Wednesday, August 31, 2011


Promoter function of the angiogenic inducer Cyr61gene in transgenic mice: tissue specificity, inducibility during wound healing, and role of the serum response element.

"The cysteine-rich angiogenic protein 61 (Cyr61) is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, stimulates cell migration, and enhances growth factor-induced cell proliferation. Cyr61 promotes chondrogenic differentiation and induces neovascularization{LSJL upregulates Cyr61 by 2.504 fold}. A 2-kb fragment of the Cyr61 promoter, which confers growth factor-inducible expression in cultured fibroblasts, is able to drive accurate expression of the reporter gene lacZ in transgenic mice. Transgene expression was observed in the developing placenta and embryonic cardiovascular, skeletal, and central and peripheral nervous systems. The sites of transgene expression are consistent with those observed of the endogenous Cyr61 gene. The transgene expression in the cardiovascular system does not require the serum response element, a promoter sequence essential for transcriptional activation of Cyr61 by serum growth factors in cultured fibroblasts. Because the serum response element contains the CArG box, a sequence element implicated in cardiovascular-specific gene expression, the nonessential nature of this sequence for cardiovascular expression of Cyr61 is unexpected. Cyr61 promoter-driven lacZ expression is inducible in granulation tissue during wound healing, as is synthesis of the endogenous Cyr61 protein, suggesting a role for Cyr61 in wound healing. purified Cyr61 protein promotes the healing of a wounded fibroblast monolayer in culture.we mapped the mouse Cyr61 gene to the distal region of chromosome 3. Cyr61 [has a role] in wound healing through its demonstrated angiogenic activities upon endothelial cells and its chemotactic and growth promoting activities upon fibroblasts."

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation.

"Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed and the CYR61/CCN1 protein was analyzed. Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation{interesting that it would increase during LSJL}. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation.  CYR61/CCN1 [plays a role in the] maintenance of the stem cell phenotype."

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells.

"Cyr61 is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis. Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis."

Cyr61 can be activated by FGF, PDGF, and TGF-Beta.

Temporal expression of the chondrogenic and angiogenic growth factor CYR61 during fracture repair.

"We employed differential display and compared messenger RNA (mRNA) populations isolated from postfracture (PF) day 5 calluses to those of intact rat femurs. One such gene in which expression was up-regulated at PF day 5 is identified as CYR61, a member of the CCN family of secreted regulatory proteins. CYR61 is a growth factor that stimulates chondrogenesis and angiogenesis. We show that its mRNA expression during fracture repair is regulated temporally, with elevated levels seen as early as PF day 3 and day 5, rising dramatically at PF day 7 and day 10, and finally declining at PF day 14 and day 21. At the highest peak of expression (PF day 7 and day 10, which correlates with chondrogenesis), CYR61 mRNA levels are approximately 10-fold higher than those detected in intact femurs. Similarly, high protein levels are detected throughout the reparative phase of the callus, particularly in fibrous tissue and periosteum, and in proliferating chondrocytes, osteoblasts, and immature osteocytes. The secreted form of CYR61 also was detected within the newly made osteoid. No labeling was detected in hypertrophic chondrocytes or in mature cortical osteocytes."

"we directly compared the mRNA populations isolated from PF day 5 calluses to those of intact femurs (included bone marrow and articular and normal growth plate cartilage) using differential display. Presently, we have identified over a dozen differentially expressed genes, some known (osteopontin{upregulated by LSJL}, bone sialoprotein{up in LSJL}, integrin-α6, major histocompatibility complex (MHC) class II antigen, phosphoglucomutase, fibronectin, versican, vimentin, hypoxia-inducible factor 1α, sec63, SM-20, EET-1, and calpain), and some novel (FxC1{down in LSJL} and FxC2)."

"CYR61 expression is activated by basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and transforming growth factor β (TGF-β)."

NOV (CCN3) regulation in the growth plate and CCN family member expression in cartilage neoplasia.

"In embryonic murine growth plates, NOV{up in LSJL} was expressed in pre-hypertrophic and early hypertrophic chondrocytes. PTHrP treatment (which inhibits terminal differentiation) decreased NOV expression in murine femurs maintained in organ culture, and decreased the activity of a NOV reporter construct in vitro. Expression of the CCN family members NOV, CTGF, CYR61, and WISP-1 was examined in 15 chondrosarcomas of various grades and in three enchondromas. Expression of all of the family members was lower in the higher-grade tumours."

"The level of expression of NOV decreases in higher-grade, less-differentiated cartilage lesions."

Monday, August 29, 2011

Water is the Key to Growing Taller

Growing Taller involves bones growing longer.  Bone growth either involves direct bone deposition or chondrocyte growth.  Bone deposition usually only results in size increase when it occurs beneath the periosteum.  The flat bone of the skull is one of the few places in the body where such periosteal driven growth can increase height.  So that means that in terms of growing taller as an adult chondrocytes are the most promising candidate to increase height.  As there are numerous osteoblasts depositing bone within the body but the only dimension that increases in size are those dimensions beneath the periosteum.

Why can chondrocytes increase height whereas osteoblasts can't?

Both chondrocytes and osteoblasts can produce Extracellular Matrix.  They differ however in that chondrocytes ECM is much more hydrophillic than the ECM of bone.
Both chondrocytes and osteoblasts proliferate.
Osteoblasts are not traditionally thought of as undergoing hypertrophy and chondrocytes however are quite famous for it.  But, most cells respond to changes in water concentration by changing size.  Osteoblasts do hypertrophy.
Both Osteoblasts and Chondrocytes can undergo apoptosis.

So the key difference between osteoblasts and chondrocytes is the hydrophillic nature of the extracellular matrix.  The mechanism of apoptosis may differ between osteoblasts and chondrocytes as well.

A study called "The Osmotic sensitivity of rats..." found that the increase in cell size in chondrocytes was likely not due to swelling(caused by water) but rather due to hypertrophy. Higher concentrations of water still increased hypertrophy.  The study did find that the extracellular matrix was influenced by water and did show evidence of responding to changes in osmolarity.

LSJL increases the hydrostatic pressure which encourages chondrocyte proliferation rather than osteoblast proliferation.  Chondrocytes of course making you taller.  Osteoblasts respond to increases in hydrostatic pressure by producing TGF-Beta which encourages chondrogenic differentiation.  Cartilage is a lot more resistant to hydrostatic pressure than bone is.  So increasing hydrostatic pressure with LSJL induces a functional adaptation for cartilage growth within the bone that happens to make you taller.

Physically injecting water into the bone wouldn't work as that would create a hole for water to leak out of reducing hydrostatic pressure.  A study done by the LSJL scientists found that drilling a hole into bone eliminated the effectiveness of their joint loading modality.  Thus, we are left with mechanical means to induce higher levels of hydrostatic pressure unless someone can come up with a way to increase water concentration in the bone while still plugging the hole left by injection.

Chondrocytes can increase height without being next to periosteum, Osteoblasts typically only increase bone dimensions when new bone is deposited beneath the periosteum.

The main differences between chondrocytes and osteoblasts is the relationship of their extracellular matrix to water and the ability for chondrocytes to release this water stored within the ECM generating force when they undergo apoptosis.  This force may be what drives bone deformation and makes your bones your longer.

The study entitled "Mechanisms of cartilage replacement in bone..." explains in more detail how apoptic chondrocytes may generate energy that results in an increase in bone size.  The opening of the chondrocytes releases water.  This release generates energy that can push apart bone.  Osteoblasts do not have an ECM that absorbs water so their apoptosis is less dramatic.

A way to test this would be to implant something like a growth plate with cells that can absorb and secrete water without creating a gap in the bone.  You'd have to just implant the device in the marrow.

Why water is key to growing taller:

High Hydrostatic Pressure(high water levels) induces chondrocyte differentiation forming growth plates
The growth plates use water to hypertrophy and then release this water pushing the bone apart making you taller

A bunch of other chemicals and genes matter but it seems that water is the primary driving force behind height increase.  Thus, it would be worth exploring ways to manipulate water like temperature(LIPUS for one thing increases temperature in the applied areas), water retention(has to be within the bone), etc.

Here's a study about chondrocyte hypertrophy and the exact mechanisms in which it occurs:

Mechanics of chondrocyte hypertrophy.

