and diacylglycerol (DAG). These signalling molecules mediated the release of intracellular calcium and activation of Protein Kinase C, respectively.Interestingly GRP54 has been shown to stimulate the arachidonic acid release and ERK1/2 , p38, as well’s Rho causing stress fibers formation. Currently scientists are examining the coupling of GPR54 receptor with other G proteins, so that evidences exist of a lateral diffusion of the receptor after binding with kisspeptin. Various inactivating mutations in GRP54 receptor have been shown to occur in humans , and these mutations have involved deletion of as many as 155 nucleotides or a few as a single nucleotide variant (L148S) in the second intracellular loop of the GPR54 receptor. Recently, on 2008 by Latronico Ana Claudia founded an activating mutation on GPR54 gene in the human, leading to precocious puberty.

Ligand

The initial product of the Kiss1 gene is a 145 aminoacid peptide, from which is cleaved a 54 aminoacid protein known such as Kisspeptin 54. In the full length protein, the sequence of kisspeptin 54 is surrounded by pair of basic residues, where furin or prohormone convertases are thought to proteolytically cleave.There are shorter peptides ( kisspeptin 10,-13, and -14 ) that share a common amidated RF motif with kisspeptin 54. Collectively they are termed “kisspeptins” and all share the same affinity for the receptor GPR54. Although all four kisspeptin peptides are biologically active, the in vivo relevance of the shorter peptides 10, 13 and 14 long peptides is unknown.

Comparative anatomy

Founded and studied in fish as regulator of reproduction, kisspeptin system has been shown to exert very different functions also due to the extraordinary range of reproductive strategies present among different species of fishes such as semelparous, iteroparous, hermaphroditic ways.

Studies in mouse (mus musculus) have provided a strong data for our understanding kisspeptin signalling in mammalian brain. KO mice for kiss1 gene provide evidence that Kiss1 gene product and its pathways are essential for the development of the murine reproductive system. Murine hypothalamus has been mapped for a detailed distribution of Kiss1 transcript (mRNA and immunoreactive kiss-1). However, because members of RF-amine family of peptides share several identical C-terminal amino-acid residues, the generation of specific antisera has been a technical challenge. So that it raises the possibility that kisspeptin antibodies should exibit cross-reactivity with other related neuroamides. In general hypothalamic areas presenting signs of the kisspeptin presence are: AnteroVentral Periventricular Nucleus (AVPV), Periventricular Nucleus (PeV), the Arcuate Nucleus (ARC), Anterodorsal Preoptic nucleus (POA), medial Amygdala, bed nucleus of Stria Terminalis.

Rat ( Rattus Norvegicus ) has been extensively studied for brain localization of kisspeptins, however the data are more unraveled compared to studies on mice due to the wide distribution across many areas of the brain of this peptide.

Molecular physiology of Kiss1 Neurons

Consistent with their role as mediators of steroid feedback, most Kiss1 neurons express alfa Estrogen Receptors (ER alfa ), beta Estrogen Receptors (ER beta ) and Progesterone Receptors (PR). Interestingly about 40% of kisspeptin expressing neurons in the Arcuate Nucleus of the mouse express the mRNA for the Leptin Receptor, thus providing the link between nutrition and reproduction.Numerous studies indicates that kisspeptin neurons are regulated by photoperiod, probably by intermediate action of melatonin secretion. Recently a subpopulation of neurons in the Arcuate Nucleus has been described coexpressing Kisspeptin, Dynorphin A, Neurokinin B and ER alfa as well PR; they are called “ KDNy neurons “. The three neuropeptides are strongly implicated into neuroregulation of GnRH secretion and similar coexpression has been found in different species including ovine, rat, mouse and humans. KDNy neurons partecipate in the regulation of pulsatile GnRH secretion.Dynorphin is an endogenous opioid peptide, which play a role in the Progesterone-mediated negative feedback control of GnRH release. On the contrary Neurokinin B is a member of the substance-P related tachykinin family; the receptor for Neurokinin B is tachykinin neurokinin 3 receptor (NK3R), expressed in GnRH neurons. Interestingly in humans loss-of-function mutations in the gene encoding neurokinin B and its receptor result in normosmic Hypogonadotropic Hypogonadism and pubertal failure. This phenotype is remarkably similar to human model defective in Kisspeptin signalling. Recently it has been demonstrated that parenteral administration of Neurokinin B or a Neurokinin B agonist to male monkeys reults in a potent stimulation of LH release, an effect abolished by administration of GnRH antagonist. Subsequent administrations of Neurokinin B is not associate with a sustained pattern of LH release, whereas monkeys remain responsive to Kisspeptin injection despite loosing responsiveness to Neurokinin B following repetitive administrations. These data suggested that Neurokinin B and Kisspeptin stimulate GnRH relelase in an independent manner.

Because GnRH neurons appear to lack both the androgen receptor (AR) and ER alfa in either sex, some intermediary neuronal systems are thought to indirectly relay the feedback signal from the gonad to GnRH neurons.Kisspeptin neurons may represent an important component of this negative feedback loop.

Many experimental evidences suggest that kisspeptin neurons in the Arcuate Nucleus (ARC) of both female and male subjects provide a tonic drive to GnRH neuronal activity, modulating by NEGATIVE feedback the effects of gonadal steroids. In female, two modes of GnRH/LH secretion occur at different times during the ovarian cycle. Tonic GnRH/LH is secreted in an episodic pattern throughout most of the cycle and it is controlled by the negative feedback actions of ovarian steroids. In particular Estradiol inhibiting pulse amplitude, whereas Progesterone inhibiting pulse frequency.Different is the behaviour on AnteroVentral Periventricular Nucleus (AVPV) where the steroid action is expressed as stimulatory firing action of these kisspeptin containing neurons. Here kisspeptin signalling is responsible of preovulatory surge in GnRH and LH release after Progesterone and Estrogen action.Some researchers argue that kisspeptin signaling is not an absolute prerequisite to sustain some degree of activity of the brain-gonaditropin axis. Some experimental evidences seems to suggest that animal without kisspeptin signals retain the capacity to show some degreee of gonadotropin secretion and ovarian cyclicity. Here Neuropeptide Y, Gabaergic and Glutamaergic pathways seems to play a relevant role, probably with intervention of Cannabinoid system in particular for GABAergic pathways.

Another important aspect of kisspeptin action is the link between nutritional status and fertility.

Leptin is a peptide hormone synthetized and secreted by adipocytes conveying information about body energy stores and nutritional status. The level of circulating leptin is proportional to fat mass and falls in both mice and humans following weight loss. The leptin deficient ob/ob mice are a well established model of hypoleptinemia. Interestingly these mice have delayed puberty and are infertile as a consequence of hypogonadotropic hypogonadism.A similar phenotype is also present in humans with mutation in leptin or its receptor and in women with hypoleptinemia as a consequence of low body weight. Leptin therapy in hypoleptinaemic mice and humans reverses these reproductive abnormalities.Leptin receptor is not expressed in GnRH neurons on Hypothalamic region of the brain; however it is expressed in kisspeptin neurons of Arcuate Nucleus. Interestingly kisspeptin expression is significantly influenced by leptin administration, so that kisspeptin release is under the influence of nutritional status.The Arcuate Nucleus contains different subpopulations of first-order leptin responsive neurons. One subpopulation express the orexigenic peptide Neuropeptide Y (NPY), while another express the anorectic peptide alfa Melanocyte Stimulating Hormone ( alfa MSH), which is derived from pro-opiomelanocortin (POMC).

References

Schally AV, Arimura A, Kastin AJ et al. Gonadotropic releasing hormone: one polypeptide regulates secretion of luteinizing and follicle stimulating hormones. Science 1971;171:1036-8.

Knobil E, Plant TM, Wildt L et al. Control of the Rhesus Monkey menstrual cycle: permissive role of hypothalamic gonadotropin releasing hormone. Science 1980;207:1371-3.

Lee JH, Miele ME, Hicks DJ et al. KISS-1, a novel human malignant melanoma metastasis suppressor gene. J Natl Cancer Inst 1996;88:1731-7.

Kotani M, Detheux M, Vandenbogaerde A et al. The metastasis suppressor gene KISS-1 enchodes kisspeptins, the natural ligands of the orphan G protein coupled receptor GPR54. J Biol Chem 2001;276:346316.

Muir AI, Chamberlain L, Elshourbagy NA et al. AXOR12, a novel human G protein coupled receptor, activated by the peptide KISS-1. J Biol Chem 2001;276:28969-75.

Ohtaki T, Shintani Y, Honda S et al. Metastasis suppressor gene KISS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001;411:613-7.

Clements MK, McDonald TP, Wang R et al. FMRF amide-related neuropeptides are agonists of the orphan G-protein-coupled-receptor GPR54. Biochem Biophys Res Commun 2001;284:1189-93.

de Roux N, Genin E, Carel JC et al. Hypogonadotropic Hypogonadism due to loss of function of the KISS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA 2003;100:10972-6.

Funes S, Hedrick JA, Vassileva G et al. The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system. Biochem Biophys Res Commun 2003;312:1357-63.

Maeda K, Adachi S, Inoue K et al. Metastin/kisspeptin and the control of estrous cycle in rats. Rev Endocr Metab Disord 2007;8:21-9.

Clarke IJ, Smith JT, Caraty A et al. Kisspeptin and seasonality in sheep. Peptides 2009;30:154-63.

Rometo AM, Krajewski SJ, Voytko ML et al. Hyperthrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys. J Clin Endocrinol Metab 2007;92:2744-50.

Oakley AE, Clifton DK, Steinmer RA. Kisspeptin signaling in the brain. Endocr Rev 2009;30:713-43.

Lehman MN, Coolen LM, Goodman RL. Kisspeptin/NeurokininB/Dynorphin (KNDy) cells of the Arcuate Nucleus: a central node in the control of gonadotropin releasing hormone secretion. Endocrinology 2010;151:3479-89.

Hameed S, Jayasena CN, Dhillo WS. Kisspeptin and fertility. J Endocrinol 2011;208:97-105.

Seminara SB, Messager S, Chatzidaki EE et al. The GRP54 gene as a regulator of puberty. N Engl J Med 2003;349:1614-27.

Beier DR, Dluhy RG. Bench and Bedside – The G Protein coupled receptor GPR54 and Puberty. N Engl J Med 2003;349:158992.

Teles MG, Bianco SD, Britto VN et al. A GRP54-activating mutation in a patient with central precocious puberty. N Engl J Med 2008;358:709-15.

Gordon CM. Functional Hypothalamic Amenorrhea. N Engl J Med 2010;363:365-71.

Caronia LM, Martin C, Welt CK et al. A genetic basis for functional Hypothalamic Amenorrhea. N Engl J Med 2011;364:215-25.

Aknowledgements

I would thanks Mrs. Ivanka for her unvaluable help in stimulating my speculative work and may be “unwanted” suggestions.

ACROMEGALY: AN EXAMPLE OF EARLY PHASES OF TUMOR DEVELOPMENT.

2011-01-23T10:22:00.000+01:00

Acromegaly

Hystory

Acromegaly was the first pituitary disorder to be recognized. Althought the first known case was possibly that of the egyptian king Akenaten in 1365 b.C., it was on 1567 that Wier J described in Codex Oporinus A virgo Gygantea with the features of an Acromegalic patient. On 1886 Pierre Marie gave an extremely accurate description of the disease, which he had observed in two patients at professor Charcot’s clinic in Paris. He suggested the term which we still use today. However, careful historical research points at the italian Andrea Verga as possibly the first to describe an unmistakable case of acromegaly. His report was published as early as 1864 in the “ Rendiconti del Reale Istituto Lombardo di Scienze e Lettere”, but had probably remained unknown to Pierre Marie as it has to most of us. “Since 1860, while visiting the chronic patients admitted to the Church of Santa Maria ai Nuovi Sepolcri, one of the houses subsidiary to te Ospedale Maggiore of Milan, I had been impressed by the look of one patient, whose waxen pallor and disproportoionately large face was almost frightening. The low personnel serving in this Hospital must have been similarly impressed, as they had nicknamed the woman “big-face”. Seeing that I was starting at her, she told me how she once had not been ugly and looked just like other girls.” Dr. Verga proceeded to collect the patient’s history, the most remarkable parts of which he reported as follows: At 35 years “…she started to realize that all parts of her body, which used to be rather thin, were progressively enlarging so that three times she had to have the rings on her fingers cut and, in particular, that her face becoming monstruous.”..” on 24 August 1856, she was declared chronic for Rheumatalgia and Amblyopia on the 16 September of the same year. Ever since, her suffering, severe joint pain in particular have not abandoned her.” The patient died on 1862 from typhus and a postmortem exam was performed during which Dr. Verga discovered on the patient’s brain “ ..a tumor, the size of a large walnut, lying over the sella tucica, pressing on and displacing the optic nerves and the mammillary processes and routing with some peduncles within the sphenoid body. The pituitary gland was not found and one wonders whether it disappeared under pressure from the tumor or the tumor itself was a true degeneration of the gland.” Dr. Verga concluded that “..this is a beautiful and exceedingly rare case of “ prosopectasia” (i.e. an elongation of the face). In contrast to Wass who attributed to Minkowsky in 1887 the first report of a pituitary enlargement among all the carefully essessed cases of acromegaly, it appears that Verga’s anatomical and clinical description of the disease was indeed the first one. In 1909 Crows, Cushing and Homans demonstrated that the complete pituitary removal is generally lethal in the dog and that growth is retarded in the few animals that survive. In 1921 Evans and Long reported in “Anatomical Record” the discovery of Growth Hormone, although at the time it was not known as true growth regulating hormone.

In 1924 Evans in his Harveian Oration stated that the anterior hypophysis is indispensable for growth to adult stature and a lesser amount of its hormone could be a direnct cause of some endocrine dystrophies whereas increased amount of the hormone are the direct cause of overgrowth. Only on 1944 Li and Evans announced on Science the isolation of growth hormone from bovine hypophysis in a highly purified form, and demonstrated that growth could be restored in hypophysectomized rats by administration of this substance. On 1956 human growth hormone was first purified and reported on Science by Li and Papkoff.

My professor Gian Michele Molinatti working at Turin University with Professor Dogliotti AM on 1959 performed the first surgical transfenoidal surgery on Acromegalic patients obtaining the first positive results of intrasellar implant of Yttrium 90 in Acromegaly, reporting also a marked improuvement of clinical and metabolic symptoms. From 1957 to 1983 a total of 138 Acromegalic patients were treated at Turin University by Professor GianMichele Molinatti equipes with interstitial radiotherapy using Yttrium 90 or Aurum 198. However due to the narrow operating field and the infective complications, the transfenoidal route have been abandoned in favour of the transfrontal and transtemporal routes. Shortly after, the advent of both antibiotic presurgical prophylaxis and operating microscopes has permitted Jules Hardy to pioneer the transnasal technique. Harvey Cushing operated originally 67 patients with a perioperatory mortality of 8.6% and a recurrence of tumor in 20 patients.

Olivecrona operated 55 cases with the intracranial route with a mortality of 9.5%. Of 31 patients operated and controlled 2 years after surgery, 17 were in good health snf the others had either hypopituitarism or recurrence of the adenoma and at his clinic many doctors interested in neurological progresses come to see the neurosurgical and treatment progressess in neurologic diseases. It was only on 1980 that French neurosurgeons such as Jules Hardy using microscopy under fluoroscopic control performed the selective adenomectomy with the complete remission of the disease and maintenance of normal pituitary function.

State of the Art

At present surgery is indicated for growth hormone secreting microadenomas, as well as for decompressing mass effects on vital structures, particularly the optic tracts. Patients with small tumors less than 10 mm in diameter and growth hormone levels less than 40 micrograms per liter should do well with transfenoidal surgery, in experienced hands. Recent surgical advances that involve the use of imaging guidance, navigation and endoscopic approaches, with perioperative pharmacotherapy, have contributed to improved outcomes. Radiotherapy is reserved for tumors that have recurred or persisted after surgery in patients with resistance to or intolerance of medical treatment. Advanced computerized imaging has permitted accurate targeting of the tumor mass, minimized radiation scatter to normal surrounding tissues, and reduced treatment times.Several centers now perform stereotactic radiosurgery with the use of the gamma knife, which delivers a single radiation fraction to a small tumor target.