"Chondrocyte hypertrophy dominates bone growth. Intra- and extracellular changes that are known to be induced by metabolically active hypertrophic chondrocytes are known to contribute to hypertrophy[these changes could produce a first that causes bone deformation]. However, it is unknown to which extent these mechanical conditions together can be held responsible for the total magnitude of hypertrophy. To address this aim requires a quantitative tool that captures the mechanical effects of collagen and proteoglycans, allows temporal changes in tissue composition, and can compute cell and tissue deformations[chondrocyte hypertrophy could induce deformation of the surrounding tissue which is bone]. After validating the numerical approach for studying hypertrophy, the model is applied to evaluate the direct mechanical effects of axial tension and compression on hypertrophy (Hueter-Volkmann principle) and to explore why hypertrophy is reduced in case of partially or fully compromised proteoglycan expression. Finally, a mechanical explanation is provided for the observation that chondrocytes do not hypertrophy when enzymatical collagen degradation is prohibited[The degradation of collagen changes the osmotic gradient] (S1Pcko knock-out mouse model). This paper shows that matrix turnover by metabolically active chondrocytes, together with externally applied mechanical conditions, can explain quantitatively the volumetric change of chondrocytes during hypertrophy. It provides a mechanistic explanation for the observation that collagen degradation results in chondrocyte hypertrophy[collagen degradation changes the osmotic balance and results in cell swelling thus possibly being the mean cause of hypertrophy], both under physiological and pathological conditions."

"During hypertrophy, collagenase activity and cellular and matrix changes are known to occur concurrently. The intracellular volumes occupied by organelles and cytoplasmic ground substance gradually increase,
together with the osmotic pressure generated by intracellular accumulation of organic osmolytes. Although quantification of the effects of cellular electro-chemo-mechanical properties has proven difficult, it is apparent that this can only be partially responsible for the ten fold volume change and four to five times increased cell height during hypertrophy"<-So the number of organic osmolytes[any compound that alters osmotic pressure] can be manipulated.  LSJL drives compounds via fluid flow so this could be part of the benefits of LSJL observed in rats.

"to keep up with such increase in cell height, the aligning extracellular matrix (ECM) needs to also stretch to 400–500% its original length."<-So chondrocyte hypertrophy stretches the ECM and possibly Type I collagen(bone as well) explaining height growth.

"The combined effect of changes in cell volume and matrix constitution is that the volume occupied by cells over the volume of ECM decreases from the early proliferative to the late hypertrophic zone. Yet, the total amount of matrix associated with one hypertrophic cell increases, with values ranging from 50 to 1000% depending on species, age and growth plate"

"The fraction of large proteoglycans per volume of matrix is largest around hypertrophic cells, which is especially interesting given that hyaluronan-mediated hydrostatic pressure causes lacuna expansion"<-LSJL increases hydrostatic pressure thus LSJL increases lacunae expansion

"Hypertrophic cells show active collagen metabolism. MMP’s are abundant in quantity and diversity around hypertrophic cells,while these cells continue synthesizing collagen"<-Why would hypertrophic cells continue to synthesize collagen if there are MMP's degrading it.  To change the osmotic pressure which results in force that exerts a stretch on the surrounding matrix(including type I collagen i.e. bone).

"We assume that the osmotic swelling pressure depends on the number of fixed charges in the proteoglycans per amount of extrafibrillar water"<-LSJL increases the fluid flow thus increasing the number of fixed charges in the proteoglycans.

"as a consequence of mechanical loading or tissue expansion, the amount of fixed charges per water volume may change."<-LSJL involves mechanical loading thus LSJL may change the number of fixed charges per water volume.

"By adding proteoglycans and/or collagen in this way, the newly derived initial solid fraction has become higher on the expense of the fluid fraction, while tissue volume does not change. During subsequent equilibration, total tissue volume increases as the fluid fraction is being restored."<-The solid part of the matrix includes type II collagen and proteoglycans.  ECM increases to restore osmotic pressure then when ECM is degraded the fluid in the matrix increases to restore osmotic pressure(thus release of fluid including water should generate some kind of force).  Then proteoglycans and type II collagen are restored once more to restore osmotic pressure.  This gradually leads to an increase in tissue volume.

"Catalytic enzymes are assumed to cleave a particular fraction of the collagen during a given time period. Meanwhile, collagen synthesis is ongoing. It is then assumed that newly formed collagen either self-assembles in an unstrained state, or otherwise induces changes in collagen fibrils, such that their strain is ameliorated."<-tissue formation occurs in such a way as to alleviate strain from newly-formed collagen this could be why bone becomes shaped in a certain way and thus applying strain to certain parts of the bone can manipulate bone growth.

"Cell hypertrophy is simulated by continuously running the model, by repeatedly adding proteoglycans
and collagen, followed by equilibration"<-so maybe you can keep the growth plates active by continuing to add proteoglycans and collagen.

This study is most promising to those with active growth plates but there are ways to increase serum proteoglycan number(by hyaluronic acid supplementation by example) and this can stimulate the cells in such a way as to be more likely to be chondrocytes.  Height growth is ultimately driven by changes in osmotic(water) pressure.

LSJL attempts to change osmotic intramedullary pressure in adult bone.

Here's a study related to osmotic pressure in articular cartilage.  We can apply some of the findings to growth plate cartilage.

Contribution of proteoglycan osmotic swelling pressure to the compressive properties of articular cartilage.

"The negatively charged proteoglycans (PG) provide compressive resistance to articular cartilage by means of their fixed charge density (FCD) and high osmotic pressure (π(PG)), and the collagen network (CN) provides the restraining forces to counterbalance π(PG)[we know that growth plate chondrocytes apply compressive resistance because the growth plate doesn't collapse to the weight of the body.  Height growth must involve the manipulation of Proteoglycans, Fixed Charged Density, Osmotic Pressure, and the Collagen network]. Our objectives in this work were to: 1), account for collagen intrafibrillar water when transforming biochemical measurements into a FCD-π(PG) relationship; 2), compute π(PG) and CN contributions to the compressive behavior of full-thickness cartilage during bovine growth (fetal, calf, and adult) and human adult aging (young and old); and 3), predict the effect of depth from the articular surface on π(PG) in human aging. Extrafibrillar FCD (FCD(EF)) and π(PG) increased with bovine growth due to an increase in CN concentration[as collagen content increased so did Fixed Charge Density and osmotic pressure], whereas PG concentration was steady. This maturation-related increase was amplified by compression. With normal human aging, FCD(EF) and π(PG) decreased[maybe this can be responsible for epiphyseal fusion, the Fixed Charged Density and Osmotic Pressure are no longer high enough to sustain the growth plate]. The π(PG)-values were close to equilibrium stress (σ(EQ)) in all bovine and young human cartilage, but were only approximately half of σ(EQ) in old human cartilage. Depth-related variations in the strain, FCD(EF), π(PG), and CN stress profiles in human cartilage suggested a functional deterioration of the superficial layer with aging. These results suggest the utility of the FCD-π(PG) relationship for elucidating the contribution of matrix macromolecules to the biomechanical properties of cartilage."

"Nearly 90% of PG is aggrecan, which complexes with hyaluronan (HA) and link protein to form large PG aggregates entrapped within the CN"<-Since so much of the proteoglycans are aggrecan that means that hyaluronic acid supplementation is event more beneficial as there's plenty of aggrecan to bind with.

"water is distributed between COL fibrils (intrafibrillar (IF)) and PG (extrafibrillar (EF)), and this water distribution varies with external stress applied to the tissue"<-LSJL provides external stress and modifies water distribution.

"Approximately twice the mass of KS[Keratan Sulfate] relative to CS[Chondroitin Sulfate] was equivalent to the same FCD. Also, πPG[osmotic pressure] increased with an increasing CS/KS ratio, reflecting the charge difference between KS and CS"<-So the more Chondroitin Sulfate you have relative to Keratan Sulfate the more osmotic pressure you generate.  Thus providing more evidence that Chondroitin Sulfate supplementation may help you grow taller.

The reason that articular cartilage does not behave like a growth plate is that chondrocyte hypertrophy is prohibited when collagen degradation is inhibited.  To grow taller by endochondral ossification you need chondrocytes[hopefully induced by LSJL stimulation of chondrogenesis in the stem cells of the epiphyseal bone marrow], ECM[type II collagen], and collagen degradation[MMPs].

Induction of re-differentiation of passaged rat chondrocytes using a naturally obtained extracellular matrix microenvironment.