The problem of radiotherapy is the slow attenuation of IGF-1 levels after treatment. The maximal control on the release of growth hormone may require more than 15 years after therapy. 10 years after radiation exposure about 50% of patients have hypopituitarism involving one or more hormonal pathways.

Biochemical knowledge of neurohomonal parhways controlling growth hormone release allows the development of new drugs now available for Acromegaly therapy. In particular Selective Somatostatin Receptor Ligands such as Octreotide and Lantreotide have been widely used to treat Acromegaly during the past two decades. These compounds are selective for SST2 and SST5 Somatostatin Receptors subtypes normally present on Somatotroph cell of pituitary. Up to 80% of patients who were followed for more than 9 years during treatment with somatostatin receptor ligands had growth hormone levels less than 2.5 micrograms per liter and normal IGF-1 levels. Eugonadism was also restored in two thirds of patients who had acromegaly with hypogonadism. Somatostatin receptors ligands are indicated after surgery has failed and after radiation therapy, during the period when growth hormone levels are elevated. Moreover primary medical treatment should be used in patients with large extrasellar adenoma whithout evidence of central compressive effects or in those declining surgery. Side effects including diarrhea, nausea, abdominal disconfort may resolve typically after 8 to 10 weeks, gallbladder sludge or gallstones develop in up tp 20% of treated patients and blood glucose levels may rise in some patients. In patients who do not have central compressive symptoms and with resistant diabetes is indicated the use of Pegvisomat a pegylated growth hormone analogue with 8 aminoacid substitutions in growth hormone binding site 1 and substitution of glycine for alanine at position 120. Daily injection of 40 mg of pegvisomat blocks the growth hormone mediated generation of IGF-1 in approximately 90% of acromegalic patients. The drug acts on peripheral tissues and affects neither the pituitary tumor nor the secretion of growth hormone. IGF-1 measurement is the biomarker for monitoring the success of treatment. Future drugs should include compounds able to recognize both the dopamine D2 receptor and somatostatin receptors (BIM-23A387), or combined treatement with Cabergoline , a receptor D2 antagonist, with other drugs, expecially somatostatin analogs.

PHYSIOPATHOLOGY

During embriogenesis development and proliferatyion of somatotrops are largely determined by a gene called the Paired-like homeodomain transcription factor of Pit-1 (PROP1), which controls the embryonic development of cells of the Pit-1 transcription factor lineage: i.e. Lactotroph, Mammo-somatotroph, Somatotroph, Thyrotroph and Gonadotroph. Prop1 can bind to its cognate site and activate target gene via the COOH terminal transactivation domain, whereas the NH2 terminal domain and the homeodomain of PROP1 posses repression function. Recent studies suggested that PROP1/catenin beta complex acts as a transcriptional activator for Pit1 and such a transcriptional repressor for Hesx1, depending from associated cofactors. Expression of Prop1 is restricted in the developing pituitary. It is initially detected at embrionic day 10 (E10) and declines after embrionic day 14.5 (E14.5). Notch signaling is required in order to maintain high levels of Prop1 expression linking to a conserved Rbp-J binding site within the first intron of the Prop1 gene. A atrget deletion of the Prop1 gene results in a failure of Pit1 gene activation, and delayed gonadotrope development. Pituitary progenitors fail to migrate to the caudomedial region of the developing gland where Pit1 is normally expressed. Genetic studies have demonstrated that a temporal relationship between regulation of Prop1 expression and a proper pituitary development. Premature expression of Prop1 in Rathke’s pouch proves to be deleterious leading to agenesis of the anterior pituitary gland, whereas persistent expression of Prop1 in thyrotropes and gonadotropes delays the gonadotrope differentiation and leads to transient hypogonadotropic hypogonadism and increased susceptibility to pituitary adenomas. The development of Corticotrops is linked to the presence of another kind of transcription factor called T-pit. Pit-1 is able to binds to the growth hormone (GH) promoter located on chromosome 17q where resides the gene coding for Growth Hormone inducing the gene tanscription. Growth Hormone is a protein secreted such as a 191 aminoacid long 4 helix bundle protein and a less abundant 176 aminoacid form. GH gene transcription is strongly stimulated by the Thyroid Hormones and Retinoid Acid through the link of TR/RXR and RAR/RXR heterodimers to TRE elements located on GH gene promoter. GH gene expression is also tightly regulated by hypothalamic factor Growth Hormone Releasing Hormone (GHRH), through activating the GHRH Receptor expressed in the somatotropes. Binding of GHRH to its receptor elicits elevated intracellular cAMP levels and subsequently activates the PKA pathway. GHRH stimulated activation of the GH promoter is mediated by PKA dependent phosphorylation of the transcriptional coactivator CBP, which interacts and synergizes with Pit1 on the Pit1 binding elements.

Glial cell Derived Neurotrophic Factor and Brain derived Neurotrophic Factor

The Rearranged during Transfection (RET) proto-oncogene is Ret protein is a part of multicomponent receptor complex formed by heterodimer with other neurotrophic factor receptors. RET is expressed in two main isoforms the short (RET9) and the long (RET51), which differ at their C terminal tail, these proteins correspond to Tyrosine kinase Receptor proteins (TrkA and TrkB). These receptors link at the inner side of plasmamembrane glycosyl-phophatidyl inositol membrane anchored coreceptors:

. Glial derived neurotrophic factor receptor (GDNF-R)

. Neurturin receptor (NTN-R)

. Artemin receptor (ART-R)

. Persephin receptor (PSP-R)

the last three able to link to growth factor belonging to “neurothrophin” family. Interestingly all these 4 receptors have in common the property to be linked to inner layer of plasma membrane through glycosyl phosphatidylinositol (GPI)-linked, so that they are also called GFR-alfa 1- to 4 receptors. Neurotropins, such as Brain Derived Neurothropic Factor (BDNF) and Glial cell Derived Neurotrophic Factor (GDNF) have been implicated in control of hippocampal dependent learning and memory ability, feeding behaviour in term of agressive response to feeding deprivation, caloric intake and expenditure through thermogenesis and muscle activity.

A small portion of our brain, called also Rhinencephalus, higly conserved through evolution in higher mammals from rats to humans, show an evelated density of neuronal networks able to control all these basic vital functions. It’s very important to remember here that this Rhinencephalic system is linked to peripheric target organs though two main mechanisms: the peripheral neural system adrenergic and cholinergic, and hormonal pituitary system. Both these systems are able to control the function of target organs, the first through direct contact with synaptic cell-to-cell structure (a sort of complex tigh-junction structure) allowing two different cell types talking each other, the second indirectly through a “wireless” connection formed by homonal factors and their receptors on target cells. Embryologically the signals that are essential for the stratification of Rathke’s pouch and for the timely and spatially organized process of endocrine cell type appearance are the same familiar factors that are involved in the patterning of many other organs, namely Shh, FGFs, BMP, Notch and Wnt families of growth factors / morphogens. Moreover the physiological function of anterior pituitary is regulated by hypothalamic releasing hormones, which they secreted by the parvocellular neurons mainly located in Anterior portion of Paraventricular nucleus (CRH, TRH, SS), in Arcuate Nucleus ( DA, GHRH) and in PreOptic region (GnRH). The parvocellular neurons project to the Medial Eminence where they are conveyed to the anterior pituitary by the hypophysial-portal vasculature. The magnocellular neurons, the second set of neuroendocrine neurons of hypothalamus, are located in two nuclei of the anterior hypothalamus, the Paraventricular and the Supraoptic nuclei from which they project to the posterior pituitary and release directly Vasopressin (AVP) and Oxytocin (OT) into general circulation. Gain and loss-of-function mutations of RET have been associated respectively with neoplasia MEN2A-MEN2B as well’s familial medullary thyroid carcinoma or a neurodevelopmental disorder called Hirschsprung disease or Megacolon Congenitus.

RET has a dual role according to the presence or absence of GDNF, providing a cellular basis of the processes of neuronal differentiation, proliferation, redifferentiation, dedifferentiation, apoptosis ( also called cell death ), accounting for so called “neuronal plasticity”. In the presence of GDNF, RET promotes survival, growth, and migration of cells such as neurons and epithelial renal cells. Converserly, in the absence of GDNF, RET is able to induce the programmed cell death by releasing a proapoptotic fragment that initiates a negative signal for apoptosis. The pathways involved in cell apoptosis induction seems to be involved into Pituitary Adenomas formation. It has been shown that RET, GDNF, GDNF receptor alfa are expressed both in human anterior pituitary and in somatotropinoma (adenoma of pituitary expressing growth hormone ). The RET proto-oncogene has a role in regulating apoptosis in somatotrophs both in vivo and in vitro. Loss-of-function of RET results in somatothops hyperplasia, which is caused by the absence of RET derived pro-death signals.

Aryl Idrocarbon Receptor pathways

Aryl Idrocarbon Interacting Protein (AIP) is also known as FKBP37, XAP2 or ARA9 and it was first identified by its interaction with hepatits B virus X protein.AIP is a 330 aminoacid protein homologous to immunophilin FKBP52. AIP forms a complex with Aryl Hydrocarbon Receptor (AHR) and two 90-Kda Heat-Shock-Protein (HSP90). AIP also binds and attenuates the activity of phosphodiesterase 4A5 (PDE4A5), phosphodiesterase 2A (PDE2A), Peroxisome Proliferation-Activated Receptor alfa (PPAR alfa), Inhibitors of Apoptosis (IAPs: ie. survivin), Rearranged during Transfection (RET) subunits of GDNF-Receptors, Translocase of the outer membrane of mitochondria 20, Thyroid Hormone Receptor beta1. AHR is a ligand activated transcription factor that regulates a variety of xenobiotic metabolizing enzymes. Dioxin-like chemicals display an high affinity to AHR, which mediates most of the toxic responses of these agents. AIP modulates the subcellular localization of AHR and prevents AHR from undergoing nucleo-cytoplasmatic shuttling. Normally the link of toxic chemicals such as “digoxin” and hydrocarbonic derivatives of tobacco smoke, which display high affinity to the AHR complex, leads to shuttling of AHR to the nucleus where it heterodimerizes with aryl hydrocarbon receptor nuclear translocator 1 (ARNT also called HIF-1 beta). ARNT is essential for the normal function of AHR, which encompasses the altered transcription and expression of various target genes, including various non heme hydroxylases (oxalate hydroxylases). AHR has an endogenous role in normal cell physiolgy in absence of xenobiotics: i.e. in cell proliferation, cell adhesion, migration, differentiation, and dedifferentiation. One interesting AHR target gene is Cyclin Dependent Kinase Inhibitor 1B (CDKN1B) encoding for p27(Kip1). P27(Kip1) is a tumor suppressor that has multiple roles in cell physiology. It acts as a cell cycle regulator and it shares overlapping pathways with Retinoblastoma (Rb) protein, a known tumor-suppressor gene linked to pituitary tumors. Recently it has been reported that germline mutations in p27(Kip1) cause a Multiple Endocrine Neoplasia Type 4 (MEN4) characterized by the presence of pituitary adenomas.Under normal coditions AHR/ARNT signaling inhibits the cell cycle through interaction with Rb and induction of p27. AIP loss-of-function mutations abolish AHR inhibition of the cell cycle leading to tumorigenesis. The interaction between RET and AIP has been demonstrated for first time both in vivo and in vitro by Romeo G. working on Policlinico S. Orsola-Malpighi, Bologna on 2009, following the report in Science on 2006 by Aaltonen LA of a Pituitary Adenoma Predisposition (PAP) linked to AIP gene germline mutation R304X located on chromosome 11q12-13. Romeo G. group suggested that RET and AIP work synergistically in regulating the somatothoph proliferation and tumorigenesis. However, neither mutation of AIP or of RET founded in pituitary adenomas can affect the ability of RET to bind to AIP. First, a possible role of mutations discoveries in AIP by Aaltonen LA group on Science on 2006 should be due to ethnicity as demonstrated by original germline mutation, ie. the so called founder effects. In other words the AIP mutations should be prevalent in particular population subgroups such as Finnish, Irish populations without any causative relation to general pathophysiology of pituitary adenomas. A germline mutation is rarely involved in the pathogenesis of sporadic non familiar tumors, and presented data are contrary to a demonstration of a generic predisposing action of the protein to somatothrops hyperplasia. Second, the involvment of Inhibitors of Apoptosis family proteins such as XIAP or Survivin, as suggested by Romeo G. group, can add another point of view on AHR pathways involvement in neoplasia formation following both aryl hydrocarbon xenobiotics acyvity, as it has been demonstrated mutations affecting primary the AHR link site at position R304X on exon 6 and so indicating a subcellular localization of the protein AIP at cytoplasmic level possibly near endoplasmic reticulum where we found many HSP90 and endocytotic vescicle system can interact with external toxic metabolites linked by AHR/ARNT complex. In conclusion the Report of Korbonitis M on AIP mutations adds more data to the so called founder effect theory; as reported by the AA AIP mutations strongly supports the hypothesis of a single, shared haplotype spanning the genome by 3.50 megabase (Mb) on chromosome 11q12-13 and carrying the same founder c910T mutation. So that Pituitary adenoma do not become manifest in every person who carries the heterozygous mutation of AIP but it can be considered a factor risk of developing pituitary adenoma, if found in germ cells.

References

Myers CS. The bones of Hen Nekht, an Egyptian king of the Third Dynasty. Man (Lond)1901;127:152-3.

Verga A. Caso singolare di Prosopectasia. Sci Lett Rendiconti Clin Sci Mat Nat 1864;1:111-7.

Minkowski O. Uber einem Fall von Akromegalie. Berl Klin Wochenschr 1887;21:371-4.

Marie P. Sur deux cas d’acromégalie; hypertrophic singulière non congénitale des extrémités supérieures, inférieures et céphalique. Rev Med Liege 1886;6:297-333.

Cushing H. Partial hypophysectomy for Acromegaly: with remarks on the function of the hypohysis. Ann Surg 1909;50:1002-1017.

Bailey P, Cushing H. Studies in Acromegaly: the microscopical structure of the adenoma in acromegalic dyspituitarism (Fugitive Acromegaly). Am J Pathol 1928;4:545-64.

Arner B, Luft R, Olivecrona H, Sjogren B. Successful treatment of a case of Cushing’s syndrome by electrocoagulation of the hypophysis. J Clin Endrocinol Metab 1953;13:1101-8.

Olivecrona H. Relation of paraventricular nucleus to the pituitary gland. Nature 1954;173:1001.

Luft R, Olivecrona H, Sjogren B. Hypophysectomy in man: experience in severe diabete mellitus. J Clin Endocrinol Metab 1955;15:391-408.

Olivecrona H, Luft R. Experience of hypohysectomy in the cancer of the breast. Ann R Coll Surg Engl 1957;20:267-79.

Luft R, Olivecrona H. Hypophysectomy in the management of neoplastic disease. Bull N J Acad Med 1957;33:5-16.

Dogliotti AM, Ruffo A, Molinatti GM. Intrapituitary fixation of Yttrium 90 in 5 cases of Acromegaly. Minerva Medica 1959;50:1575-78.

Dogliotti AM, Ruffo A, Molinatti GM. Our experience in pituitary surgery expecially in relation to cancer. Minerva Medica 1959;50:4257-62.

Dogliotti AM, Ruffo A, Molinatti GM. Radiobiologycal and surgical hypophysectomy in the treatment of cancer. Panminerva Med 1960;2:153-8.

Melmed S. Acromegaly. N Engl J Med 2006;355:2558-73.

Zhu X, Gleiberman AS, Rosenfeld MG. Molecular physiology of pituitary development: signaling and transcriptional networks. Physiol Rev 2007;87:933-63.

Pellegata NS, Quintanilla-Martinez L, Siggelkow H et al. Germ-line mutations in p27Kip1 cause a multiple endocrine neoplasia syndrome in rats and humans. Proc Natl Acad Sci USA 2006;103:15558-63.

Georgitsi M, Raitila A, Karhu A et al. CDKN1B/p27Kip1 mutation in multiple endocrine neoplasia. J Clin Endocrinol Metab 2007;92:3321-5.

Michaud JL, DeRossi C, May NR et al. ARNT2 acts as the dimerization partner of SIM1 for the development of the hypothalamus. Mech Dev 2000;90:253-61.

Hosoya T, Oda Y, Takahashi S et al. Defective development of secretory neurones in the hypothalamus of Arnt2-knockout mice. Genes Cell 2001;6:361-74.

Keith B, Adelman DM, Simon MC. Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene revelas partial redundancy with Arnt. Proc Natl Acad Sci USA 2001;98:6692-7.

Vargiolu M, Fusco M, Kurelac I et al. The tyrosine kinase receptor RET interacts in vivo with Aryl Hydrocarbon Receptor interacting protein to alter Survivin availability. J Clin Endocrinol Metab 2009;94:2571-8.

Yu R, Bonert V, Saporta I et al. Aryl Hydrocarbon Receptor Interacting Protein variants is sporadic pituitary adenomas. J Clin Endocr Metab 2006;91:5126-9.

Vierimaa O, Georgitsi M, Lehtonen R et al. Pituitary adenoma predisposition caused by germline mutations in the AIP gene. Science 2006;312:1228-30.

Raitila A, Georgotsi M, Karhu A et al. No evidence of somatic aryl hydrocarbon receptor interacting protein mutations in sporadic endocrine neoplasia. Endocr Relat Cancer 2007;14:901-6.

Hellovaara E, Raitila A, Launonen V et al.The expression of AIP-related molecules in elucidation of cellular pathways in pituitary adenomas. Am J Pathol 2009;175:2501-7.

Chahal HS, Stals K, Unterlander M et al. AIP mutation in pituitary adenomas in the 18^th Century and Today. N Engl J Med 2011;364:43-50.

Aknowledgments:

I would thanks my Professor Gian Michele Molinatti and his collaborators for their unvaluable help in stimulating and nurish my learning activity, part of Continual Medical Education, as important part of our work of Medical Doctors. A great role was played by Ivy for her unwanted but precious help in modulating, and sometime stopping in the right time, my otherwise unresting working activity.

Understanding dental and oral physiology: the way to learn aging process.

2011-01-04T23:22:00.000+01:00

Dental tissues

Between mineralized tissues, teeth and periodontal tissues represent a very important and peculiar kind of mammalian stuctures.

Three of the five tpes of human mineralizations occurs in human teeth, namely:

enamel
dentine
cementum

Like the deposition strata present in wood, their structure represent an hi-fi system record of physiological conditions present at time of their formation through fetal life to adulthood.

Enamel is a surface tissue of epithelial origin characterized by an extreme resistance to work load, whereas dentine and cementum are of mesenchymal origin and formed by avascular tissues.

Tooth development is controlled by regulatory genes determining tooth type:

- incisor

- canine

- premolar

- molar

It has been demonstrated that a crucial role in tooth morphogenesis is playd by interactions between epithelium derived structures sush as enamel and cranial neural crest derived mesenchymal cells.

In particular some proteins have been demonstrated to be involved into such as interactions:

Hedgehog proteins members
Wingless (Wnt) protein family
Tumor Necrosis Factor family members
Bone Morphogeneic Protein family

Skeletal structures of the mouth and supporting structures of orophopharynx and trachea represent so called “viscerocranium” or “splacnocranium” and are formed by bones undergoing either transmembranous and endochondral ossification.

Membranous Viscerocranium is formed by:

- maxilla

- palatine bones

- zygoma

- squamous temporal bones

- mandible

Interestingly mandible is formed by ossification of Neural Crest Cells – derive mesemchyme condensing aroud a mesodemal core of the Meckel’s cartilage (also called mandibular prominence).

Endochondral viscerocranium is fomed from the first two pharyngeal arches consisting of:

- middle ear bones

- styloid process of temporalis

- hyoid bone

- laringeal catilages deriving by arches 4 and 6.

The relevance of pharyngeal arch tissue is determining future bone formation is exeplified by the presence between structures derived from these primordial archs of:

cells of inferior paratyroid gland deriving from arch 3 and pouch 3 (dorsal tissue)
cells of superior parathyroid gland deriving from pouch 4 (dorsal tissue)

Teeth are formed by ectodermal (oral epithelium) and mesenchymal (neural crest derived) tissues of the mandibular and maxillary prominence. Each tooth bud is composed of an ectodermal dental lamina (forming later enamel) and a basal mesenchymal (neural crest derived) dental papilla (giving rise to dentine and cementum).

The embryonic tooth germ passes through three morphological states:

bud
cap
bell

and there are three main components the enamel organ, dental papilla, and the dental follicle.

The enamel organ differentiates into four layer structure, within which the enamel knot is the signaling center that regulates tooth shape and size.

A complex sequence of epithelial-mesenchymal interactions results in waves of differentiation that start at the eventual enamel-dentine junction underlying the cusp tips so determining by the spatio-temporal induction of secondary enamel knots. Incisal central mammellons , a rounded prominences on biting edge when incisors first erupt and cup tips enamel-dentine junctions are the starting points of two tissues interactions, eventually delineating the whole junction between tissues as the tooth germ growth.

Secretory signaling molecules varied continously during tooth initiation and construction in the different cell types.

Odontoblasts which make dentine are postmitotic cells differentiating from mesenchymal neural crest derived precursors present on the dental papilla at the interface between inner enamel epithelial cells of the enamel organ, which themselves differentiated into pre-ameloblasts. So that dentine formation triggers the pre-ameloblasts to differentiate into mature ameloblasts, the cells rsponsible for enamel deposition.

A bilayer structure formed by epithelial cells, called epithelial root sheath, extends from the enamel organ at the base of the developing crown to map out the dentine-cementum junction and to initiate the differentiation of the odontoblasts of the root.

The third tissue type, called cementum, is the product of both fibroblasts and cementoblasts, which differentiate from mesenchymal cells of the dental follicle adjacent to the dentine once the epithelial cells of the root sheath have moved away from the interface.

In human teeth a small amount of afibrillar cementum may form on enamel surface close to the junction between the crown and the root, if there are interruptions in the covering layer of epithelial cells once enamel formation has been completed.

Within developing tooth, a core of loose connective tissue forms the dental pulp.

The dental follicle structure gives rise to three components of peridontium

cementum
alveolar bone
periodontal ligament

Some embryological structures have been identified:

- tooth germs partially enclosed into the developing alveolar bone, typically a woven bone , formed by osteoblasts, with enclosed osteocytes, and it is remodelled to accomodate growing teeth by osteoclasts.

- The follicle, a sac of loose connective tissue separating developing tooth from bony crypt, it is essential for tooth eruption and it will become the periodontal ligament on tooth eruption.

Enamel

Enamel matrix when first seceted is a delicate structure, protected firmly by enamel organ.

The mature enamel is the hardest tissue present in human body, it is acellular and may contain 98% by weight or 93% by volume of an apatitic calcium phosphate .

Dentine mold embeds ameloblasts tubules once they contact odontoblasts; the extracellular proteinaceous matrix of enamel is secreted by ameloblasts, presented such as higher polarized cells. The main component of matrix enamel is represented by some proteins thought to control crystal growth:

- amelogenin

- enamelin

- tuftelin

- ameloblastin (amelin and sheathlin )

In order to achieve the high grade of mineralization, most portion of dentine matrix mold is degraded by neutral metalloproteinases and serine proteinase and removed, even while the ameloblasts are still secretory.

Initially enamel crystals are rich in carbonate, presenting a structure long and slender; hwever during growth carbonate content falls and crystals becomes thicker.

In humans, an high level of mineral accumulates during early stage of development, and a long postsecretory period that may last 5 or more years accounts for an hardening process due also to the presence of life ameloblasts.

The most notable feature of enamel is the organization of crystals into “prisms” about 6 micron in length, growing mainly parallel to each other and their hexagonal cross-section is parallel in each group.

In the superficial enamel, where formation rate is low the secretory interface is flat, and there is no change in crystal orientation.

However during most of enamel formation the scretory process of each ameloblast is maintained at relatively constant shape between a belt of intercellular attachment at interface between cells and matrix. In this way the cystals have their long axis perpendicular to the general plane of the developing enamel surface.

In humans enamel is non uniform on its crystal prisms junction orientation, and dividing lines are present at the prism junctions, so that interlocking prisms are described such as keyhole-shaped. The discontinuity in crystal orientation increases relatively during enaml maturation.

Finally ameloblasts move away from dentine surface and travel in groups across the surface they make. This result in decussation of the enamel prisms due to ameloblast crossing at X shaped line and forming Hunter-Schreger bands.

The prims size shows varicosities with the same period as cross-striations in the prism, thought to be related to circadian changes in composition of the mineral components. A prominence cross striations occurs at 7 to 10 days period called “regular striae of Retzius “ and following major life events such as birth (neonatal line), severe illness during enamel formation.

At finished enamel surface imbrucation lines block the access to internal growth layers.

Once tooth erupts the surfaces of the enamel become abraded, microcracks develop particularly along developmental faults, and the chemistry of mineral exposed to the oral environment changes.

Dentine

Forms the main part of the tooth and it extend from crown to the root. Its color is pale and yellow in contrast to much whiter harder enamel. Dentine is tought to be elastic and odontoblasts tubules radiates out from dental pulp.

References

Thesleff I. Genetic basis of tooth development and dental defects. Acta Odontol Scand 2000;58:191-4.

Miletich I, Scharpe PT. Neural crest contribution to mammalian tooth formation Birth Defects Res C Embryo today 2004;72:200-12.

Thesleff I, Mikkola M. The role of growth factors in tooth development. Int Rev Citol 2002;217:93-135.

Laurikkala J, Mikkola M, Mustonen T et al. TNF signalling via the ligand receptor pair ectodysplasin and edar controls the function of epithelial signalling centers and is regulated by Wnt and activin durign tooth organogenesis. Dev Biol 2001;229:443-55.

Rickels MR, Zhang X, Mumm S et al. Oropharyngeal skeletal disease accompanying high bone mass and novel LPR5 mutations. J Bone Miner Res 2005;20:878-85.

Michalowicz BS, Hodges JS, DiAngelis AJ et al. Treatment of periodontal disease and the risk of preterm birth. N Engl J Med 2006;335:1885-94.

Tonetti MS, D’Aiuto F, Nibali L et al. Treatment of periodontitis and endothelial function. N Engl J Med 2007;356:911-20.

Bashutski JD, Eber RM, Kinney JS et al. Teriparatide and osseous regeneration in the oral cavity, N Engl J Med 2010;363:2396-2405.

Aknowledgments: I would like to thanks Ivy for her unvaluable and “unwanted “ help in writing.

Osteoporosis: an unsolved diagnostic clinical problem.

2010-12-07T14:45:00.001+01:00

The recent paper of Murray J. Favus published on New England Journal of Medicine on November 18 2010 raises many questions on optimal bisphosphonate use in clinical settings.

First of all the identification of patients to be treated with bisphosphonates has not universallly accepted rules. The clinical and laboratory investigations that have been used to this pourpose are very different, depending from clinicians experience.

Modified from Kanis Pic

Recently WHO group of John Kanis introduced FRAX index (Fracture Risk Assessment Tool index ) as diagnostic tool, freely available on the web, in order to help physicians of different countries to treat osteoporotic patients. This interesting tool requires a well defined clinical exam to answer to possible risk factors for bone disease; two kind of choices are available with or without the DEXA hip value insertion. Finally the have an estimated 10-year probability of hip fracture or major osteoporotic fractures.

What’s is not at present clear is the threshold above which we can safetly and correctly use a given medical therapy, such as bisphosphonates.

Another important unsolved question is difference between male and female affected patients. It is quite clear today that the main difference between sexes is the different mechanism of trabecular bone losses leading to bone fracture. In men trabecular bone volume declines similarly as women over life, however the microstructural basis for the decrease in trabecular volume differ between sexes.

In women there appear to be loss of trabeculae with decrease in trabecular number and incresed intertrabecular space, whereas in men the primary mechanism for the decrease in trabecular volume is trabecular thinning.

This mechanism in turn is likely to have a significant impact on age related changes in bone strength in women compared to men, because the reduction in trabecular number has a 2 or 5 times greater impact on bone strength compared with reduction in trabecular thickness that result in similar decreases in bone volume.

These changes cannot be solved with DXA techniques, but only with HR-pQCT.

Another important point are the different bone changes in osteopenic compared to osteorotic patients. In particular trabecular thickness and cortical thickness have been found to be decreased in osteoporotic fractured women compared to osteopenic ones. On the contrary cortical bone density is not different between osteopenic and osteoporotic women.

The demonstration of the complexity of this apparently easy answer to the presence of a previous osteoporotic fracture is the absence on amnestic clinical history of an asymptomatic vertebral fracture, later identified only by proper radiographic exams.

Concerning bisphosphonate use in particular we have to remember some basic points for a good clinical therapy.

First, bisphosphonates can be used only if the subjects have normal Vitamin D3 levels, so above 30 ng/ml of 25 hydroxycholecalciferol. We know from previous epidemiologic studies how frequent, if not universal, is the deficit of Vitamin D3 in population at risk for bone diseases. From this consideration is a safetly rule to use bisphosphonates in association with adequate levels of Vitamin D supplementation (880 UI at day).

Second, the clinical steps in follow-up of patients treated with bisphosphonates should include, according to my experience, measures of bone density with DEXA techniques possibly at 1 year and unavoidably at 2 years after initiation of therapy. Our expectations from these higly sophisticated computerized exams, today fortunately widely available in developed countries, should not include an incrrease in bone density (BMD) values compared to basal values. A good result should be considered the maintainement of bone density values previously present two years before, whereas negative results due patient’s low compliace or unresposive subjects should be considered if we observe a decrease in BMD values.

Concerning other laboratory parameters I think that measurements of plasma calcium levels, alkaline phosphatase levels, phosphate plasma levels should be enough to follow the bisphosphonates treated patients once a year. Concerning the clinical utility of osteocalcin and serum C-terminal telopeptide of type 1 collagen, I think that they should be measured only in clinical studies before the bisphosphonate therapy in order to have some laboratory evaluation of bone turnover rates. The variations in their values during therapy, also during first 6 months, cannot add more informations on bone health status; providing the experimental evidence of a net decrease during this period of these markers due to “freezing actions” of bisphosphonates on bone turnover indexes.

Third and more problematic is the question concerning the duration of the bisphosphonate therapy. Many postmarketing studies adressed in the past the question: “ How long we have to maintain the patients under bisphosphonate therapy safetly? “, I think the Author MJ Favus is right when he stated that 5 years long therapy should be considered the optimal and safe therapy duration. And I would add not only for a “ drug holiday” period, after that we can reintroduce the drugs, but forever.

Too many adverse effects have been described in medical scientific litterature to prolong this “ chemiotherapy “ of bone disease more than 5 years.

Besides the Osteonecrosis of the jaw (ONJ) demontrated after their wide use using parenteral routes in bone metastatic patients and probably due to higher plasma levels reached during too short time period ( 15 minutes infusion ), I think that more attention should deserve the recent identification of Atypical Fractures of femoral subthrocanteric and more generally diaphyseal femur fractures in postmenopausal women taking bisphosphonate orally.

Bisphosphonates’ action on bone tissue has a natural equivalent in the genetic defects causing osteopetrosis. These rare genetic diseases are due to a defect in bone osteoclasts resorption.

A century ago, Albers-Schonberg described the radiographic findings in a patient with increased bone density

Autosomal dominant osteopetrosis is characterized by a defect in gene coding for chloride channel (CLCN7).

An autosomal recessive osteopetrosis form si also linked to partial alteration of chloride channel (CLCN7) (15%) linked to the proton pump ATPase.

Other autosomal recessive forms are linked to carbonic anidrase II gene defect (<5%), typically linked to renal tubular acidosis; proton pump human subunit A3 of osteoclast ATPase proton pump (TCIRG1, also termed ATP6i) (60%).

Whereas acquired forms of osteopetrosis have been now described after the therapeutic use of bisphosphonates, initially by Whyte MP.