"Cell-derived matrix (CDM) [are] a physical microenvironment for chondrocyte re-differentiation. Once different cell types were cultured for 6 days and decellularized using detergents and enzymes, fibroblast-derived matrix (FDM), preosteoblast-derived matrix (PDM), and chondrocyte-derived matrix (CHDM) were obtained. From SEM observation, each CDM was found to resemble a fibrous mesh with self-assembled fibrils. Both FDM and PDM showed a more compact matrix structure compared to CHDM. numerous matrix proteins were quite different from each CDM in quantity and type. Specific matrix components, such as fibronectin, type I collagen, and laminin were detected. the water contact angle suggests that FDM is more hydrophilic than PDM or CHDM{Gelatin is also hydrophilic}. The proliferation of rat primary chondrocytes growing on CDMs was better than those growing on a plastic coverslip (control) or gelatin. Meanwhile synthesis of glycosaminoglycan (GAG) was more effective for passaged chondrocytes (P4) cultivated on CDMs, and the difference was significant compared to cells grown on the control or on gelatin. As for the gene expression of cartilage-specific markers, CDMs exhibited good chondrocyte re-differentiation with time: the dedifferentiating marker, type I collagen (Col I) was restrained, whereas the ratio between Col II and Col I, and between aggrecan and Col I, as an indicator of re-differentiation, was greatly improved. Col II showed a very positive signal in chondrocytes cultivated for 2 weeks on the CDMs. when 3D cell pellets made from either plate-grown or matrix-grown dedifferentiated chondrocytes (P5) were cultured for 4 weeks, matrix-grown cells were significantly better in the induction of chondrocyte re-differentiation, than those grown on the plate."

Type I collagen and Laminin was more abundant in FDM.

Water channel activity of plasma membrane affects chondrocyte migration and adhesion.
"Aquaporin-1 (AQP1){AQP5 is upregulated 3 fold in LSJL} water channel is expressed in human and equine articular chondrocytes. We investigated the expression of the AQP1 water channel in cultured articular chondrocytes from wild-type (AQP1(+/+)) and AQP1-knockout (AQP1(-/-)) mice and characterized its function in chondrocyte proliferation, migration and adhesion. Expression of AQP1 mRNA and protein was identified in freshly isolated neonatal AQP(+/+) chondrocytes. AQP1 protein [is localized] to the plasma membrane of AQP(+/+) chondrocytes in primary cultures. Relative plasma membrane water permeability of AQP1(+/+) chondrocytes was approximately 1.6-fold higher than that of AQP1(-/-) chondrocytes. The chondrocyte proliferation rate was not affected by AQP1 deletion. However, the serum-induced transwell migration rate of AQP1(-/-) chondrocytes was markedly reduced compared with AQP1(+/+) chondrocytes (16.2 +/- 0.2 vs 27.1 +/- 0.3%, respectively). Cell adhesion to type II collagen-coated plates was also significantly reduced in AQP1(-/-) chondrocytes compared with AQP1(+/+) chondrocytes (38.1 +/- 0.3 vs 51 +/- 1%, respectively). AQP1-mediated plasma membrane water permeability plays an important role in chondrocyte migration and adhesion."

"The aquaporins (AQPs) are a family of water-transporting proteins with 13 homologous members"

"AQP-mediated high plasma membrane water permeability enhances cell migration"

Increased differentiation capacity of bone marrow-derived mesenchymal stem cells in aquaporin-5 deficiency., states that chondrogenic differentiation increases upon AQP5 deficiency which is consistent if AQP5 enhances migration and proliferation both of which have inverse relationships to differentiation.

Thursday, August 25, 2011

Grow taller by inhibiting PTEN?

LSJL has no effect on PTEN(at least over or under threshold) and PTEN is a PI3K pathway inhibitor.  LSJL stimulates the PI3K pathway so  if anything that inhibits PTEN increases height then that could mean that stimulating the PI3K pathway increases height which would support height increasing applications of LSJL.

Characterisation of the PTEN inhibitor VO-OHpic.

"PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a phosphatidylinositol triphosphate 3-phosphatase that counteracts phosphoinositide 3-kinases. VO-OHpic is an extremely potent inhibitor of PTEN with nanomolar affinity in vitro and in vivo.  Inhibition of recombinant PTEN by VO-OHpic is fully reversible. Both K(m) and V(max) are affected by VO-OHpic, demonstrating a noncompetitive inhibition of PTEN. The inhibition constants K(ic) and K(iu) were determined to be 27 ± 6 and 45 ± 11 nM, respectively."

"PTEN hydrolyses the phosphate group in the 3′ position from phosphatidylinositol 3,4,5-triphosphate (PIP3) to form phosphatidylinositol 4,5-biphosphate and, in doing so, is counteracting phosphoinositide 3-kinases (PI3K). The balance of PTEN and PI3K activities determines the cellular PIP3 levels, which in turn is recognised by other signalling components such as Akt"

"we generated mice lacking this key phosphatidylinositol 3'-kinase pathway regulator in their osteo-chondroprogenitors. A phenotype of growth plate dysfunction and skeletal overgrowth was observed.
We used mice deficient in a negative regulator of PI3K signaling, the tumor suppressor, Pten.
Pten gene deletion in osteo-chondrodroprogenitors was obtained by interbreeding mice with loxP-flanked Pten exons with mice expressing the Cre recombinase under the control of the type II collagen gene promoter (Pten(flox/flox):Col2a1Cre mice).
Pten(flox/flox):Col2a1Cre mice exhibited both increased skeletal size, particularly of vertebrae, and massive trabeculation accompanied by increased cortical thickness. Primary spongiosa development and perichondrial bone collar formation were prominent in Pten(flox/flox):Col2a1Cre mice, and long bone growth plates were disorganized and showed both matrix overproduction and evidence of accelerated hypertrophic differentiation (indicated by an altered pattern of type X collagen and alkaline phosphatase expression). Consistent with increased PI3K signaling, Pten-deficient chondrocytes showed increased phospho-PKB/Akt and phospho-S6 immunostaining, reflective of increased mTOR and PDK1 activity. No significant change in growth plate proliferation was seen in Pten-deficient mice, and growth plate fusion was found at 6 months."

"Specific ligand—receptor interactions recruit PI3K into proximity with its substrate, phosphatidylinositol(4,5)-bisphosphate, PI(4,5)P2, to generate the “second messenger,” PI(3,4,5)P3. The phosphatidylinositol 3′-phosphatase, Pten (phosphatase and tensin homolog deleted on chromosome 10), a well-known tumor suppressor, reverses the effects of PI3K by dephosphorylating PI(3,4,5)P3 at the D-3 position, to yield PI(4,5)P2"

"in cells lacking Pten, PI(3,4,5)P3 levels are higher and/or sustained, leading to overactivation of the PI3K pathway"

"By 15 wk of age, Ptenflox/flox:Col2a1Cre mice were an average of 1.15 times longer than control mice"

"Femoral lengths [from] Ptenflox/flox:Col2a1Cre femurs were 1.07 times longer than those of control mice"

"in contrast to wildtype mice whose epiphyseal growth plates do not fuse with age, the tibial growth plates of Ptenflox/flox:Col2a1Cre mice were fused by 6 mo of age"

"Compared with controls, expression of type X collagen protein in 1-wk-old Ptenflox/flox:Col2a1Cre mouse growth plates was not only increased but was also more widespread, with expression of this protein encroaching well into the proliferative zone"

"HIF-1α protein stability and transcriptional activity are both stimulated by PI3K → Akt pathway activation"

"Ptenflox/flox:Col2a1Cre mice [had] 13% increase in body length caused by the enlargement of vertebrae and intervertebral disk spaces[so this can increase spinal height in adults without inducing new growth plates]. Interestingly, long bones only exhibited a 4.6% increase in the Ptenflox/flox:Col2a1Cre mice"

PTEN deficiency causes dyschondroplasia in mice by enhanced hypoxia-inducible factor 1alpha signaling and endoplasmic reticulum stress.

"Chondrocytes within the growth plates acclimatize themselves to a variety of stresses that might otherwise disturb cell fate. The tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) has been implicated in the maintenance of cell homeostasis. We have created chondrocyte-specific Pten knockout mice (Pten(co/co);Col2a1-Cre) using the Cre-loxP system. Following AKT activation, Pten mutant mice exhibited dyschondroplasia[a mass of hypertrophic cartilage] resembling human enchondroma[a cartilage bone marrow cyst]. Cartilaginous nodules originated from Pten mutant resting chondrocytes that suffered from impaired proliferation and differentiation, and this was coupled with enhanced endoplasmic reticulum (ER) stress. ER stress in Pten mutant chondrocytes only occurred under hypoxic stress, characterized by an upregulation of unfolded protein response-related genes as well as an engorged and fragmented ER in which collagens were trapped. An upregulation of hypoxia-inducible factor 1alpha (HIF1alpha) and downstream targets followed by ER stress induction was also observed in Pten mutant growth plates and in cultured chondrocytes, suggesting that PI3K/AKT signaling modulates chondrocytic adaptation to hypoxic stress via regulation of the HIF1alpha pathway."

"The HIF1α/VEGF axis supports chondrocyte survival in the interior growth plate, where oxygen tension is much lower than in the exterior region. In addition, this axis may also modulate chondrocytic size and proliferation, cartilaginous matrix accumulation and blood vessel invasion during endochondral bone formation"

"AKT phosphorylates key intermediate signaling molecules, including glycogen synthase kinase 3β (GSK3β), murine double minute 2 (MDM2) and mammalian target of rapamycin (mTOR; FRAP1)"

You can see possible new growth plate formation in this image:

"At postnatal day (P) 40, the tails of mutant mice were 11-14% longer than those of PtenCo/+;Col2a1-Cre and Pten+/+;Col2a1-Cre controls".  However the length of the bones of the temur and tibia were the same.

"The non-hypertrophic chondrocytes that extended into the bone marrow cavity began to proliferate ectopically and to exhibit hypertrophy, gradually forming a proliferating pseudo-growth plate within the bone marrow cavity"

"The proliferation of Pten mutant chondrocytes was in fact decreased in the neoplastic core as compared with controls"

"Chondrocyte-specific S1P (Mbtps1) knockout mice exhibit chondrodysplasia and a complete lack of endochondral ossification caused by defects in Col II secretion and increased apoptosis"

"The upregulation of BiP, together with the downregulation of Ppr Ihh and Col10a1, indicated that ER stress was triggered upon Pten deletion in chondrocytes and resulted in decreased differentiation."

"ER stress was significantly increased in Pten mutant chondrocytes under conditions of hypoxia, suggesting that a PTEN deficiency together with hypoxia synergistically triggers ER stress and subsequent de-differentiation of Pten mutant chondrocytes."

"the activation of the PI3K/AKT pathway in chondrocytes might serve multiple functions during the progression of hypoxia-induced ER stress; it perturbs the ER but may save the cell. As a consequence, dysplastic chondrocytes develop."<-So PTEN may cull the herd of weak chondrocytes.

"Inactivation of HIF1α in chondrocytes leads to increased cell death, accelerated proliferation and reduced yield of Vegf, Pgk and p57Kip2"

"inactivation of murine von Hippel-Lindau tumor suppressor protein (VHL) in chondrocytes causes enlarged cell size, accumulation of ECM, decreased proliferation and increased expression of Vegf, Pgk and p57Kip2 through a mechanism that facilitates the stabilization and accumulation of HIF1α protein"

Conditional loss of PTEN leads to skeletal abnormalities and lipoma formation.

"We have generated chondrocyte specific PTEN deletion mice using Col2a1Cre and PTEN(loxp/loxp) mice. PTEN mutant mice are viable and fertile, nonetheless, develop kyphosis over time. Histological analyses show mutant vertebrae and intervertebral discs are larger and therefore the spines are longer than in control mice. In addition, the growth plates are thicker, invading trabecular bone areas are deeper, and marrow adipocyte populations are higher in PTEN mutant mice. Furthermore, the growth plates, not normally fused in mouse long bones, are fused in PTEN mutants."

"PTEN is required for maintaining normal growth plate structures in older mice."<-Mice with loss of PTEN had growth plate fusion at 5-8 months.

Wednesday, August 24, 2011

Frat and LSJL

LSJL upregulates FRAT.  It was called GBP on the pathway.

Re-evaluating the role of Frat in Wnt-signal transduction.

"Frat proteins are potent activators of canonical Wnt-signal transduction. By binding to GSK3, Frat prevents the phosphorylation and concomitant degradation of beta-catenin and allows the activation of downstream target genes by beta-catenin/TCF complexes[this is consistent to what happens in LSJL]. The identification of the Xenopus Frat homologue GBP as an essential component of the maternal Wnt-pathway during embryonic axis formation suggested that Frat might fulfill a similar role in higher vertebrates. As a result most, if not all, studies addressing Frat function have focused on its ability to bind GSK3 and induce signaling through beta-catenin/TCF. Consequently, Frat has been advocated as the "missing link" that bridged signaling from Dishevelled to GSK3 in the canonical Wnt-pathway. Recent mouse-knockout studies however, call for a reevaluation of the physiological role of Frat. Mice that lack all Frat-family members appear to be normal and display no obvious defects in beta-catenin/TCF signaling."

Mice have Frat3 which is not present in humans.  FRAT1 binds to LRP5.

LSJL and integrin alpha 11

LSJL induces integrin alpha 11 expression.

BMP-2 and TGF-beta1 differentially control expression of type II procollagen and alpha 10 and alpha 11 integrins in mouse chondrocytes.

"The expression of alpha 10 and alpha 11 integrin subunits and the IIA/IIB spliced forms of type II procollagen have been monitored for the first time in parallel in the same in vitro model of mouse chondrocyte dedifferentiation/redifferentiation. We demonstrated that TGF-beta1 stimulates the expression of the non-chondrogenic form of type II procollagen, IIA isoform, and of a marker of mesenchymal tissues, i.e. the alpha 11 integrin subunit. On the contrary, BMP-2 stimulates the cartilage-specific form of type II procollagen, IIB isoform, and a specific marker of chondrocytes, i.e. the alpha 10 integrin subunit. Collectively, our results demonstrate that BMP-2 has a better capability than TGF-beta1 to stimulate chondrocyte redifferentiation and reveal that the relative expressions of type IIB to type IIA procollagens and alpha 10 to alpha 11 integrin subunits are good markers to define the differentiation state of chondrocytes. In addition, adenoviral expression of Smad6, an inhibitor of BMP canonical Smad signaling, did not affect expression of total type II procollagen or the ratio of type IIA and type IIB isoforms in mouse chondrocytes exposed to BMP-2. This result strongly suggests that signaling pathways other than Smad proteins are involved in the effect of BMP-2 on type II procollagen expression."

"the shift from type IIA [Collagen] to type IIB is a sign of chondrocyte differentiation."

"α11 integrin is more characteristic of mesenchymal tissues"

"chondrocytic differentiation of mesenchymal stem cells is associated with an increase in α10 expression and a decrease in α11 expression "<-unfortunately this wasn't observed in LSJL.  Although this could be indicative of pre-chondrogenesis induced by LSJL.  Expression of alpha10 was normal and alpha11 increased.

"BMP-2 primarily stimulates expression of a well-differentiated chondrocytic phenotype whereas TGF-β1 stimulates a pre-chondrocytic phenotype"

Monday, August 22, 2011

What can growth plate models teach us about height growth?

Understanding growth plates and growth plate mechanics by helping us try to form new ones.  LSJL attempts to form new growth plates by increasing the hydrostatic pressure in the bone marrow to induce chondrogenic differentiation.  Is this enough to form new growth plates?  What can a growth plate model teach us about what's needed for growth plate formation?

A mathematical model of epiphyseal development: hypothesis of growth pattern of the secondary ossification centre.

"This paper introduces a 'hypothesis about the growth pattern of the secondary ossification centre (SOC)', whereby two phases are assumed. First, the formation of cartilage canals as an event essential for the development of the SOC[Cartilage canals are formed by MMPs and VEGF; LSJL does upregulate MMP expression(specifically MMP-3), VEGF is regulated by Estrogen which is why you need an equilibrium of Estrogen or to inhibit the bad effects of Estrogen like ER-alpha]. Second, once the canals are merged in the central zone of the epiphysis, molecular factors are released (primarily Runx2 and MMP9) spreading and causing hypertrophy of adjacent cells[Low concentrations of diosigen increase Runx2 and BMP-2 and ultrasound increases Runx2 expression as well; MMP-9 can be increased by tensile strain(stretching)]. In addition, there are two important molecular factors in the epiphysis: PTHrP and Ihh. The first one inhibits chondrocyte hypertrophy and the second helps the cell proliferation. Between these factors, there is negative feedback, which generates a highly localised and stable pattern over time. From a mathematical point of view, this pattern is similar to the patterns of Turing. The spread of Runx2 hypertrophies the cells from the centre to the periphery of the epiphysis until found with high levels of PTHrP to inhibit hypertrophy. This mechanism produces the epiphyseal bone-plate. Moreover, the hypertrophy is inhibited when the cells sense low shear stress and high pressure levels that maintain the articular cartilage structure[LSJL induces high pressure in the epiphyseal bone marrow thus inhibiting hypertrophy]."