Given these similarities, it can be supposed that aminobisphosphonates alter prenylation of proteins present normally on plasma membrane of osteoclasts, shown to be important for their reabsorbing activity and in particular to those forming chloride channels(CLCN7) and Proton Pump ATPase (TCIRG1), inhibiting their plasmamembrane exposure inside osteoclasts ruffled border and so acidification process required for hydroxyapatite dissolution in Howship lacunae.

New more physiological therapeutic modulations of bone metabolism should come from the study of RANKL/RANK pathway (DENOSUMAB), SCLEROSTIN secreted by osteocytes inhibitor (AMG-785), Wnt/DKK1 pathway inhibitor (BHQ880), Catepsin-K inhibitor (ODANACATIB, RELACATIB, BALICATIB), Src-tyrosina kinase inhibition (SARACATINIB), CLCN7 inhibition (NS3736), Calcium sensing Receptor 1 inhibitors (MK-5442), Androgen Receptor modulators SARM (OSTARINE), Estrogen Receptor modulators SERM (Raloxifene, Arzoxifene, Basodoxifene, Lasofoxifene) and Calcitonin Receptor modulators (Salmon Calcitonin).

Refereces

Smith RW, Walker RR. Femoral expansion in aging women: implications for osteoporosis and fractures. Science 1964;145:156-7.

Ruff CB, Hayes WC. Subperiosteal expansion and cortical remodeling of the human femur and tibia with aging. Science 1982;217:945-8.

Fleisch H, Russell RGG, Straumann F. Effect of pyrophosphate on hydroxyapatite and its implications in calcium homeostasis. Nature 1966;212:901-3.

Francis MD, Russell RGG, Fleisch H. Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science 1969;165:1264-6.

Fleisch H, Russell RGG, Francis MD. Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture in vivo. Science 1969;165:1262-4.

Khosla S, Riggs BL, Robb RA et al. Relationship of volumetric bone density and structural parameters at different skeletal sites to sex steroid level in women. J Clin Endocrinol Metab 2005;90:5096-5103.

Khosla S, Melton LJ III, Robb RA et al. Relationship of volumetric BMD and structural parameters at different skeletal sites to sex steroid level in men. J Bone Miner Res 2005;20:730-40.

Khosla S, Riggs BL, Atkinson EJ et al. Effects of sex and age on bone microstructure at the ultradistal radius: a population based noninvasive in vivo assessment. J Bone Miner Res 2006;21:124-31.

Albers-Schonberg HE. Rontgenbilder einer seltenen Knockenerkrankung. Much Med Wochenschr 1904;51:365-8.

Teitelbaum SL. Bone resorption by osteoclasts. Science 2000;289:1504-8.

Tolar J, Teitelbaum SL, Orchard PJ. Osteopetrosis. N Engl J Med 2004;351:2839-49.

Whyte MP, Wenkert D, Clements KL et al. Bisphosphonate-induced osteopetrosis. N Engl J Med 2003;349:457-63.

Lenart BA, Lorich DG, Lane JM. Atypical fractures of the femoral diaphysis in postmenopausal women taking alendronate. N Engl J Med 2008;358:1304-6.

Odvina CV, Zerwekh GE, Rao S et al. Severely suppressed bone turnover: a potential complication of alendronate therapy. J Clin Endocrinol Metab 2005;90:1294-301.

Follis RH Jr, Park EA. Some observation on bone growth, with particular respect to zones and transverse lines of increased density in the metaphysis. AJR Am J Roentgenol 1952;68:709-24.

Isaia GC, Lala R, Defilippi C et al. Bone turnover in children and adolescents with McCune-Albright syndrome treated with pamidronate for bone fibrous dysplasia. Calcif Tissue Int 2002;71:121

Many thanks to Ivy for her unvalued ( may be also unwanted help ) in my daily scientific work and to Novartis Foundation for his help in my scientific formation.

see to SCIENCE references for conclusiomn i?ve drown.

Intracellular kinases: to scientific basic reseacrh to patients beds

2010-11-20T13:40:00.001+01:00

Src oncogene

The non receptors tyrosina kinases Src are very important in many aspects of cell’s physiology.The viral src gene was the first to be identified, and its cellular counterpart was the first prooncogene to be discoverd into vertebrate genome.Peyton Rous 90 years ago described the visurs now bears his name; and 25 years ago it was demonstrated that viral –src originates from a cellular progenitor, the protoncogene-src. Rous reports that a solid tumors could be induced by a filtrable agent, but Rous was told “this can’t be cancer, because you know its cause.”

In 1955 Harry Rubin showed that each cell in a Rous sarcoma virus (RSV) induced tumor could release infectious virus, and that the cells producing virus survived.

The 1960 was the golden age for RSV biology. From the laboratories of Harry Rubin, Peter Vogt and Howard Teming, Hidesaburo and T Hanafusa it was demonstrated that:

1. the replication of compentent viruses permit to isolate virus clearly related to RSV but not transforming.

2. One strain of virus RSV showed to be replication-defective and malignant transforming.

These two important findings indicated that virus replication and malignant transformation might be separable genetic properties of RSV.

The 1970 were the golden age of genetic of RSV; in particular Peter Vogt and Peter Duesberg showed that the RNA genome of transforming, replication competent strains of RSV (called “a”) was larger that those of non transforming mutants (called “b”). So that they coined the memorable equation:

a = a + x

were x was the transforming gene. This gene was finally sequenced by Bishop, Hanafusa and Gilbert on early 1980’ after genetic crosses between different mutant strains of viruses.

Nobel Laureate Harol Varmus

In 1981 Harold Varmus identified a fusiform mutant of RSV that behaved as a host-range mutant, transforming the mammalian cell line, but not chicken embryo fibroblasts. Clearly the host dependent properties of these mutants indicates that these mutations might affect the interaction between the src gene product and host target cells. As we see later this idea was found to be correct, because this properties lie within region of the Src protein (Scr homology domain –SH) involved in protein-protein interaction.In fact retroviruses can be transmitted as genetic elements within the germ line. And it has been proposed by Huebner and Todaro in 1969 that animal cells might carry transforming viruses that could cause cancer when they latent transforming genes were induced (oncogenes). Was in 1976 that in collaboration with Harold Varmus, Peter Vogt and Mike Bishop a complementary DNA probe specific for viral-src recognized an homologous sequences in various normal avian DNA. The conservation of this sequence in various vertebrates indicates that it was a normal cellular gene, not a part of a genetically trnasmitted virus.The term “proto-oncogene” was coined to identify the cellular precursor of a retroviral transforming gene; the term was chosen to distinguish these genes from the “oncogenes” postulated by Huebner and Todaro. c-src was the first of several proto-oncogenes to be discovered in the vertebrate genome and in 1989, this epochal discovery earned Bishop and Varmus the Nobel prize in Physiology and Medicine.

With the recent demonstration that a humanized antibody against Erb-B2 (Herceptin) is beneficial in the treatment of breast cancer and that “abl” inhibitor ( Imatinib ) is effective in the treatment of chronic myelogenous leukemia, these discoveries are now finally yielding a practical reneward.

Shortly after the identification of SRV, Hanfusa discovered that infected chiken with mutant RSV clonatining large deletions in v-src led to recovery of avian sarcoma viruses with a reconstitued transforming gene.

The cellular origin of c-src was formally confirmed in the early 1980s when Hanafusa et al. Reported that the cloned c-src gene had the typical structure of a cellular gene containing 10 intronic sequences and 11 exons. The sequences of c-src revealed also that it differed from that of virus-src by a carboxyterminal substitution and several point mutations.

The genomic stucture of viral RNA of RSV was demonstrated to be composed starting from 5’ nucleotide sequence to 3’ nucleotide RAN by:

1. “gag” gene

2. “pol” gene

3. “env” gene

4. “src” or “transforming” gene

as for most post of retroviruses including HIV.

Moreover Hanafusa demonstrated that the normal “c-src” has a low transforming activity in both mammalian cell lines and chiken embryo fibroblasts.

To address the question about the mechanism of “c-src” activation in normal cells, it was necessary to know the protein sequence of both genes “viral src protein” and “normal c-src protein”.

The history of experimental studies that lead to protein sequences identification, started fom 1977 with the initial work of Erikson and Brugge leading to identification of so called “pp60^v-src” now simply called “v-src”.

They induced tumors in newborn rabbits with a mammalian tropic strain of RSV and later they used the serum from these tumor bearing rabbits to immunoprecipitate a 60 kDa phosphoprotein from RSV-transformed fibroblasts.

In 1979 Tony Hunter analysing phosphoaminoacids composition of polyoma middle T antigen wich is phosphorylated later by c-Src kinase discovered the phosphorylation site at Tyrosina that become phosphotyrosine. In the same year Sam Cohen found that Epidermal Growth factor Receptor (Erb pro-oncogene) was in fact a tyrosina kinase.

So that Scr and Erb were the prototypes of a large family of kinases that we know to be involved in the regulation of cell growth and differentiation.

Later it was demonstrated step by step the molecular structure of Scr protein, those tridimensional structure was definitely clarified only recently by Xu W.

Normally the cytoplasmic tyrosine kinases are linked to inner side of plasma membrane through their N terminal side and resides into cytoplasm in its inactive globular conformation.

The molecular monains present on cytoplasmic tyrosine kinases are from N terminal to C terminal portion:

1. membrane linking domain

2. SH-3 polyproline binding domain

3. SH-2 phosphotyrosine liking domain

4. Linker region polyproline rich

5. SH-1 or cathalitic domain with tyrosine kinase activity and posses an autophosphorylation site in its Tyrosine 416

6. Regulatory inhibiting domain with a Tyrosine 527 that is subjected to phosphorylation by other tyrosine kinase (formerly called C terminal tyrosine kinase or Csk)

The importance in cellular biology of the discovery of these molecular domains into Scr protein is due to the fact they were the first demonstration of how happen a protein-protein interaction “in vivo”. The first to be discovered was SH-2 domain in 1990 for Src Homology-2 domain by Moran et al. Only three years later in 1993 proximal to this region was discovered another region called SH-3 for Src homology-3 region also medianting protein-protein interactions, in proline rich target sequence.Was in 1997 that was finally clear the three dimensional structure of the naive protein at its inactive state, cearly indicating that “phopshotyrosine 527 interact with SH-2 domain, whereas the Linker region proline rich is able to link SH-3 domain by the group of John Kuriyan.

Concernign the role exerted normally by Src tyrosine kinase it was at first time clear its implication in cell proliferation control.In fact Src like kinases are regulated by a series of plasma membrane-associted receptors such as:

1. Receptor tyrosine kinses such as Erb family receptors, FGFs receptors, PDGFs receptors, IGF-1/Insulin receptors through autophosphorylation site liking to SH-2 domain

2. Integrin receptors through Focal Adhesion Kinases (Rho-kinases) and phosphotyrosine able to link to SH-2 domain of what now is called Integrin Linked Kinases (ILK)

3. Seven transmembrane G protein coupled receptors such as beta adrenergic receptor, PTH receptor, Calcitonin receptor all coupled with “arrestin “ molecule providing the link with SH-3 domain with its proline rich region.

4. Cytokine rceptors such as TNF family receptors, IL-1 receptor probably through phosphotyrosine linking to SH-2 domain

5. Fc receptor activated by th formation of complex between Antigen/antibody/Complement proteins for B lymphocytes and other heamatopoietic cells is associated through tyrosine phosphorylated with SH-2 domain of Src like cytoplasmic tyrosine kinase (Blk)

6. T cell receptor through Zed portion of its Complex is associated to an Src like tyrosine kinase at SH-2 domain (p56 Lck)

Mice models

Src KO mice: osteopetrosis

Src/Fyn double KO mice die prenatally

Src/Yes double KO mice die prenatally

Src/Fyn/Yes triple KO mice die prenatally

Related cytoplasmic tyrosine kinases

Src, Fyn, Yes1(ubiquitarious)

Lck, Blk, Hck, Fgr, Lyn (hematopoietic)

As RET oncogene was the first Tyrosina Kinase Receptor discover during transfection on rodents and chiken with retrovirus, Src was the first Cytoplasmic tyrosina kinase discovered as viral oncoprotein able to induce Sarcoma Rous in avian through a viral transfection. Normally there is a tight control of cytoplasmic tyrosina kinase activity that reside near the plasmamembrane in its inactive state.

Its molecular structure is composed by a so called Src-like Homology domains (SH domains) the first near the plasmamembrane called SH3 able to bind to Pro-X-X-Pro motif on target proteins; the second in mid portion of the mlecule called SH2 able to link phosphotyrosine aminoacid in particular the phosphotyrosines resulting from autophosphorylative process on Receptor Tyrosina Kinases.Finally at C terminal tail of Src it is present the true tyrosina kinase loop containing a Tyrosine at position 416 involved into the phosphorylation by other kinases (Receptor and Cytoplasmic) leading to stabilization of active conformation. The final C terminal site of cytoplsmic tyrosina kinases show a C terminal Tyrosine residue in position 530 in humans that is important in control of the transition from active to inactive state. If Tyr 530 is phosphorylated it stabilizes the Src like proteins into its inactive state interacting with SH2 domain itself and so forming a globular conformation able to render the cathalitic loop inacessible to other protein interactions.

Between first discovery on 1976 and the identification of molecular structure of on 1997 many other cytoplasmic tyrosina kinases were found in normal cells of many tissues showing the same domains (SH2 and SH3 preceeding the true kinase loop). Some of them have been involved into signalling transmission between Receptors tyrosina kinases and production of second messengers such as cAMP by adenylcyclase enzymes providing a mechanical demostration of the long sought link between tyrosina kinase receptor activation and adenyl cyclase activity.These are called Adaptor proteins and are placed on inner surface of plasmamembrane; the function of adaptor proteins is normally those to phosphorylate GDP into GTP tehy are:

1. Shc protein

2. Grb-2 protein ( Growth Factor receptor binding protein 2)

3. Sos1 protein

They are intracellular proteins located on inner cell membrane and these three proteins act in concert as a phosphorylative complex that in utimate analysis activated the small weight G-protein (ras oncogne) located on inner surface membrane.

GRBs conatined almost an SH2 domain with or without an SH1 domain and all are Growth factor Receptor Binding protein:

GRB-1 is also called “p85” the regulatory subunits of PI(3)K and it posses two SH2 domains.

GRB-2 is a 25 kDa polypeptide composed by a SH2 domain flanked by two SH3 domains

SOS for Son of Sevenless conatin the C-terminal segment with the tru tyrosine kinase cathalitic loop.

Other have showed to be implicated into lymphocyte signal trasmission through TCR (Tcell rceptor or B cell rceptor) interaction and their proper ligands so mediating the intracellular pathways leading to immune response of both kind of lymphocytes T and B cells.

References

Huebner RJ, Todaro GJ. Oncogenes of RNA tumor viruses as determinant of cancer. Pro Natl Acad Sci USA 1969;64:1087-1094.

Stehelin D, Varmus HE, Bishop JM, Vogt PK. DNA related to the transforming gene(s) of avian sarcoma viruses is present in normal avian DNA. Nature 1976;260:170-3.

Sicheri F, Kuriyan J. Structures of Src-family tyrosine kinases. Curr Opin Struct Biol 1997;7:777-785.

Yarden Y, Sliwkowsli MX. Untangling the ErB signalling network. Nature Rev Mol Cell Biol 2001;2:127-137.

Druker BJ et al. Efficacy and safety of specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia, N Engl J Med 2001;344:1031-7.

Xu W, Harrison SC, Eck MJ et al. Three dimensional structure of the tyrosine kinase c-Src. Nature 1997;385:595-602.

Sicheri F, Moarefi I, Kuriyan J. Crystal stucture of the Src family tyrosine kinase Hck. Nature 1997;385:602-609.

Moran MF et al. Src homology region 2 domains direct a protein-protein interactions in signal transduction. Proc Natl Acad Sci USA 1990;87:8622-6.

Renal kidney stones: from Randall’s particles to calcifying nanoparticles.

2010-09-22T13:19:00.000+02:00

With the advent of postgenomic era, more data are available on molecular pathogenesis of kidney stones, a disease very frequent in human population affecting an increasing number of patients. Important risk factors for development of renal calcifications are obesity and hypertension both increasingly present in developed world in such a way assumed to be of epidemiologic proportion.