Note that the secondary ossification center is actually within the epiphysis rather than in the metaphyseal growth plate.  Thus, LSJL may form growth plates within the epiphysis itself thus increasing the size of the epiphysis.  Tweaking may be needed to get LSJL effective for the metaphyseal area.

"The mechanical loads induce deformation of the cartilage matrix, causing alterations in interstitial fluid hydrostatic pressure and changes in ionic and osmotic composition of both the tissue and interstitial fluid. The chondrocytes perceive these variations and generate specific responses to these stimuli. Besides, they can coordinate changes in expression and synthesis of matrix proteins and finally, in the biomechanical performance of the tissue"<-Now in adults there is no cartilage matrix, however the bone marrow is still deformed and there is still a change in hydrostatic pressure that can induce chondrogenic differentiation.

"The high octahedral stresses (or shear) accelerate the hypertrophy and high pressure levels maintain the cartilaginous structure, that is, the mechanical environment is of vital importance in epiphyseal growth. Mechanical loads play a relevant role in endochondral development. During bone growth, cartilage is particularly sensitive to mechanical factors for its deformation facility to sustain loads over time. The octahedral stresses play an important role in the growth of cartilage canals towards the centre of the epiphysis, where they release, especially Runx2 and MMP9s."<-shear stress helps form cartilage canals whereas hydrostatic pressure helps maintain the cartilage structure.  Both shear stress and hydrostatic pressure are likely needed in sufficient quantities to form new growth plates.

"This stress is the mechanical factor influencing the growth of cartilage canals towards the central area. The results of the formation and growth of the canals in the random sites of perichondrium are shown"<-shear stress did result in the formation of new cartilage canals from the perichondrium(immature periosteum).  Can shear stress be used to get cartilage canals to form from the periosteum into the metaphyseal area?

An equilibrium quantity of shear stress versus hydrostatic pressure may be needed for optimal growth plate formation?

Developmental regulation of the growth plate

"Bone formation begins when mesenchymal cells form condensations — clusters of cells that adhere through the expression of adhesion molecules. In a few areas, most notably the flat bones of the skull, the cells of these condensations differentiate directly into bone-forming osteoblasts[thus osteoblasts can increase height directly in the flat bone of the skull]. These cells lay down a matrix particularly rich in type I collagen in a process named intramembranous bone formation. But this straightforward process is the exception.  In most condensations, the cells become chondrocytes, the primary cell type of cartilage; cells at the border of condensations form a perichondrium. Chondrocytes have a characteristic shape, secrete a matrix rich in type II collagen and the proteoglycan aggrecan, and, more generally, express a characteristic genetic program driven by SOX9"<-the goal of LSJL driven hydrostatic pressure is to get stem cells to adopt that chondrogenic cell shape.

"The hypertrophic chondrocyte, simply through its size, is the principal engine of bone growth"<-so most of the increase in size in bones is due to hypertrophic chondrocytes.  However, osteoblasts can hypertrophy but do not result in taller bones(except for intramembranous ossification).

"While hypertrophic chondrocytes perform multiple tasks in the centre of the cartilage mould, the mould enlarges further through continued proliferation of chondrocyte"<-hypertrophic chondrocytes are the regulators while proliferating chondrocytes provide the growth.  Osteoblasts do not have coupled hypertrophy and proliferating cells which is likely why osteoblasts do not make you taller.

""As bones enlarge further, so-called secondary ossification centres are established when chondrocytes in
characteristic, new locations stop proliferating, hypertrophy, and attract vascular invasion along with osteoblasts. In the long bones of the limb, growth chondrocytes continue to proliferate between regions of bone of the primary and secondary ossification centre"<-So if you could keep those growth plate chondrocytes proliferating in the primary ossification centre you would grow in baby-like proportions.

"The proliferative effect of Ihh is likely to be a direct action on chondrocytes, because the expression of Ptc-1 and of other general targets of hedgehog signalling is stimulated by Ihh in chondrocytes"<-so increase serum levels of Ihh increase chondrocyte proliferation and grow taller.

"PTHrP acts primarily to keep proliferating chondrocytes in the proliferative pool"<-so you can also possibly grow taller and keep growth plates open by increasing levels of PTHrP.

"When chondrocytes are no longer sufficiently stimulated by PTHrP, they stop proliferating and synthesize Ihh. Ihh can then, by mechanisms that are still unknown, stimulate the production of PTHrP at the ends of bone"<-so you need specific ratios of PTHrP and Ihh to optimize height growth.

"Knockout of Fgfr3 in mice leads to an increased rate of proliferation of chondrocytes and an expansion of
the length of chondrocyte columns"<-Inhibitors of FGFR3 would be a great way to grow taller.  Transgenic expression of FGFR3 causes dwarfism.

"FGF signalling through FGFR3 inhibits proliferation; this inhibition is at least partly through activation of the Janus kinase–signal transducer and activator of transcription-1 (JAK–STAT1) pathway"<-so inhibiting parts of the JAK-STAT1 pathway could serve to increase height as well.

"knockout of Fgf18 leads to an increase in chondrocyte proliferation that closely resembles the effect of  Fgfr3"<-inhibiting FGF18 is another potential height increase therapeutic option.

"knockout of the Fgfr3 gene increases Ihh expression"<-thus increasing Ihh expression may be one of the ways that knocking out FGFR3 therefore increasing Ihh expression like knocking out FGFR3 may be a way to grow taller.

"Addition of BMPs to bone explants increases proliferation of chondrocyte"<-thus BMPs are likely a very effective way to grow taller especially BMPs 2,4, and 7.

"BMP signalling increases the expression of Ihh by prehypertrophic chondrocyte"<-so BMP may too increase height by an Ihh mechanism so it may be best to directly increase Ihh levels.

"SOX9 is expressed in cells of mesenchymal condensations and in proliferating chondrocytes, but not in hypertrophic chondrocytes."<-if we can stimulate SOX9 expression in mesenchymal condensations(the bone marrow) then we can stimulate new growth plate information and height growth.

According to the study "TNF-alpha upregulates expression of BMP-2 and BMP-3 genes in the rat dental follicle--implications for tooth eruption", TNF-alpha can upregulate BMP-2 and BMP-3.  TNF-alpha is an inflammatory cytokine so it has negative effects as well but it's possible it might help you grow taller if it can upregulate those genes.  TNF-alpha is upregulated by exercise.

Follistatin is a BMP agonist but Follistatin may have beneficial effects.  Too much BMP-3 can cause weaker bones for example.

A mechanobiological model of epiphysis structures formation

"Developing bone consists of epiphysis, metaphysis and diaphysis. The secondary ossification centre (SOC) appears and grows within the epiphysis, involving two histological stages. Firstly, cartilage canals appear; they carry hypertrophy factors towards the central area of the epiphysis. Canal growth and expansion is modulated by stress on the epiphysis. Secondly, the diffusion of hypertrophy factors causes SOC growth. Hypertrophy is regulated by biological and mechanical factors present within the epiphysis. Cartilage canal spatial-temporal growth patterns were obtained as well as the SOC formation pattern."

"Cartilage canal formation is due to invagination developing at specific sites on the perichondrium. The canals carry nutrients, blood vessels, growth factors and hormones to the central area of the epiphysis"<-If we can form new cartilage canals we can get new height growth.

"These canals expand in response to the mechanical stress supported by bone due to a load being exerted by the surrounding muscles in motion"<-but there must be a limit or perhaps cartilage canal expansion does not affect adult height?

"The canals release molecular factors in the inner area of the epiphysis triggering the chondrocyte hypertrophy phase. These factors are mainly matrix metalloproteinase-9 (MMP9), also known as gelatinase-B, and the RUNX2 transcription factor. RUNX2 is a positive modulator of hypertrophy and is released by hypertrophic chondrocytes "

"Parathyroid hormone-related protein (PTHrP) and Indian hedgehog (Ihh) protein are found in the epiphysis and are critical for future bone development. PTHrP inhibits Ihh expression whilst Ihh stimulates PTHrP expression; these two molecules interact with each other in a negative loop. Ihh action stimulates proliferation and PTHrP inhibits hypertrophy"<-So Ihh is more powerful than PTHrP which may be why Ihh genes are more associated with height.

"PTHrP is expressed in the articular perichondrium and Ihh in pre-hypertrophic and hypertrophic chondrocytes"

"Hypertrophy rate of chondrocytes is positively modulated by RUNX2 transcription factor expression and dissemination and is inhibited by the presence of PTHrP"<-also of note that hypertrophic chondrocytes are located at the region of greatest octahedral(shear) stress.

"SOC[Secondary Ossification Center] growth thus initially follows a radial pattern from the centre of the epiphysis where RUNX2 is released from the canals and spreads outwards. Once RUNX2 induces hypertrophy, the hypertrophic cells collaborate in forming RUNX2. PTHrP inhibits hypertrophy and decreases RUNX2 expression"

"cartilage maturation and ossification are inhibited by intermittent hydrostatic compression stress and accelerated by intermittent non-destructive octahedral shear caused by physiological loading"

"hydrostatic pressure increases cartilage tissue expression"

"Cartilage canals appear randomly and become extended due to octahedral mechanical stresses occurring in the epiphysis."<-Thus the body is capable of forming more than two cartilage canals.

"An experiment applied 200 mN cyclical forces at 1 Hz for 12 h. Their results showed average formation of eight cartilage canals and the appearance of SOC."<-Like LSJL.

"The 200 mN mechanical cyclic loading at 1 Hz applied for 12 h at a single firmly attached condyle explant in an organ culture. Shear forces were generated due to the convex curvature of articular surface. High magnification of the formation of a canal correlates with the applied mechanical load."

Epiphyseal growth plate and secondary peripheral chondrosarcoma: the neighbours matter

"Chondrocytes interact with their neighbours through their cartilaginous extracellular matrix (ECM). Chondrocyte–matrix interactions compensate the lack of cell–cell contact and are modulated by proteoglycans and other molecules. The epiphyseal growth plate is a highly organized tissue responsible for long bone elongation. The growth plate is regulated by gradients of morphogens that are established by proteoglycans. Morphogens diffuse across the ECM, creating short- and long-range signalling that lead to the formation of a polarized tissue. Mutations affecting genes that modulate cell–matrix interactions are linked to several human disorders. Homozygous mutations of EXT1/EXT2 result in reduced synthesis and shortened heparan sulphate chains on both cell surface and matrix proteoglycans. This disrupts the diffusion gradients of morphogens and signal transduction in the epiphyseal growth plate, contributing to loss of cell polarity and osteochondroma formation. Osteochondromas are cartilage-capped bony projections arising from the metaphyses of endochondral bones adjacent to the growth plate. The osteochondroma cap is formed by cells with homozygous mutation of EXT1/EXT2 and committed stem cells/wild-type chondrocytes. Osteochondroma serves as a niche (a permissive environment), which facilitates the committed stem cells/wild-type chondrocytes to acquire secondary genetic changes to form a secondary peripheral chondrosarcoma."

"Cell–cell interactions and the transcription factor Sox9 regulate the formation of [mesenchymal] condensations"

"N-cadherin and N-CAM, are important in establishing an aggregation centre by recruiting mesenchymal cells from surrounding tissue"

"Down-regulation of N-CAM by the binding of syndecan to fibronectin and activation of homeobox genes (ie Msx-1 and Msx-2) by the presence of BMP-2 and BMP-4 stop condensation growth and initiate pre-chondrocyte differentiation "

" In the resting zone, chondrocytes are non-polarized and irregularly arranged. Resting chondrocytes serve as precursors (committed stem cell pool) for proliferative chondrocytes. In the proliferating zone, cell division of chondrocytes occurs perpendicular to the long axis of the growing bone. Proliferating chondrocytes have to undertake a series of cell movements/rotations (intercalation) and shape changes to align one on top of the other to generate the typical chondrocyte columns of the growth plate. Once acquired, this columnar organization is maintained. Chondrocytes require adhesion to cartilaginous ECM for all types of shape changes. Integrins are an important family of receptors that mediate chondrocyte–matrix adhesion. Integrins regulate centrosome function, the assembly of the mitotic spindle and cytokinesis. In β1-null growth plates, chondrocytes display mitotic figures perpendicular to the long axis, but they stay side-by-side and failed to move over each other and form columns, suggesting that β1 integrins regulate chondrocyte shape and rotation. In the hypertrophic zone, chondrocytes stop proliferating and change their expression profile to synthesize type X collagen and to prepare for mineralization of the surrounding cartilaginous ECM"

"Proteoglycans, such as heparan and chondroitin sulphate, function in concert to establish an Indian hedgehog (Ihh) gradient, either through affecting its diffusion or by protecting it from degradation"

"Planar cell polarity pathway has been demonstrated to regulate chondrocyte polarity in the epiphyseal growth plate. Non-polarized resting chondrocytes, which are committed progenitor cells responsible for the generation of proliferating chondrocytes, become polarized proliferating chondrocytes assuming a precise position and orientation in the epiphyseal growth plate, creating columns of stacked cells. Planar cell polarity pathway comprises molecules such as Wnt5A, the Rho family of GTPases and Gpi-anchored proteins. These molecules are shown to regulate orientated cell division and movements (intercalation) of chondrocytes"

"conditional deletion of Kif3a in the growth plate chondrocytes results in the depletion of cilia and loss of columnar organization"->Will overexpression of Kif3a result in more cilia?

"Areas with a growth plate-like distribution of proteoglycans in gradients possibly contain chondrocytes with functional EXT1/EXT2. Areas with reduced amount of proteoglycans with no gradient formation have probably chondrocytes with homozygous inactivation of EXT1/EXT2."

The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification.

"Chondrocytes secrete factors that regulate the behaviour of the invading bone cells, including vascular endothelial growth factor and receptor activator of NFκB ligand."

"The growth plate chondrocyte orchestrates the invasion of its own domain by the ossification front not only through preparation of the cartilage tissue, but also by secreting soluble molecules that regulate the behaviour of the invading cells."

"In the presence of IHH signalling, [Gli] proteins promote transcription of IHH target genes, but when IHH is absent, Gli2 and Gli3{up in LSJL} are subject to proteolytic conversion into transcriptional repressors. IHH stimulates chondrocyte proliferation through inactivation of the repressor form of Gli3, in particular, as demonstrated by the phenotype of double IHH-null/Gli3-null mice"

"IHH binds to aggrecan through its chondroitin sulphate side chains, and in the mouse growth plate normal sulphation of chondroitin sulphate is required for normal IHH protein distribution and signalling, and for chondrocyte proliferation. IHH signalling, and thus proliferation of growth plate chondrocytes, is also dependent on the presence of an intact primary cilium, a structure consisting of a basal body and a ciliary axoneme that extends several micrometres from the surface of the chondrocyte and most other cells"

"Phosphorylation of Smads 1 and 5 in chondrocytes is dependent on the presence of neogenin, which is a receptor for the neuronal axon guidance cues netrins and repulsive guidance molecules "

"mice that lack both Shn2 and Shn3 (but not each gene individually) show defects in endochondral ossification including inadequate chondrocyte proliferation, an effect that is likely to be due to failure of BMP signalling"

"FGFR3 is a tyrosine kinase receptor, and its suppression of chondrocyte proliferation in the growth plate appears to be mediated by STAT1. The activity of FGFs, like that of IHH, is modulated by glycosaminoglycans. Mice that lack sulphatase-modifying factor 1, which activates sulphatases (and thus proteoglycan desulphation) show dwarfism, which can be rescued by crossing with FGF18-null mice. Thus, it appears that FGF-induced repression of chondrocyte proliferation is limited by the desulphation of glycosaminoglycans. The relevant glycosaminoglycan is most likely to be heparan sulphate, found in proteoglycans such as perlecan in the cartilage ECM. BMP signalling antagonises the inhibition of chondrocyte proliferation caused by activation of FGFR3"

"TRPS1-null mice demonstrate abnormally low levels of proliferation in growth plate chondrocytes, which can be attributed to a role for TRPS1{up in LSJL} in repression of STAT3 expression, which in turn allows for elevated expression of cyclin D1"

"TRPS1 binds the transcriptional activator form of Gli3"<-So maybe the form of Gli3 is the transcription activator form in LSJL.

"Cartilage proteoglycans, in addition to aggrecan, include the small leucine-rich proteoglycans decorin, biglycan{up} and fibromodulin, as well as the large proteoglycan perlecan. Other non-collagenous proteins found in cartilage ECM include the matrilins and thrombospondin family members, such as thrombospondin-5, also known as cartilage oligomeric matrix protein (COMP)."