From rare monogenic diseases characterized by single gene mutations scientists should have important insight on pathogenesis of kidney stone formers also in so called more common Idiopatic Hypercalciuria.

First of all we have to remember that a common criteria unifies stone former people: presence of hypercalciuria, and altered PH values. These factors are responsible of metastability level reached by urinary ions.

It seems quite evident at present that the first nucleation complex is represented by calcium phosphate crystal in biological form of hydroxyapatite. Apatite crystal nucleation is responsible of formation of more common forms of kidney stones such as calcium oxalate and also of stones present in hyperuricemia. Also after bacerial infection, struvite crystal deposition, is preceeded by formation of hydroxyapatite crystal nucleation.

Dietary behaviours is of paramount importance as demonstrated by relevance of low water introduction, high dietary oxalate containing products, high dietary sodium intake

Molecular structure of kidney stones

Nanobstructing stones produce no symptoms or signs apart from hematuria. Stone with diameter less than 5 mm have a great chance of passage, those with a dimeter between5 and 7 mm have a 50% chance to passage, those with a diameter comprised greater than 7 mm almost always require urological intervention. Stone anlysis ideally is performed by X ray diffraction or by infrared spectroscopy.

CT radiographs taken with a 5 mm cuts, without infusion of contrast agents, is the most sensitive method to detect kidney stones at present. Radiographs appearance and density is used to determine the composition of stones.

Supersaturation

Stones result from a phase change in which dissolved salts condense into solids, and all phase chnages are driven by supersaturation, which is usually approximated for such salts by the ratio of their concentration in the urine to their solubilities and calculated by computer algorithms.

At supersaturation values greater than 1, crystals can form and grow, if supersaturation values are less than 1, crystals of a substance dissolve. Composition of stones that a patient forms correlates with supersaturation values of urine he produces. Although increasing urine volume is an obvious way to lower supersaturation, patients examined in a variety of practice settings have been found to be able to increase their urine volume by an average of only 0.3 l/d.

For unclear reasons, sodium intake and urinary calcium excretion has been found to increase with increased urine volume, partially offsetting the fall in supersaturation.

Urinary calcium and oxalate concentrations, with urine volume, are the main determinants of calcium oxalate supersaturation values.

Urinary calcium concentration and urinary PH are the main determinants of calcium phosphate supersaturation values.

Urinary PH is the main determinant of uric acid supersaturation value.

Metastability

Urine with supersaturation levels greater than 1 is referred such as “Metastable “ because the excess of dissolved materials, at concentration above its solubility, must eventually precipitate. The supersaturation value needed to produce a solid phase of crystals is called the “ Upper Limit of Metastability “ (ULM) and varies with urine supersaturation and it is lower among stone former patients compared to normal patients.

Urine contains some molecules that retard the formation of solid phase crystals and it is precisely this retardation that can allows the existence of transient Supersaturation level. Between substance known to inhibit crystal growth we have:

- Citrate able to reduce supersaturation by binding calcium and inhibiting the nucleation process and calcium crystal growth. Urine citrate levels are measured clinically and low levels are considered a risk factor and a possible cause for stone formation.

- Osteopontin

- Prothrombin F1 fragment

- Inter-alfa-trypsin inhibitor

- Calgranulin

- Tamm-Horsfall glycoprotein (uromodulin)

- Albumin

- RNA ad DNA fragments

- Glycosaminoglycans

All have been identified as urine inhibitors of calcium phosphate and calcium oxalate crystallization. They present as a common feature long stretches of polyanion chains that can bond with with surface calcium atoms and prevents crystal growth; such blockade prevents measurable phase changes and raises the upper limit of metastability, because newly formed crystals cannot grow beyond extremely minute dimensions. In particular these molecules also prevent clumping of small particles into larger ones (i.e. aggregation). The contribution of these molecular inhibitors to pathogenesis of renal stones is of great interest and topics of intense scientific research.

Pathogenesis of Oxalate Calcium stones

Calcium oxalate stone forms on the surfaces of the renal papillae over collections of interstitial suburothelial Calcium Phosphate particles named Randall plaque. It is believed that Randall plaques promote Calcium Oxalate overgrowth beause some data suggest that the number of Calcium oxalate stones varies directly with plaque surface coverage. Plaques begins in the basement membranes of the thin Henle loops. Basement membrane plaque comprises a myriad of particles in which crystal and organic layers alternate; the outer surface of all particles is the organic layer. The crystal in plaque is always biological apatite, the mineral pahse found in bone. Outside basement membrane, in the interstitium plaque particles coalesce so that islands of crystals float in an organic sea. It is this coalescent material that extends to the suburothelial region and over which calcium oxalate stones grow. The identity of the organic molecules surrounding apatite in plaque are unknown except for osteopontin, which coats the surface of apatite and position itself precisely at the apatite organic layer interface. In that way oxalate calcium stones form over the organic coating of apatite particles.

Randall seventy years ago examined papillae of cadaveric renal units and demonstrated that interstitial crystal plaques in the papillary tip were common in stone formers. These crystals were composed not of calcium oxalate the most common solid phase found in patients with nephrolithiasis, but of calcium phosphate. Randall believed that the calcium phosphate crystals serve as a nucleation surface for calcium oxalate stones.

All pathological calcifications contain snowball-like calcium phosphate spheres 200 nm in size. Structures similar to the snowballs were discovered over a decade ago in blood and blood products. These structures called calcifying nanoparticles were detected in numerous pathological cacifications, such as kidney stones, in atherosclerotic plaques, in psammoma bodies of cancer, in prostatic stones, and in gallbladder.

Calcifying nanoparticles are calcified self propagating entities, morphologically very similar in mineral composition to spherical bodies observed in Randall’s plaques. Also referred as nanobacteria, these nanoparticles are controversial agents due to lack of their genomic evidence. Calcifying nanoparticles (CNP) are able to cause specific infections, and they are detected in pathological calcificiations.

However, general debate over their existence continues caused by methologic problems in their detections; usually immuno-detection using anti-CNP antibodies, culture-techniques and electron microscopy. Using these methods of detection, it has been suggested that CNPs are tiny, cell-like spheres (80-200 nm in diameter) precipitating apatite crystals from media forming apatite-protein complexes on their exterior membrane. This protein-associated mineralization reach a diameter of one to several micrometers.

Interestingly 14% of healthy adults in Scandinavia have anti-CNP antibodies; in comparison 75% of patients with kidney disease have CNP antigen in blood.

In vitro destruction of the calcified apatite shell of CNP with EDTA chelation reveals numerous, 50 to 100 nm in diameter, membranous cells similar to those observed in Randall plaques. Moreover electron microscopy studies revelaed that renal plaques show 1-5 micron in diameter apatite spheres, similar to the structure of CNP.

The anatomy and microstructure of the Randall’s plaque reveals loss of urothelial cells. The initial site of plaque formationis always in the papillary tip and must be in the basement membrane of the thin loop of Henle, because that is the one site always involved with plaques formation. Within the basement mambrane plaques are individual particles of alternatin mineral and organic layers in a tree ring configuration.

Plaque clearly migrates from the basement membrane into the surrounding interstitium and when it does so, the individual particles can be found associated in an orderly way on type I collagen fibers.

Subsequently, particles associated with type I collagen fuse into a syncytium in which islands of mineral appear to float in an organic sea. It si generally believed that that the plaque organic matrix, mineral and collagen fibers are tightly associated. Accordingly, the mineral phase of plaques are always overlaid with organic matrix, so that uncoated mineral is never present.

The mineral phase of plaque is invariably biological apatite. Within plaque osteopontin is abundant and it is localized preferentially at the interface between the apatite and the adjacent organic matrix. The third heavy chain (H3) of the inter-alpha trypsin molecule localizes into the organic matrix layers, whereas the hyaluronin is present in the interstitium with H3 chain of inter-alpha trypsin molecules. The last molecules are so present in a totally different site compared to osteopontin located as previously described at the interface between apatite crystals and organic matrix layer. All moleculae previously described that can influence crystallization process tend to adhere to crystals, so that the localization of osteopontin at the mineral interface is not surprising.

Genetic disorders of renal calcium transport

Monogenic Hypercalciuric stone-forming diseases

Dent disease complex (OMIM 300008, 300009, 310468 ) Ch Xp11.22 Endosomal chloride channel 5 (CLCN5) hypercalciuria with phosphaturia and proteinuria, variable rickets

Bartter syndrome type I (OMIM 601678, 600839 ) Ch. 15q15-21 Sodium Potassium cotransporter (Na absorption) (SLC12A1) Hypercalciuria with hypokalemic alkalosis and sodium wasting.

Bartter syndrome type II (OMIM 241200, 600359 ) Ch. 11q24-25 Potassium channel (K supply for SLC12A1) (KCNJ1) Hypercalciuria with hypokalemic alkalosis and sodium wasting.

Bartter syndrome type III (OMIM 607364, 602023 ) Ch. 1p36 Basolateral chloride channel (CLCNKB) Hypercalciuria with hypokalemic alkalosis.

Bartter syndrome type IV Barttrin defect Chloride channel subunit (BSNL) Hypercaiuria with deafness

Bartter syndrome type V (OMIM 601199) Ch. 3q13.3-q21 Calcium Sensing Channel Receptor (CASR) with severe gain of function. Hypercalciuria with hypokalemic alkalosis and hypomagnesemia, chronic renal failure. Seizures, hypocalcemia and tetany.

Hypocalcemia hypercalciuria autosomal dominant (OMIM 146200, 601199 ) Ch. 3q13.3-q21 Calcium sensing receptor (CASR) with gain of function. Hypercalcemia with chronic renal failure. Hypocalcemia and tetany, hyperphosphatemia, hypoparathyroidism.

Hypercalciuria and nephrocalcinosis with familial hypomagnesemia (OMIM 248250, 603959 ) Ch. 3q27 Paracellin-1 (or claudin-16) (PCLN1) Hypercalciuria with hypermagnesiuria, polyuria, chronic renal failure and distal Renal Tubular Acidosis. Hypomagnesemia with tetany, seizures.

Distal Renal Tubular Acidosis

dRTA Autosomal dominant (OMIM 179800, 109270) Ch. 17q21-q22 Chloride bicarbonate exchanger (SLC4A1) Osteomalacia with hypokaliemia.

dRTA Autosomal recessive (OMIM 267300, 192132 ) Ch. 2q13 Proton secretion. Hydrogen ATPase B1 (ATP6V1B1) Rickets with hypokaliemia, growth failure and sensorineural hearing loss.

dRTA Autosomal Recesive (OMIM 602722, 605239 ) Ch. 7q33-34 Proton secretion . Hydrogen ATPase (ATP6V0A4) Rickets with hypokaliemia, growth failure.

Cystinuria

Cystinuria type A Autosomal Recessive (OMIM 104614) Ch. 2p16.3 Heteromeric aminoacid transport (heavy unit) (SLC3A1) Heterzygotes: normal urine cystine excretion.

Cystinuria type B Autosomal Recessive (OMIM 604144) Ch. 19q13.1 Heteromeric aminoacid transport (light unit) (SLC7A9) Heterozygotes: elevated urine cystine excretion

Primary Hyperoxaluria

PH type I (OMIM 259900) Ch. 2q36-q37 Alanine-glyoxylate aminotransferase (AGXT) Conversion of glyoxylate to glycine with increasing oxalate concentration. Hyperoxaluria with increasing urinary glycolate excretion and chronic renal failure.

PH type II (OMIM 260000, 604296) Ch. 9cen Glyoxylate reductase/Hydroxypyruvate reductase (GRHPR) Conversion of glyoxylate to glycolate. Hyperoxaluria with increasing urinary L-glyceric acid excretion, and chronic renal failure.

References

Randall A. The origin and growth of renal calculi. Ann Surg 1937;105:1009-27.

Randall A. Papillary pathology as precursor of primary renal calculus. J Urol 1940;44:580-9.

Coe FL, Parks JH, Asplin JR. The pathogenesis and treatment of kidney stones. N Engl J Med 1992;327:1141-52.

Evan AP, Lingeman JE, Coe FL et al. Randall’s plaque of patients with nephrolithiasis begins in basement membrane of thin loops of Henle. J Clin Invest 2003;111:607-16.

Evan AP, Coe FL, Rittling SR et al. Apatite plaque particles in inner medulla of kidneys of calcium oxalate stone formers: osteopontin localization. Kidney Int 2005;68:145-54.

Evan AP, Lingeman JE, Coe FL et al. Role of interstitial apatite plaque in pathogenesis of the common calcium oxalate stone. Semin Nephrol 2008;28:111-9.

Coe FL, Evan A, Worcester E. Kidney stone disease J Cln Invest 2005;115:2598-2608.

Worcester EM, Coe FL. Calcium kidney stones. N Engl J Med 2010;363:954-63.

Kajander EO, Ciftcioglu N. Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation. Proc Natl Acad Sci USA 1998;95:8274-9.

Carson DA. An infectious origin of extraskeletal calcification. Proc Natl Acad Sci USA 1998;95:7846-7.

Vali H, McKee MD, Ciftcioglu N et al. Nanoforms: a new type of protein associated mineralization. Geoch Cosmoch Acta 2001;65:63-74.

Asplin JR, Arsenault D, Parks JH et al. Contribution of human uropontin to inhibition of calcium oxalate crystallization. Kidney Int 1998;53:194-9.

Stapleton AM, Ryall RL. Blood coagulation proteins and urolithiasis are linked: crystal matrix protein is the F1 activation peptide of human prothrombin. Br J Urol 1995;75:712-9.

Marengo SR, Resnick MI, Yang L et al. Differential expression of urinary inter-alpha-trypsin inhibitor trimers and dimers in normal compared to active calcium oxalate stone formers. J Urol 1998;159:1444-50.

Pillay SN, Asplin JR, Coe FL. Evidence that calgranulin is produced by kidney cells and is an inhibitor of calcium oxalate crystallization. Am J Physiol 1998;275:F255-F261.

Hess B, Nakagawa Y, Parks JH et al. Molecular abnormality of Tamm Horsfall glycoprotein in calcium oxalate nephrolithiasis. Am J Physiol 1998;29:F569-F261.

Ettinger B, Tano A, Citron JT et al. Randomized trial of allopurinol in the prevention of calcium oxalate calculi. N Engl J Med 1986;315:1386-9.

Emmerson BT. The mangement of gout. N Engl J Med 1996;334:445-51.

Priè D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:2399-409.

Epidermolysis bullosa: natural model for cancer cells study.

2010-08-31T21:47:00.000+02:00

Epidermolysis Bullosa

Epidermolysisi bullosa is a group of rare genetic diseases in which bullous lesions such as fluid filled cavities, or blisters larger than 0.5 cm affecting primary the skin arise after exposure to traumatic agents.

Three major forms of epidermolysis bullosa have been described to date:

- Dystrophic

- Junctional

- Simplex

Characterized by loss of tissue integrity respectiverly at upper dermis, dermo-epidermal interface, within epidermis.

Simplex

With rare exception epidermolysis bullosa simplex is inherited with an autosomal dominant fashion.The EB simplex is the most frequent form occurring approximateli at a rate of 1 case per 25.000 live births; however it is also the least severe form. In EB simplex, trauma induced loss of tissue integrity ccurs within the basal layer of epidermal keratinocytes. In these patients inborn errors render basal keratinocytes fragile, and more susceptible to rupture when epidermis is subjected to a mechanical stressor event. Most cases of EB simplex result from mutations affecting either Keratin 14 (K14) or Keratin 5 (K5), these two kind of proteins are expressed respectively in basal keratinocytes and in related complex epithelia, forming the type I and type II intermediate filaments (IF) proteins. Because of heteropolymeric structure of keratin filaments, mutations in either the K5 or the K14 genes can elicit a substantial fraction of the broad spectrum clinical pictures seen in EB simplex.

Epidermolysis Bullosa Simplex has being the first disorder shown to be caused by mutations in a gene encoding an intermediate filament (IF) protein and also the first epithelial skin fragility condition to have its etiology revealed, in the early 1990s.