"Pseudoachondroplasia in humans is caused by mutations in COMP, and the milder multiple epiphyseal dysplasia can be caused by mutations in the genes encoding COMP, collagen type IX{up} or matrilin-3{up}"  "these proteins are at least partly able to substitute for each other"

"Mice in which the gene for hyaluronan synthase 2 (Has2) is inactivated in tissues derived from limb bud mesoderm possess abnormally short limbs"

"SOX9-activated transcription appears to be modulated by epigenetic mechanisms, since it occurs predominantly in hyperacetylated chromatin; the histone acetyltransferase p300 associates with SOX9 and enhances SOX9-dependent transcription. Moreover, inhibition of histone deacetylases (HDACs) stimulates expression of SOX9-activated cartilage ECM genes and induces histone acetylation in the region of the Col2a1 enhancer in primary chondrocyte cultures"

"Overexpression of HDAC1 or 2 in chondrocytes results in down-regulation of expression of Aggrecan and Col2a1"

" aggrecan degradation does not appear to be required. There are no morphological defects in the growth cartilage of mice in which aggrecan is rendered resistant to MMP cleavage"

"the role of hyaluronan may be to initiate hypertrophy-inducing intracellular signalling in chondrocytes emerging from the proliferative phase."

"Hypothyroidism in humans results in slowing of longitudinal bone growth, with abnormally thin growth plates and impaired chondrocyte hypertrophy. Studies in genetically manipulated mice have demonstrated that the receptor responsible for these effects is thyroid hormone receptor α and not thyroid hormone receptor β"

"In chondrocytes in vitro, T3 up-regulates Wnt4 mRNA and protein expression as well as cellular accumulation of β-catenin, and inhibition of WNT signalling by secreted WNT antagonists inhibits T3-induced hypertrophy"

"PTHrP stimulates cyclin D1 expression in chondrocytes, and is unable to down-regulate RUNX2 expression in chondrocytes from cyclin D1-null mice, apparently because cyclin D1 contributes to proteasomal degradation of RUNX2. The transcriptional co-regulator Zfp521, which is induced by PTHrP, has also recently been identified as an effector of PTHrP's actions in the growth plate; the growth cartilage of mice with chondrocyte-specific deletion of Zfp521 resembles that of PTHrP-null mice, and PTHrP is unable to stimulate cyclin D1 expression or inhibit RUNX2 expression in the absence of Zfp521"

"Mice with a heterozygous in-frame deletion of the DNA-binding domain of TRPS1 show elevated expression of RUNX2 in the growth plate; moreover, TRPS1 directly interacts with RUNX2 to inhibit its function. These observations suggest that TRPS1 acts to fine-tune cell cycle exit and progression to the hypertrophic state, by limiting both the antihypertrophic activity of PTH and the pro-hypertrophic activity of RUNX2, while at the same time supporting proliferation in a cyclin D1-dependent manner"

"growth plate mineralisation is not required for normal function of the growth plate itself. The importance of growth plate mineralisation may lie in provision of an appropriate composition for the remnants of cartilage matrix on which bone is deposited in the primary centre of ossification, since bone volume in the newly formed bone of the metaphysis is reduced in the absence of carminerin"

"light chondrocytes appear to disintegrate within their cell membrane and dark chondrocytes progressively extrude their cytoplasm into the extracellular space."

"growth plate cartilage degradation is not dramatically altered by failure to express cathepsin K"

"there is abnormal retention of growth plate cartilage in MMP9-null mice, it appears that osteoclasts depend more on this enzyme than on cathepsin K for their degradation of cartilage matrix"

"blood vessels appear to precede osteoclasts as the first cells that enter the lacunae recently vacated by dying hypertrophic chondrocytes, thus it is not particularly surprising that they are able to invade the growth plate in the absence of osteoclast activity. Another cell type that has recently been described as accompanying the blood vessels at the ossification front is the ‘septoclast’, a perivascular cell that expresses cathepsin B; it has been proposed that septoclasts assist in the degradation of the transverse cartilage septa, thus allowing entry of capillaries"

"High-mobility group box 1 protein (HMGB1) is secreted by hypertrophic chondrocytes, and acts as a chemoattractant for endothelial cells, osteoclasts and osteoblasts; HMGB1-null mice display delayed invasion of the growth plate by cells of the ossification centre"

Regulatory mechanisms for the development of growth plate cartilage.


"RhoA signaling through its main effector ROCK inhibits chondrogenesis by suppressing the expression of Sox9"

"Sox9 suppresses the expression of Runx2 and β-catenin signaling and thereby inhibits the progression from proliferation to prehypertrophy of chondrocytes. Twist-1 is a basic helix-loop-helix-type transcription factor, which represses the expression of Runx2 in the perichondrium. Runx2 enhances the expression of fibroblast growth factor 18 (Fgf18) and exerts an indirect negative effect on chondrocyte maturation. Osterix regulates the calcification and degradation of cartilaginous matrix through MMP13 expression in association with Runx2 "

"Genetic deletion of Mef2c in endochondral cartilage impairs hypertrophic maturation, while the forced expression of a superactivating form of Mef2c resulted in precocious chondrocyte hypertrophy. The activity of Runx2/3 and Mef2c/d is inhibited by the histone deacetylase HDAC4. Other transcription factors, such as Msx2, the AP1 family member Fra2, and FoxA family transcription factors, also positively control chondrocyte hypertrophy"

"chondrocytes express several integrin subunits including fibronectin receptors (α5β1, αnβ3, αnβ5), a laminin receptor (α6β1) and collagen receptors (α1β1, α2β1, α10β1). The importance of β1 integrin-mediated signaling in chondrogenesis was demonstrated by the chondrodysplasia-like phenotype of chondrocyte-specific β1 integrin-knockout mice"

"Inactivation of the α10 integrin gene resulted in growth plate dysfunction, which was associated with an abnormal cell shape and increased apoptosis of chondrocytes"  Knockout of alpha1 integrin resulted in no growth plate defects.

"HDAC1 and HDAC2 were shown to repress the expression of some cartilage-specific genes including Col2a1, and the Snail transcription factor was identified as a mediator of the repression. The up-regulation of HDAC7 expression was suggested to contribute to the cartilage degradation by promoting the expression of MMP13"

Tuesday, August 16, 2011

Grow Taller with Cnidium

Cnidium is similar to Icariin but it's another way to increase BMP-2 levels which are necessary for initial chondrogenesis and may be a way to help boost LSJL results(note that neither is advised for those who are not skeletally mature due to both of their tendencies to accelerate terminal chondrogenic differentiation).  She Chuang Zi - Cnidium Fructus, 100 grams,(MinTong).  It's also unclear what the optimal interaction dosages would be between Cnidium and Icariin.

Osthol, a Coumarin Isolated from Common Cnidium Fruit, Enhances the Differentiation and Maturation of Osteoblasts in vitro.

"Primary osteoblastic cells isolated from newborn Wistar rats [were treated with Osthol]. Osthol was supplemented into cultured medium at 10(-7), 10(-6), 10(-5) and 10(-4) mol/l, respectively. No stimulating effect was found on cell proliferation, but 10(-5) mol/l osthol caused a significant increase in alkaline phosphatase (ALP) activity. Osteogenic differentiation markers were examined over a period of time at this concentration, and compared with control cells that were not supplemented with osthol. ALP activity, osteocalcin secretion and calcium deposition level in cells treated with osthol were 1.52, 2.74 and 2.0 times higher, respectively, than in the control cells. The number and area achieved in osthol-treated cells were 1.53-fold higher than in control cells. The gene expression of the growth and transcription factors basic fibroblast growth factor, insulin-like growth factor I, bone morphogenetic protein 2 (BMP-2), runt-related gene 2 (Runx-2) and osterix, which are associated with bone development, were also investigated. The increase in mRNA expression was 1.94, 1.74, 1.68, 1.83 and 2.31 times, respectively, higher compared to the control. Furthermore, osthol increased the protein expression of p38 mitogen-activated protein kinase (MAPK) and type I collagen. p38MAPK protein and collagen in osthol-treated cells were 1.42 and 1.58 times higher in osthol-treated cells compared to the control.  Osthol could stimulate the osteoblastic differentiation of rat calvarial osteoblast cultures by the BMP-2/p38MAPK/Runx-2/osterix pathway."<-Icariin had best effects on BMP-2 expression with 1X10(-9)mol/L.  Whereas the most effective concentration of osthol was 1X10(-5)mol/L.  Osthol increased IGF-1 expression by 1.74 which is nearly double.  And it also increased BMP-2 expression by 1.68 which is quite significant.  Cnidium definitely looks promising as a supplement to LSJL due to it's significant effects on IGF-1 and BMP-2.  However, dealing with the interactions with Icariin which seems similar is tricky at this point.

However Osthol has been studied only on bone and not on chondrocytes or chondrogenesis.  However, bFGF and BMP-2 may have spillover effects on chondrogenesis.