Many genetic findings have now formalized a new role for keratin in regulating skin pigmentation. Skin pigmentation is the result of an intimate partnership between keratinocytes and neural-crest derived melanocytes, which are located on the basal layer of epidermis and hair matrix. Memebrane-bound melanosomes are manufactures in melanocytes and transferred to the cytoplasm of proximal keratinocytes, where their cargo, ie. pigmented melanin granules, are released. There is growing evidences that melanin pigments functionally interact with keratin filaments; the dynein light chain, partecipating directly to transport of melanin pigment to form a supranuclear cap in pigmented keratynocytes, interacts with head domain of keratin 5 protein.

Dystrophic

Autosomal recessive dystrophic epidermolysis bullosa (DEB) is a severe skin disorder beginning at birth and characterized by recurrent blistering at the level of the sublamina densa beneath the cutaneous basement membrane. This results in mutilating scarring and contractures of the hands, feet, and joints. Patients also developed strictures of the gastrointestinal tract from mucosal involvement, which can lead to poor nutrition. Affected individuals have an increased risk of developing aggressive squamous cell carcinoma.

Hovnanian et al (1992) demonstrated linkage between a PvuII polymorphic site in the COL7A1 gene on chromosome 3p21 and recessive dystrophic epidermolysis bullosa in 19 informative families (maximum lod score of 3.95).

Ryynanen et al. (1991) and Uitto et al. (1992) demonstrated linkage between a PvuII RFLP of the COL7A1 gene and dominant DEB, suggesting that the autosomal dominant and autosomal recessive disorders are due to mutations in the same gene.

Molecular Genetics

In an African American family in which 4 individuals related as first cousins once removed had autosomal recessive epidermolysis bullosa dystrophica, Christiano et al. (1993) identified a homozygous mutation in the COL7A1 gene. The unaffected mother and half-brother were heterozygous for the mutation.

In 3 Japanese brothers with autosomal recessive DEB, Christiano et al. (1995) found compound heterozygosity for 2 truncating mutations in the COL7A1 gene.The unaffected parents were each heterozygous for 1 of the mutations.

Christiano et al. (1996) identified glycine substitution mutations in the COL7A1 gene in affected members of 4 unrelated families with RDEB. Two families were compound heterozygous for a glycine substitution and a premature termination mutation, whereas the other 2 families were homozygous for a glycine substitution. In all 4 recessive families, the glycine substitution mutation was silent in heterozygous carriers who had no disease manifestations. Christiano et al. (1996) stated that the COL7A1 gene is thus unique among the collagen genes in that different glycine substitutions can be either silent in heterozygotes or can result in a dominantly inherited DEB. Inspection of the location of the glycine substitutions did not show a positional effect in terms of phenotype or pattern of inheritance.

Varki et al. (2007) analyzed the COL7A1 gene in 310 patients with dystrophic epidermolysis bullosa. Mutations were found in 1 or both alleles in 243 (78.4%) patients, comprising 355 mutant alleles of the anticipated 438 (81.1%) mutant alleles. The authors reviewed the spectrum of COL7A1 mutation and genotype-phenotype correlations, noting that patients with severe recessive DEB tended to have premature truncating mutations, whereas those with milder dominant DEB tended to have glycine substitutions. Seven patients had features of both dominant and recessive forms of disease and were found to carry both dominant and recessive mutations.

Modifier Genes

A defect in collagenase (MMP1) was implicated early on in the pathogenesis of dystrophic epidermolysis bullosa. Type VII collagen is susceptible to degradation by collagenase. Bauer (1977) found that procollagenase purified from fibroblasts of 2 patients with DEB was more thermolabile, showed decreased calcium affinity, and had decreased activity in vitro compared to control values. Bauer postulated a structural gene mutation, defective posttranslational modification of the enzyme, or a mutation in a gene regulating normal degradation of collagenase. Bauer and Eisen (1978) observed enhanced collagenase production by cultured skin fibroblasts in 8 of 10 patients with autosomal recessive dystrophic epidermolysis bullosa. Increased levels of immunoreactive collagenase were found in unaffected and affected areas of the skin. Bauer et al. (1986) found that enhanced expression of collagenase by fetal recessive dystrophic epidermolysis bullosa skin fibroblasts could serve as a biochemical adjunct and possibly an alternative to morphologic examination of tissue for antenatal diagnosis.

Titeux et al. (2008) found a significant association between a functional SNP (rs1799750) in the MMP1 gene and disease severity in 3 affected members of an RDEB family who were discordant for the SNP. The observations were confirmed in a cohort of 31 unrelated French RDEB patients: the functional SNP resulting in increased collagenase activity was associated with more severe phenotype (p = 6.27 x 10(-5)). Titeux et al. (2008) concluded that increased MMP1 leads to increased collagen degradation and worsening disease severity, suggesting that MMP1 is a modifier gene in RDEB.

Animal Model

In an inbred breed of golden retriever dogs with RDEB and aberrant expression of collagen type VII, it was isolated and analyzed the 9-kb dog COL7A1 cDNA and identified a 5716G-A transition in exon 68, resulting in a gly1906-to-ser (G1906S) substitution at a conserved residue. Highly efficient transfer of the wildtype COL7A1 cDNA to both dog RDEB and human primary RDEB COL7A1-null keratinocytes, using recombinant retrovirus vectors, achieved sustained and permanent expression of the transgene product. The expression and posttranslational modification profile of the recombinant collagen type VII was comparable to that of the wildtype counterpart. The recombinant canine collagen type VII in human RDEB keratinocytes and dog cells corrected the observable defects caused by RDEB keratinocytes in cell cultures and in vitro reconstructed skin. Not only infection efficiency but also high expression levels may be required to ensure therapeutic efficacy in the presence of mutated gene products.

Transgenic mouse model with conditional inactivation of Col7a1 expression resulting in a Col7a1 hypomorphic animal expressing about 10% of normal Col7a1 levels. Homozygous mice appeared normal at birth, but developed blisters on the paws by 24 to 48 hours after birth. Hypomorphic mice showed poor general condition resulting from poor nutrition and blisters of the tongue. A liquid diet resulted in increased survival. Mitten deformities of the paws were found to result from soft tissue accumulation and contraction due to aberrant fibrosis that accompanied wound healing. The phenotype resembled the human recessive disorder, including skin fragility, nail dystrophy, pseudosyndactyly, and growth retardation. Intradermal injection with wildtype fibroblasts restored Col7a1 deposition and function and resulted in phenotypic improvement.

From DEB to cancer

Normally injury to an adult tissue initiates a sophisticated repair process aimed to restore the damaged body site. The series of events involved into repair process must be tightly regulated and synchronized to re-establish the tissue integrity and its homeostatic equilibrium. Defects and excesses in healing process are quite simple examples of cellular physiology derangements.

In 1863 Rudolf Virchow postulated that chronic irritation and previous injuries are precondition for “cancer growth”.

Important examples supporting Virchow view in developing cancer hypothesis are known precancerous lesions such as chronic viral hepatitis, Helicobacter pylory induced gastric inflammation, inflammatory bowel disease and skin blistering diseases, such as Recessice Dystrophic Epidermolysis Bullosa (RDEB) predisposing to squamous cell carcinoma early in life.

DEB is an heterogeneous group of disorders characterized by chornic epithelial fragility; evident such as trauma-induced skin blistering. It is caused by mutations of 1 of 14 genes encoding proteins forming the dermal-epidermal junction, a specialized basement membrane zone that attaches the epidermis to the dermis. Between these genes we have keratin 14 and keratin 5 responsible of type I and type II intermediate filaments formations, plectin, a cytolinker protein responsible of integrating various cytoskeletal and cell-adhesive elements in a functionally unified network, Collagen type XVII, alfa 1 (COL7A1), a hemidesmosomal plaque protein required for tight adherence of basal keratinocytes to the basal lamina.

In a classic publication, Harold Dvorak postulated that “tumors are wounds that do not heal”. Dvorak recognized that the composition of the tumor stroma strongly resembles the granulation tissue of healing skin wounds, so that epithelial tumor cells promote the formation of their stroma by activating the same process of wound healing. This process in cancer tissue environment is uncontrolled leading to cell proliferation, invasion and metastasis.

The heterotypic interactions operating within tumors are higly complex and involve the exchange of distinct molecular species mediating cell-to-cell contact signaling, many of these signals are not routinely exchanged by normal cell types, by undamaged tissue and therefore seem unique to tumors.

This complexity raises some fundamental questions: How do cancer cells learn to release and respond to so heterotypic signals ? Are these molecular pathways invented anew, being assembled together piece by piece, each time normal cells evolve progressively into cancer cells? Or do cancer cells exploit preexisting biological programs that are normally used by cells for other purposes?

One possible solution came from studies of Harold Dvorak on 1986 showing strict resemblance between histological appearance of tumors and wounds, both in signalling processes and molecular pathways used. Accordingly, cancer cells simply activate a complex, normal physiological program, wound healing, that is already encoded in their genomes.

Wound healing has been studied most extensively in the context of the skin. Following the formation of a superficial wound to the skin and underlying tissues, blood platelets aggregate and release granules containing PDGF, TGF beta among other growth factors. Wounding also causes release of vasoactive factors, increasing the permeability of blood vessels near the wound. This helps the wound site to acquire fibrinogen molecules from blood plasma, which, when converted to fibrin, create a scaffolding of the blood clot.

PDGF released from platelets attracts fibroblasts and stimulates their proliferation. Thereafter, TGF beta activates these fibroblasts, inducing the release of a class of secreted proteases termed Matrix Metalloproteinases (MMPs). Unlike most secreted proteinases, which have a serine in their catalytic site, MMPs carry a zinc ions to aid in catalysis, so that these ions inspired the name. Fibroblasts activated also secrete mitogens such as various Fibroblasts Growth Factors (FGFs) that can stimulate the proliferation of some epithelial cells.

The finalistic function of secreted MMPs is the degradation of specific components of the extracellular matrix (ECM), reaching three targets; first remodelling ECM structure and forming new space available for new cells; second releasing a variety of new growth factors embedded in an inactive form into proteoglycans matrix and now become solubilized and activated. On this group of growth factors we have FGF, TGF beta, PDGF, EGF and INF gamma. Third releasing proenzymes converted by MMPs into their active forms.

Finally monocytes attracted into wound sites are the source of FGF and VEGF able to stimulaye endothelial cells proliferation and new capillaries formation in the sites proximal to loss of tissue: a process called neoangiogenesis.

In order to reconstructing epithelial sheet , epithelial cells reduce their adhesion to the ECM, especially to the basement membrane that separe them from stromal compartement. To do so, epithelial cells increased their mobility and suppress the synhtesis of E-cadherin with N-cadherin, normally expressed in fibroblasts, as main structure of “adherens junctions”.While shifting from one type of cadherin to another, epithelial cells undergo a major chnage in their phenotype, which causes them to assume a fibroblastic appearance. This profound shift is termed “epithelial-mesenchymal transition” (EMT) and enable epithelial cell to become motile and invasive. Interestingly EMT is reinforced by latent TGF beta released from ECM and, importantly, it represent only a temporary shift in cell phenotype. So that when they covered the wound site reconstructing the epithelium, they revert to an epithelial state via the program termed “Mesenchymal-epithelial transition” (MET).

If we compare these processes with the interactions of epithelial cancer cells and their stromal neighbors, we find striking parallels between wound healing and tumorigenesis. One clear similarity between the two processes derives from the presence of clumps of fibrin in the tumor-associated stroma, in the case of tumor due to constitutively lack from capillaries within tumors of fibrinogen. This fibrinogen lack is the cause of formation inside the tumor of large bundles of fibrin strands, forming an important scaffold that helps these recruited mesenchymal fibroblasts cells to attach, through integrins into fibrin matrix and migrate. The invading stromal cells remodel this “provisional matrix” by degrading many initially formed fibrin molecules and replacing them with a more permanent matrix that is assembled from collagen secreted by fibroblasts. This step closely parallels the sequence of wound healing.

In wound healing one task of stromal cells involves the physical contraction of wound site in order to close wounds. This contraction is mediated by a specialized class of fibroblasts termed myofibroblasts, capable of using the actin-myosin contractile system to generate a mechanical tension needed fro wound to closure.

Essentially identical myofibroblasts are prominent components of the stroma present in the majority of advanced carcinomas. TGF beta released by many types of carcinomas is the major factor responsible for progression of myofibroblasts in the tumor associated stroma. Interestingly, the tumor stroma formed by myofibroblasts differ substantially in appearance from the stroma present in normal epithelial tissue; so that pathologists label it such as “reactive” or “ desmoplastic” stroma. Referring to hardness of the tumor mass as a whole due to deposition of extracellular matrix by myofibroblasts. As the progression of a carcinoma advance to higher , more aggressive grade, the proportion of stroma that is desmoplastic often increases in parallel. Myofibroblasts construct the desmoplastic stroma through the secretion of large amount of collagen type I and type III, fibronectin, proteoglycans, and glycosaminoglycans, . In addition these cells secrete urokinase plasminogen activator (uPA), MMPs, so that as desmoplastic stroma matures stromal cells disappeared, being replaced progressively by the dense, acellular, collagenous ECM that is the hallmark of this type of tumor associated stroma.

Carcinoma associated fibroblasts is a term used to describe a mixed population of fibroblasts and myofibroblasts that are present in the stroma of epithelial tumors. Gene expression analyses of these cells underscore the strong similarities between these cells and the fibroblasts present in wound sites, providing a further indication of the role of these cells as key players in tumor progression.

One of most relevant differences is the so called Warburg Effect describing the strict dependance from anaerobic glycolysis in energy production of cancer cells.

References

Virchow R, Virchow R. Aetiologie der neoplastischen geschwulste/Pathogenie der neoplastischen Geschwulste. Verlag von August Hirschwald, Berlin Germany 1863.

Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 1986;315:1650-9.

Dolberg DS, Hollingsworth R, Hertle M et al. Wounding and its role in RSV-mediated tumor formation. Science 1985;230:676-8.

Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182-6.

Wagner JE, Ishida-Yamamoto A, McGrath JA et al. Bone marrow transplantation for recessive dystrophic epidermolysis bullosa. N Engl J Med 2010;393:629-39.

Friedl P, Gilmour D. Collective cell migration in morphogenesis, regeneration and cancer. Nature Rev Mol Cell Biol 2009;10:445-57.

The inhibitory endocrine actions of arachidonic acid derivatives present in plants and in human too.

2010-08-13T21:10:00.000+02:00

The Endocannabinoid system

The first document in the discovery of therapeutic and psychotropic actions of the plant Cannabis sativa date back to 4000 years ago present in a document writed in India.

Gaoni and Mechoulam on 1964 described the actions of a purified component of hashish; from this documents a stunning amounts of researchs revealed that endocannabinoid system as a central modulatory role in animal physiology.

But what’s are the endocannabinoid elements present in animals? These compounds comprise lipid ligands called “endocannabinoids” of specific receptors; compounds undergoing a specific metabolism.

In general, the endocannabinoid system is involved in many different physiological functions, many of which relate to stress recovery systems and to the maintenance of homeostatic balance of physiological very important functions such as :

- Neuroprotection

- Nociception

- Regulation of motor activity

- Control of memory processing

- Modulation of immune and inflammatory processes

- Control of heart rate, blood pressure, bronchial activity

- Finally they exert an important function with an antiproliferative actions in tumor cells

In 1992 the first endogenous endocannabinoid the so called “anandamide” (AEA), was identified. Subsequently a second endocannabinoid called 2-arachidonoyl glycerol (2-AG) was discovered both by Mechoulam’s group. Both these compounds are derivatives of arachidonic acid and are able to bind to Cannabinoid receptor 1 and 2 (CB1 and CB2), with differences in affinity and efficacy activation.

During the last few years, several other bioactive lipid mediators have been described, these compounds are:

- 2-arachidonoyl-glyceryl-ether (noladin ether)

- O-arachidonoyl-ethanolamine (virodhamine)

- N-arachidonoyl-dopamine

- Oleamide

Endocannabinoids are very lipophilic and thus cannot be stored into vescicles like other neurotransmitters. Consequently, the regulation of endocannabinoid signaling is tightly controlled by their synthesis, release, uptake, and degradation.

Several extracellular stimuli, including membrane depolarization and increased intracellular calcium or receptor stimulation, can activate complex enzymatic machineries, which lead to the cleavage of membrane phospholipids and to the synthesis of endocannabinoids. Endocannabinoids can activate their receptors either after previous into the extracellular space or directly moving within the cell membrane. The enzymes able to degrade endocannabinoids are quite well characterized. They are fatty acid amide hydrolase (FAAH) and monoglycerol lipase. An interesting aspect of endocannabinoid activity is the rapid introduction of their synthesis, receptor activation, and degradation. So that it has been suggested that they act on demand, with a tightly regulated spatial and temporal selectivity. In other words the system exerts its modulatory actions only when and where it is needed.

The hormonal stimulation with glucocorticoids can lead to the synthesis of endocannabinoids in the hypothalamus through a rapid nongenomic mechanisms.

Concerning degradation of endocannabinoids, endocytic processes are involved in the uptake of endocannabinoids and found that about half of the anandamide uptake occurs via a caveola/lipid raft-related process.

In the central nervous system (CNS), endocannabinoids can act as neurotransmitters transferring information from one neuron to the next.

Postsynaptically released endocannabinoid travel to the presynaptic site where they activate CB1 receptors, mediating a retrograde signal. The overall effect is a decrease in the release of neurotransmitters such as glutamate and GABA.

The decrease in neurotransmitters release is present in many brain regions including Hypothalamic areas and Ventral Tegmental Areas where they regulates the release of dopaminergic neurons. In the cerebral cortex endocannabinoid can mediate an autocrine signaling that induces a self-inhibitory effect on neuronal activity in particular in GABAergic neurons of the cerebral cortex.

It has been known for a long time that the exogenous cannabinoids are able to affect secretion of pituitary hormones, thus having a strong effect on peripheral target organ functions.

The hypothalamus is generally considered as the main site of cannabinoid action on neuroendocrine functions. An elegant study supported this view showing that endocannabinoids act as a retrograde messengers activating CB1 receptors expressed at presynaptic glutamaergic terminals in the hypothalamus. The subsequent inhibition of the release of the excitatory neurotransmitter glutamate into neuroendocrine cells of the Para Ventricular Nucleus and at the Supraoptic Nucleus leads to final inhibitory effect on all neuroendocrine functions.

The role of cannabinoid in modulation of the hypothalamic pituitary gonadal axis and fertility is probably the most important function exerted by this endocrine neuromodulators system in humans.

While FSH secretion seems to be unaffected by cannabinoids secretion, several evidences attributes to cannabinoids a strong activity of down-regulation of blood LH levels.

This effect is due to a complete suppression of the secretory pulse of LH.

A general consensus attributes LH-inhibitory action of cannabinoids to a suprapituitary site of action. Cannabinoids indirectly modify GnRH secretion by negatively modulating the activity of neurotransmitters known to facilitate GnRH secretion, such as norepinephrine and glutamate, and by stimulating neurotransmitters known to down-regulate GnRH secretion such as dopamine, GABA , opioids, and Corticothropin Releasing Hormone.

The stimulatory effect exerted by cannabinoids on dopaminergic neurons varies in function of the gonadal status, as demonstrated by progesterone receptors, dopamine receptor type 1D and CB1 receptor activation on activating sexual behaviour in female rats.

However it is not well known where and under what circumstances the endocannabinoids are produced to do so.

It has been speculated that endocannabinoids may influence hormonal secretion and sexual behaviour by directly targeting to the CB1 receptor.

Moreover, an important source of endocannabinoid haas been found into the ovaries in particular during ovulation, making possible hypothesize that the endocannabinoids may help to regulate follicular maturation and development of the ovary.

The uterus contains the highest levels of anandamide detected so far in mammalian tissues, and it is the only tissue where anandamide is the main component (up tp 95%) of N-acetylethanolamides. High levels of maternal anandamide are detrimental to early placental and fetal development as demonstrated by high level of FAAH degradating enzyme in cytothrophoblastic cells and we known that levels of FAAH are under control of several hormones including progesterone, leptin, and FSH very well known regulator of fertility.

In summary, all the steps starting from fertilization up to pregnancy seem to be tightly controlled by endocannabinoid system, reinforcing the notion that this endocrine system should be considered not only as a central neuromodulator but also as a phisiological actor of fertility in a wider scenario.

In males CB1 receptor activation by anandamide reduce sperm mobility by affecting mitochondrial activity, and inhibiting the capacitation-induced acrosome reaction. It is therefore reasonable to hypothetize that anandamide levels might be increased in different pathological conditions of the male reproductive tract. In these cases, tha pharmachological blockade of the ndocannabinoid system might be helpful in the treatment of some forms of male infertility.

The second important role exerted by endocannabinoids in human physiology is Modulation of Energy Balance.

First is the finding of an high degree of evolutionary conservation of the role of this system in the regulation of feeding responses. Second is the observation that high levels of endocannabinoids in maternal milk are critically important for the initiation of the suckling response in newborns.

The stimulating effect of cannabinoids on appetite observed in healthy subjecs promoted the assessment of the efficacy of a cannabinoid treatment for clinical syndromes showing loss of appetite or weight, such as cancer or AIDS-associated anorexia, as well’s as associated therapy to limit nausea and vomiting symptoms associated with most chemotherapeutic drugs. In 1985, the US Food and Drug Administration officially approved the use of Delta9-Tetrahydrocannabinol ( Dronabinol ) for the treatment of chemotherapy-induced nausea and vomiting refractory to other drugs. Recently, Dronabinol was also proposed as an orexogenic drug in patients suffering from Alzheimer’s disease.

More recent experiments make it possible to attribute the endocannabinoid system with an important role in the process underlying the motivation to obtain food. It is suggested that endocannabinoids gradually increase during intermeal intervals, reaching a critical level where motivation to eat is triggered. Accordingly, the longer the time since the last meal, the greater the activity in relevant cannabinoid circuits, and consequently the higher the motivation to eat. In the nucleus accumbens increased levles of anandamide and 2-AG in the fasting conditions, and a concomitant decline of 2-AG with the feeding state strongly support this hypothesis.

Some experimetal data suggested that the activation of endocannabinoid system may alter the appetitive value of ingested substances. This hypothesis is consistent with the evidence that this endocrine system exerts a facilitatory function on brain reward circuits. Some findings seems to support the hypothesis of incentive action increasing the incentive vallue of the food regardless of the quality of the macronutriens. Other data however resembling the “marshmallow effect” in marijuana smokers, have been interpreted in terms of eat highly palatable food (“orosensory reward hypothesis” ).

The reward / reinforcement circuitry of the mammalian brain consists of a series of synaptically interconnected brain nuclei associated with the medial forebrain bundle, linking Ventral Tegmental Area, the nucleus accumbens and the ventral pallidum. This circuit is implicated in the pleasure produced by natural rewards, such as food, addictive drugs, and sex and it is the neural substrate of drug addiction and addiction-related phenomena, such as craving and dysphoria induced by withdrawal.

In such framework, food intake acts on dopamine, opioid, serotonin, and noradrenaline neuronal fibers, which connect the hindbrain and midbrain to the hypothlamus to modulate the action of feeding and satiety factors.

Most important pathways involved into neuronal circuits of natural rewards are:

- Mesolimbic dopaminergic pathways (nucleus accumbens)

- Hypothalamic opioid pathways (Naloxone experiments)

- Serotoninergic system (Dexfenfluramine , Sibutramine experiments)

All three systems are controlled by and interact with endocannabinoid pathways.

We now know that at genetic level a nuclear receptor plays a very important action throughout the hypothalamic-pituitary-steroidogenic axis, it is called steroidogenic factor-1 (SF-1 or NR5A1). SF-1 is a key transcription factor in establishing the cytoarchitecture of the VMH. The SF-1 neurons in the VMH have been implicated in energy homeostasis and contribute to the sensing of important regulators of energy homeostasis such as leptin and glucose. SF-1 KO mice exibited delayed onset obesity, exibited marked increase in anxiety-like behaviour and as a result of the absence of VMH structures these mice had a decreased hypothalamic expression of Brain-Derived Neurotrophic Factor (BDNF).

It has been recently demonstrated that disruption of VMH neurons is associated with a markedly decreased expression of CBR1 (cannabinoid receptor type 1). In particular SF-1 directly regulates CBR1 linking to its 5’ flanking region acting as transcription promoter agent at gene coding for CBR1.

A complex redundant neuronal hypothalamic network provides high level of adaptability of feeding behavior to various central and peripheral stimuli. Redundancy in appetite-stimulating signaling is conceivable in view of the vital importance of feeding for survival.

References

Gaoni Y, Mechoulam R. Isolation, structure and partial synthesis of an active constituent of hashish. J Am Chem Soc 1964;86:1646-7.

Kolodny RC, Masters WH, Kolodner RM et al. Depression of plasma Testosterone levels after chronic intensive marihuana use. N Engl J Med 1974;290:872-4.

Matsuda LA, Lolait SJ, Brownstein MJ et al. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990;346:561-4.

Devane WA, Hanus L, Breuer A et al. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 1992;258:1946-9.

Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993;365:61-5.

Marsicano G, Goodenough S, Monory K et al. CB1 cannabinoid receptors and on-demand defense against excitotoxicity. Science 2003;302:84-8.

Mani SK, Mitchell A, O’Malley BW. Progesterone receptor and dopamine receptors are required in Delta9 Tetrahydrocannabinol modulation of sexual receptivity in female rats. Proc Natl Acad Sci USA 2001;98:1249-54.

Stella N. How might cannabinoids influence sexual behavior? Proc Natl Acad Sci USA 2001;98:793-5.

Schuel H, Burkman LJ, Lippes J et al. N-Acetylethanolamines in human reproductive fluids. Chem Phys Lipids 2002;121:211-227.

Rossato M, Ion Popa F, Ferigo M et al. Human sperm express cannabinoid receptor CB1, the activation of which inhibits motility, acrosome reaction, and mithocondrial function. J Clin Endocrinol Metab 2005;90:984-91.

De Petrocellis L, Melck D, Bisogno T et al. Finding of endocannabinoid signalling system in Hydra, a very primitive organism: possible role in the feeding response. Neuroscience 1999;92:377-87.

Fride E, Ginzburg Y, Breuer A et al. Critical role of the endogenous cannabinoid system in mouse pup suckling and growth. Eur J Pharmacol 2001;419:207-14.

Pagotto U, Marsicano G, Cota D et al. The emerging role of the endocannabinoid system in endocrine regulation and energy balance. Endocr Rev 2006;27:73-100.

Van der Lely AJ, Tschoep M, Heiman ML, Ghigo E. Biological, physiological, pathophysiological, and pharmachological aspects of ghrelin. Endocr Rev 2004;25:426-57.

Age-related macular degeneration and Diabetic Retinopathy: two sides of the same coin ?

2010-08-05T10:14:00.000+02:00

Somatostatin

On 1952 Sheenan syndrome was observed such as result of pituitary apoplexy after partum, the pituitary apoplexy was able to revert the diabetic retinopathy.

From years between 1950 to 1970 pituitary ablation was observed to be useful in blocking the progression of diabetic retinopathy.

The level of Growth Hormone measured in plasma with the first radioimmuno assays during the 1960 to 1970 were found to be increased in diabetic patients.

Next level of awareness of involvment of GH pathway was the demonstration during the 70-1980 years that the plasma levels of Insulin-like Growth Factor I also called somatomedin were found to be increased in vitreous of eyes affected by diabetic retinopathy.

The synthetic production of Somatostatin, a peptide able to block the secretion of GH from pituitary, allows the demonstation that Somatostatin administration to diabetic patients was able to block the progression of diabetic retinopathy ( from 1980 to 1990 ).

Last on late 1990 it was demonstrated a role of growth hormone and Insuli-like Growth Factor I in the angiogenesis processes involved into neovascularization of vitreous present in diabetic patients affected by diabetic retinopathy.

Role of IGF-1 in diabetic retinopathy

It was demonstrated by experimental evidences that elevated levels of IGF-1 are able to:

- Induce the leakage of fluids across blood retinal barrier

- Stimulate neovascularization of retinal tissue

- Stimulate the contraction of Muller’s cells

- Upregulates the expression of VEGF in retinal pigmented epithelial cells

Moreover the inhibition of IGF-1 action at retinal level was able to block the neovascularization process.

In vitro experimental data demonstrated that:

- On mammalian retinal microvascular cells are present IGF-1 Receptors

- On human retinal endothelial cells are present Somatostatin Receptors

- IGF-1 linking to its receptors is able to induce the release of plasminogen activatorand increase the DNA sythesis in retinal endothelial cells.

- GH linking to its receptors can stimulate the proliferation of human retinal endothelial cells

- Ocreotide ( a somatostatin inhibitor ) is able to inhibit the proliferation of retinal endothelial cells produced by both IGF-1 and by b-FGF

The mechanism of action of IGF-1 on diabetic retinopathy is explained at molecular level with involvment of Retinal Pigmented Epithelial Cells that are able to link IGF-1 and through PIK3-HIF1 alfa to induce the gene expression by these cells of Vascular Endothelial Growth Factor (VEGF). Once secreted both VEGF and IGF-1 act on Retinal Endothelial Cells in order to stimulate their proliferation through MAPKinases pathways.

These data suggested that Somaostatin analogs like Octreotide (Sandostatin LAR 20 and 30 mg) can be used to arrest diabetic retinopathy. Two large randomized phase III studies have been developed to date in order to demonstrate the clinical utility of Sandostatin one in European countries and another on american countries.

Activation of PKC

Hyperglycaemia has been shown to increase the levels of cellular Diacylglycerol (DAG) by thee mechanisms: directly, througth increased oxidants production, and througth increased levels of AGE (Advanced Glycation End Products). We know from biochemistry that the intracellualr production of DAG is linked to activation of Phospho Kinase C (PKC), a well known intracellular kinase pathway found to be present not only in retinal tissues but also in vascular cells, muscle cells, kidney, heart and neurons.

In vascular tissue the activation of PKC pathways leads to different reactions such as:

- Increased synthesis of basement membrane proteins

- Leukocytes activation with increased release of cytokines and of other growth factors such as Endothelin, VEGF and TGF beta.

- Increase in endothelial cell permeability with simultaneous stimulation of endothelial cells proliferation

- Smooth muscle cells contraction due to concomitant leakage of calcium stores from endoplasmic reticulum.

In summary diabetic retinopathy can be linked to activation of PKC pathways accounting for many common clinical aspects of disease such as: vascular retinal injury and disfunction with vascular fluid leakage and following Macular Edema. Retinal Capillary vessels non perfusions with subsequent stimulation of neovascularization and Proliferative Retinopathy.

A drug initially called LY333531 given to dosing regimen of 25 mg/kg/day by oral route is able to block PKC activation, an effect demonstrated by reduction of VEGF expression in retinal tissue. This heterocyclic compound, now called Ruboxistaurin (RBX) is higly selective for inhibiton of PKC, higly selective for beta isoform of PKC, able to reduce retinal neovascularization, reduce vascular permeability and to normalize retinal blood flow.

Two phase II trials have been developed in order to clinically evaluate the benefit of RBX at three different dosages 8, 16, 32 mg at day on Non Proliferative Diabetic Retinopathy on Diabetic Macular Edema. Aiello LP on 2003 demonstrated the dose related effects of RBX on Diabetic Retinopathy and Laser Photocoagulation; he also demonstrated that visual loss was decreased with the sue of RBX at increasing dosages in patients affected by diabetic retinopathy.

One phase III trial has to be published on 2005 and the aim of the study is to demonstrate a reduction in visual loss on patients with both Macular Edema and Non Prliferative Diabetic Retinopathy.

Renin-Angiotensin system

Angiotensinogen product by liver is transformed into blood by the enzyme Renin into angiotensin I and lated by Angiotensin Converting Enzyme (ACE) on angiotensin II that explaine its action linking to two kind of receptors called AT1 and AT2. The first one explaining the vasoconstriction of muscle vessel cells, inducing renal kidney resorption through aldosterone stimulation, inducing cell growth and remodelling on cardiovascular cells both endothelial and muscular cells. The second one , AT2, explaine its action inducing apoptosis, vasodilatation, and explaining an antiproliferative and anti defferentiation action.

At present we have two kind of drugs able to interfere with renin-angiotensin system: ACE-Inhibitors and ARBs or Angiotensin Receptor Blockers active only on type 1 angiotensin receptors.

Interestingly RAS is present also on Retinal tissue beginning at postnatal day 1 and increasing progressively after birth. It has been demonstrated in particular the presence on retinal rat tissue of both Renin, Angiotensin Converting Enzyme, and AT1 receptors.

Diabetic patients affected by diabetic reninopathy show an increased levels of angiotensin II concentration at vitreous level; suggesting that diabetic patients have an activation of retinal RAS. The same diabetic patients show also an increase in vitrous concentration of VEGF proportional to levels of vitreous Angiotensin II.

Moreover diabetic patients show an increased in retinal vessel cells proliferation proportional to activation of RAS, demonstrated hystologically in rat iris and inner retinal tissue. Iris cells as well inner retinal cells of diabetic rats proliferate much more when exposed to renin activity, compared to iris and inner retinal cells of non diabetic mice.

Exposing retinal cells to ARBs so blocking AT1 receptors at retinal levels we can have different reactions:

- Retinal endothelial cells reduction in VEGF-R2

- Retinal pericyte cells reduction in VEGF secretion and Reactive Oxigen Species (ROS) production

- All retinal tissue in vivo show beside decresed expression of VEGF-R2 and secretion of VEGF, also decreased intracellular activation of PKC.

Experimetal evidences on rat demonstrated that retinal rat cells exposed to candesartan or angiotensin converting enzyme inhibitors show a decreased production and transcription of VEGF and decreased retinal neovascularization.

The ADVANCE trial using Perindopril and Indapamide demonstrated that in patients affected by diabetes type 2 there is a non significant trend to reduce visual deterioration in patients affected by diabetic retinopathy. (Lancet September 2007)

The DIRECT trial was designed to demonstrate the effect of Candesartan on diabetic retinopathy.

DIRECT-1 prevent study the prevention of diabetic retinopathy in patient affected by type 1 Diabetes but without retinopathy.

DIRECT-2 protect study patient with type 1 diabetic retinopathy mild to moderate

DIRECT-3 protect study patient with type 2 diabetic retinopathy mild to moderate

ETDRS retinopathy scale is based on 7 field stereo photographs and measure the severity and levels of Early Treatment Retinopathy Study scale

Severity Level ETDRS

Absent 10

Microaneurysm 20

Step 2 change

Mild Non Proliferative 35

Step 3 change

Moderate Non Prolifer 43

Moderately Severe NPDR 47

Severe NPDR 53

Proliferative DR 61-65-71- 75-81

17 stages of ETDRS have been detected in this way and they are used now in large scale randomized trials in order to evaluate the grade of DR.

ARB in Clinical Practice

On DIRECT-3 study 29% of patients presented level 20 ETDRS, 54% of patients presened 35 ETDRS level, and finally 17% presente 43-47% level of ETDRS.

DIRECT-3 study demonstrated that the use of Candesartan at doses of 16 to 32 mg at day

Is able to block the pregression from step 3 Mild NPRD in a way non different from placebo, whereas regression from RD stable on step 2 or step 3 was superior in patients reated with Candesartan. In partivcular at retinopathy level raanging between 20 and 35 the regression activity of Candesartan was significantly different from palcebo whereas on patients affected by more serious levels of retinopathy (about > 35) the effect of Candesartan was not evident and equal to placebo.

Fibrate

The FIELD trial published on The Lancet on 2007 demonstrated that the use of Fibrate for 5 years is able to reduce the Laser need treatment in diabetic retinopathy, reduces step-2 progression to greater retinopathy grade in patients with pre-existing retinopathy but not in patients without retinopathy, is not able per se to reduce the levels of HbA1c, blood glucose and blood pressure.

Practical view

In summary the key player in DR seems to be VEGF that is able to destroy the expression of proteins maintaining tight junctions between Retinal capillary endothelial cells and retinal pigmented epithelial cells. At vessel levels these tight junctions are able to form the so called retinal blood barrier; between pro-barrier factors we have Factor X of coagulation, between permeability factors we have VEGF and hystamine.

As a result of increased expression of VEGF we have an increase in vascular permeability and compromised retinal blood barrier.

At present two kind of drugs are used intravitreally to prevent DR:

- Triamcinolone intravitreal administration: used to block the production of PKC

- Anti-VEGF monoclonal antibody such as Bevacizumab and Ranibizumab and Pegaptanib sodium.

References

de Joung PTV. Age-related Macular degeneration. N Engl J Med 2006;355:1474-85.

Aiello LP. Targeting intraocular neovascularization and Edema – One drop at a time. N Engl J Med 2008;359:967-9.

Klein BKE. Reduction in risk progression of Diabetic retinopathy. N Engl J Med 2010;363:287-8.

The ACCORD Study Group amd ACCORD EYE Study Group. Effects of medical mtherapies on retinopathy progression in type 2 diabetes . N Engl J Med 2010;363:233-44.

Sacks FM. After fenofibrate intervention and event lowering diabetes (FIELD) study: implications for fenofibrate. Am J Cardiol 2008;102(12A):34L-40L.

Many thanks to Massimo Porta MD, PhD, Full Professor of Internal Medicine at Institute of Internal Medicine for his suggestions and his unvaluable experience in the firld of Diabetic Retinopathy.

Klotho more than “ninphae”

2010-07-12T22:08:00.000+02:00

From Greek mythology “Klotho” was one of three Moirae. She is responsible for spinning the thread of human life, so that she mades major decisons when a person is born. She controls who born through his life she also decide who has to be saved or put to death.

Two other sisters , Lachesis and Atropos, are responsible of human destiny and influence their misery and suffering.

Clotho assisted Hermes to create the alphabet, and forced the goddess Afrodite into making love with other gods, killed the Titan Typhon with poison fruits and persuaded Zeus to kill Asclepius with a bolt of lightning.

As you know Asclepius, the Roman Esculapius, is the god of Medicine and Healing. Asclepius has a daughter Epione ( Goddess of soothin of pain) and he his the father of : Hygieia (Hygiene), Panacea (Universal Remedy), Aceso (Goddess of Healing process), Leso or Laso (Goddess of recuperation from illness), Aglaea or Aglaia (Shining one, splendor, brillant, Healthy Glow) wife of Hephaesto and mother of Eucleia (good repute), Eupheme (Acclaim), Euthenia (Prosperity), Philophrosyne (Welcome).

The rod of Asclepius, a snake-entwined staff, remains a symbol of medicine today, also if sometimes a staff with two snakes (the caduceus) is mistakenly used instead.

He was one of the Apollo’s sons.

Asclepius

God of Medicine

Is KLOTHO, an anti-ageing hormone ?

The KLOTHO gene was identified serendipitously through a hypomorphic allele that results in severe early degenerative changes and short lifespan ( Nature 1997). The homozygous mutant animals develop normally until 3 weeks of age, then exhibit severe growth retardation, osteoporosis, ectopic calcification, aterosclerosis, emphysema and atrophy of the skin, thymus, testes and ovaries, and die at an average age of 61 days.

KLOTHO prolong lifespan at least in part by inhibiting insulin-IGF-1 signalling. KLOTHO may be secreted by KIDNEY cells blocking both IGF-1 and Insulin receptor action at adipocytes and target tissue levels.

Levels of calcium and phosphate are elevated in KLOTHO deficient mice, demonstrating that this protein has a role in calcium and phoshate homeostasis. The altered calcium and phoshate levels are due to elevated 1,25 OH Vitamin D3 levels, which result from increased expression of 1alfa hydroxylase gene activity at kidney level.

Normalization of 1,25 OH Vitamin D3 with a Vitamin D deficient diet partially rescued some of the KLOTHO deficient phenotypes, including slow growth, ectopic calcification and early death. This suggests that the putative pro-ageing effect of KLTHO deficiency is not associated with ageing itself, but rather that the pathology is related to altered Vitamin D metabolism.

FGF23 KO mice share many features with the KLOTHO deficient mouse, including hypercalcemia, hyperphosphatemia, ectopic calcification, hypoglycaemia, infertility, and very short lifespan.

FGF23 is a circulating factor that is produced in the bone and inhibits phosphate transport in renal proximal tubular cells. FGF23 deficiency results in phosphate retention and hyperphosphatemia, constitutively elevated expression of 1 alfa hydroxylase, elevated levels of 1,25 OH Vitamin D3 and hypercalcaemia.

Interestingly without KLOTHO the functions of FGF23 is literally abolished.

Many tissues express FGF Receptors subtypes that interact with the KLOTHO-FGF23 complex, therefore it is possible that KLOTHO exerts his anti-ageing action through the activation of his enzymatic activity i.e. beta glucoronidation.

We know that all steroids enzymes including all lipophilic vitamins such as Vitamin D, with a steroid like structure, can be glucuronized in order to achieve a better hydrophility and flow into the blood vessels. The pool of glucuronide-linked steroids hormones is an inactive quote of hormones, those destiny is in normal condition to be metabolized further into hepatic cells or recycled by endocrine organs.

KLOTHO can hydrolyse STEROID GLUCURONIDES, including estradiol, estrone, estriol and vitamin D so that some effects of KLOTHO can occur through processing of inactive streroid glucuronides to active steroids hormones. Steroid hormones could have a role in the regulation of ageing in mammals.

References

Arking DE, Krebsova A, Macek M Sr et al. Association of human aging with a functional variant of klotho. Proc Natl Acad Sci USA 2002;99:856-61.

Duce JA, Podvin S, Hollander W et al. Gene profiling analysis implicates klotho as an important contributor to aging changes in brain white matter of rhesus monkey. Glia 2008;56:106-117.

Arking DE, Becker DM, Yanek LR et al. KLOTHO allele status and the risk of early-onset occult coronary artery disease. Am J Hum Genet 2003;72:1154-61.

Mitani H, Ishizaka T, Aizawa T et al. In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage. Hypertension 2002;39:838-43.

Ogata N, Matsumura Y, Shiraki M et al. Association of klotho gene polymorphism with bone density and spondylosis of the lumbar spine in postmenopausal women. Bone 2002;31:37-42.

Yamada Y, Ando F, Niino N et al. Association of polymorphisms of the androgen receptor and klotho genes with bone mineral density in Japanese women. J Mol Med 2005;83:50-7.

Mullin BH, Wilson SG, Islam FM etr al. Klotho gene polymorphisms are associated with osteocalcin levels but not bone density of aged postmenopausal women. Calcif Tissue Int 2005;77:145-51.

Kawano K, Ogata N, Chiano M et al. Klotho gene polymorphisms associated with bone density of aged postmenopausal women. J Bone Miner Res 2002;17:1744-51.

Kuro-o M, Matsumura Y, Aizawa H et al. Mutation of the mouse klotho gene leads to a syndrome resembling ageing. Nature 1997;390:45-51.

Roth GS, Lane MA, Ingram DK et al. Biomarkers of caloric restriction may predict longevity in humans. Science 2002;297:811.

Kurosu H, Yamamoto M, Clark JD et al. Suppression of aging in mice by the hormone Klotho. Science 2005;309:1829-33.

Chang Q, Hoefs S, van der Kemp AW et al. The beta glucuronidase Klotho hydrolyzes and activates the TRPV5 channel. Science 2005;310:490-3.

Russell SJ, Kahn CR. Endocrine regulation of aging. Nat Rev Mol Cell Biol 2007;8:681-91.

Sohal RS, Weindruch R. Oxidative stress, caloric restriction, and aging. Science 1996;273:59-63.

Ingram DK, Cutler RG, Weindruch R et al. Dietary restriction and aging: the initiation of a primate study. J Gerontol 1990;45:B148-B163.

Sohal RS, Argarwal S, Candas M et al. Effect of age and caloric restriction on DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76:215-24.

Lee C-K, Klopp RG, Weindruch R et al. Gene expression profile of aging and its retardation by caloric restriction. Science 1999;285:1390-3.

Kujoth GC, Hiona A, Pugh TD et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 2003;309:481-4.

Weindruch R, Sohal RS. Caloric intake and aging. N Engl J Med 1997;337:986-94.

Priè D, Friedlander G. Genetic disorders of renal phosphate transport. N Engl J Med 2010;362:2399-2409.

Neuroblastoma : the gateway to cancer ?

2010-07-02T15:57:00.001+02:00

A very interesting paper has been published on the issue of The Journal of June 10 2010 by Maris John M. working at the Children’s Hospital of Philadelphia, Division of Oncology. Colket Translational Research Building, Rm. 3060, 3501 Civic Center Boulevard, Philadelphia, PA 19104 – 4318, USA, concerning Neuroblastoma news. Very interesting is the Supplemental Material downlodable on the web site of the Journal concerning the Historical steps on the discovery of Neuroblastoma pathogenesis and the following bibliographic references.

Most important I read the original papers writed bu Alfred J Knudson Jr. on ‘70 and ‘80 years based on “ second hits” pathogenetic hypothesis of cancer mutation and the following studies on sympathetic derived cancers in humans including Paragangliomas, Pheocromocytomas, Retinoblastoma, Medullary Cancer of The Thyroid, Neuroblastomas, Neurofibromas and related complex syndromic disease such as Von Hippel-Lindau disease, von Reklinghausen disease and endocrine tumors associations I first studied on years of my degree as Medical Doctor (1979-1985) as Amino Precursor Uptake and Decarboxylation Syndromic diseases ( ADUP diseases ). For many years these syndromic complexes have been largely studied in searching the demonstration of clonal or multiclonal origin of cancer, and of mutations intervening in these rare hereditary forms of cancers. Revisiting these studies after 25 years we can see how’s the profound influence of Genome Project studies on inherited diseases affecting in particular children and young patients. Now we can easly affirm that the “ two hits “ pathogenetic hypothesis of Alfred J Knudson Jr. is a wrong hypothesis of work, that has been hypothetized only from statistical data. Whereas genomics, proteomics, and all “omics” , new sciences derived from Human Genome Project Map allowed us to depict a very complex picture of gene alterations in the pathogenesis of cancer disease. Knudson has anyway the important role in discovering for the first time the carcer hereditary forms we have to study, with more adequate and sophisticate technologies such as Microarray techniques. It seems quite clear now that the picture of genetic alterations present in cancer pathogenesis is not so simple, and we can say with Mozart replying to Prussian Emperor about the complexitie of his music: “ We are sorry, but we have only musical notes we need ! ”.

References

Diskin SJ, Hou C, Glessner JT et al. Copy number variation at 1q21.1 associated with neuroblastoma. Nature 2009;459:987-91.

Brinster RL, Chen HY, Messing A et al. Transgenic mice harboring SV40 T-antigen genes develop characteristic brain tumors. Cell 1984;37:367-9.

Brodeur GM, Seeger RC, Schwab M et al. Amplification of N-myc in untreated human neuroblastomas correlates with advanced disease stage. Science 1985;224:1121-4.

Knudson AG. Hereditary cancer, oncogenes and antioncogenes. Cancer Res 1985;45:1437-43.

Knudson AG, Meadows AT, Nichols WW et al. Chromosomal deletion and retinoblastoma. N Engl J Med 1976;295:1120-3.

Vandepoele K, Van Roy N, Staes K et al. A novel gene family NBPF: intricate structure generated by gene duplication during primate evolution. Mol Biol Evol 2005;22:2265-74.

Mefford HC et al. Recurrent rearrangements of chromosome 1q21.1 and variable pediatric phenotypes. N Engl J Med 2008;359:1685-99.

Poliseno L, Salmena L, Zhang J et al. A coding-independent function of gene and pseudogene mRNA regulates tumor biology. Nature 2010;465:1033-8.

Maris JM. Recent advances in Neuroblastoma. N Engl J Med 2010;362:2202-10.

Weinberg RA. The Biology of cancer. Garland Science, Taylor & Francis Group, LLC Eds. 2007.