Monday, August 15, 2011

growing tall with interleukins

In vitro engineering of cartilage: effects of serum substitutes, TGF-beta, and IL-1alpha.

"We used porous collagen sponges to assess the effects of serum substitutes and exogenous TGF-beta1 and IL-1alpha on chondrocytes (bovine articular chondrocytes, bACs) and on chondroinduced human dermal fibroblasts (hDFs). We determined the effects of low concentrations of FBS[fetal bovine serum] and two serum substitutes, Nutridoma and ITS(+3), on cellularity and matrix production. After culture for intervals, sponges were harvested for histological and biochemical measurement of cartilage-specific chondroitin 4-sulfate proteoglycan (C 4-S PG).
Cultured bACs showed equivalent growth in Nutridoma (1%) and 10% FBS. Both TGF-beta1 and IL-1alpha significantly stimulated accumulation of C 4-S PG by bACs in 3D porous collagen sponges. Many endogenous growth factors were upregulated in hDFs cultured with chondroinductive DBP. Addition of TGF-beta1 and IL-1alpha for 11 days significantly stimulated accumulation of C 4-S PG by hDFs cultured in DMEM with 1% Nutridoma.
Porous collagen sponges are supportive of chondrogenesis and of chondroinduction by DBP. Optimization of serum-free culture conditions, including growth factors, matrix components, and mechanical stimuli will expedite translation to wider clinical applications. Use of autogenous dermal fibroblasts pre-cultured with DBP and induced to chondrocytes offers an alternative to autogenous chondrocytes."

"In the samples treated with 54 nM IL-1α, the metachromatic matrix around the cells and between the particles of DBP appeared denser and more granular than in samples that were not treated with IL-1α."

" IL-1α is an endogenous, autocrine factor in human osteoblasts: first, osteoblast conditioned medium is a mitogen for other osteoblasts even in the presence of indomethacin but only partially in the presence of IL-1α-neutraling antibody; second, that osteoblasts secrete IL-1α; and, third, that exogenous recombinant human IL-1α stimulated proliferation of normal human osteoblasts"

This study provides contradictory results

Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.

"We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis"

"Addition of IL-1α at day 0, 3, or 7 resulted in a total block of the chondrogenic differentiation as no increased expression of type II collagen and aggrecan mRNA was detected"

The role of IL-6 in bone marrow (BM)-derived mesenchymal stem cells (MSCs) proliferation and chondrogenesis.

"we isolate MSCs from the murine bone marrow, and induce MSCs chondrogenesis with different concentrations of IL-6{up} in vitro.  IL-6 inhibited the differentiation of MSCs into chondrocytes in the dose-dependence manner."

Couldn't get full study.

The inhibition by interleukin 1 of MSC chondrogenesis and the development of biomechanical properties in biomimetic 3D woven PCL scaffolds.

"a three-dimensionally (3D) woven poly(ε-caprolactone) (PCL) scaffold [was] seeded with bone marrow-derived mesenchymal stem cells (MSCs). Pro-inflammatory cytokine interleukin-1 (IL-1) [was present], which is found at high levels in injured or diseased joints. MSC-seeded 3D woven scaffolds cultured in chondrogenic conditions synthesized a functional ECM rich in collagen and proteoglycan content, reaching an aggregate modulus of ~0.75 MPa within 14 days of culture. However, the presence of pathophysiologically relevant levels of IL-1 limited matrix accumulation and inhibited any increase in mechanical properties over baseline values. On the other hand, the mechanical properties of constructs cultured in chondrogenic conditions for 4 weeks prior to IL-1 exposure were protected from deleterious effects of the cytokine. IL-1 significantly inhibits the chondrogenic development and maturation of MSC-seeded constructs; however, the overall mechanical functionality of the engineered tissue can be preserved through the use of a 3D woven scaffold designed to recreate the mechanical properties of native articular cartilage."

"Chondrogenic culture conditions resulted in a reduction in apparent hydraulic permeability of 3 orders of magnitude to approximately 0.0008 mm4/N s"

"Chondrogenic culture conditions containing TGF-β3 resulted in a dense ECM rich in both s-GAGs (as stained by Safranin-O or chondroitin-4-sulfate) and collagens (as stained by Fast Green) that accumulated and completely filled the interstitial voids within the scaffold."

"IL-1 appears to act through the activation of nuclear factor kappa B (NF-κB), as inhibition of this pathway using a dominant-negative suppressor of I-κB, curcumin, or resveratrol abrogates the effects of IL-1 on MSCs."

"relatively high concentrations (100 ng/ml) of bone morphogenetic proteins (BMPs) such as BMP-2 or BMP-9 can partially overcome the effects of IL-1"

"MSCs may play an anti-inflammatory role when reintroduced in vivo and enhance repair by producing IL-1 receptor antagonist (IL-1Ra)"

Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells.

"Low doses of IL-1β (50 pg/mL) enhanced colony-forming units-fibroblastic (CFU-f) and -osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks."

But it is also mentioned that IL1B can inhibit MSC chondrogenesis.

1000pg/mL of IL-1B resulted in catabolic effects.  Doses between 50-1000 were generally not tested. In some metrics catabolic effects were noted at 100pg/mL. There was a slight increase in Type II collagen at 50pg/mL(with a large inhibition at 1000pg/mL illustrating the importance of inhibiting IL-1B for height growth if levels are too high).  So slight IL1B may still be beneficial for mesenchymal chondrogenesis.

"MSC were also induced to differentiate in pellet in the absence or presence of IL-1β during the entire culture time. Histological analyses indicated that tissues formed by BM-MSC exposed to low doses of IL-1β (10 and 50 pg/mL) were more intensely stained for cartilage specific matrix than tissues formed in absence of IL-1β, while those exposed to high doses (≥ 250 pg/mL) were less intensely stained"

A study showing normal non-periosteal bone growth in adults

Many people have noticed a large width gain in the epiphysis after performing LSJL.  There are stem cells in the synovial joint and new osteophytes could be formed from there.  Although this is a different mechanism than endochondral ossification, it does prove LSJL's ability to induce chondrogenic differentiation of a stem cell type(if it is in fact osteophyte formation).  Since bone is more sturdy than the synovial capsule, more hydrostatic pressure should be needed to induce sufficient pressure to induce chondrogenic differentiation there.  If you are not feeling interestitial fluid flow in your bone and your bone is not increasing in width than it may be safe to say that you are not providing enough pressure as both of those events should occur before sufficient pressure for endochondral ossification.  However, it is unknown how much pressure after this point is needed to induce endochondral ossification.  In a few days, I will be stopping LSJL on the finger of my left hand and begin performing it on my right hand.  To make sure that that the increase in width is due to osteophyte formation and not some sort of callus.

Surprising evidence of pelvic growth (widening) after skeletal maturity.

"Following an increase in length and width during childhood and adolescence, skeletal growth is generally assumed to stop. This study investigates the influence of aging on the dimensions of the pelvis and the L4 lumbar vertebra during adulthood. The dimensions of the pelvis, L4 vertebra, and femoral heads were calculated for 246 patients who had received pelvic and abdominal Computed Tomography scans. Linear regression analysis determined the significance of relationships between age and width of the pelvis. There was a strong correlation between increasing patient age and increasing width of the pelvis at the trochanters, (0.333 mm/year of age){the trochanter is part of the femur}, at the iliac wings, (0.371 mm/year of age){the iliac wing is the horizontal-most flaps at the end of the pelvic on the iliac}, and between the femoral heads{the femoral head is part of the femur}, indicating that the bony pelvis widens over 20 mm between the ages of 20 and 80. The pelvic inlet did not enlarge over time while the distance between the hips and the femoral head diameter did significantly increase[the femoral head is an epiphyseal type bone which should not be able to increase in width without periosteal deposition]. The height of L4 did not increase over time, but the L4 width did increase. These correlations were seen in both genders. Surprisingly, our results suggest that the pelvis and L4 vertebra increase in width after skeletal maturity and cessation of longitudinal growth."

In the study it mentioned that L4 vertebral height did increase by 0.9mm.  However, it was done between the ages of 20to80 and some say that vertebral height growth does not cease until the mid 20s.  So this increase could have occurred before then.  It is also possible of course that the growth occurred later as well.  There was a positive slope for age indicating that there was an increase in vertebral height even into the 80s.

"the femoral head data, which we expected would also show no increase (because there is no known mechanism for enlargement of the femoral heads) did show enlargement of the femoral heads with age"<-indicating the possibility of something like osteophyte based bone formation.

"We also were unable to confirm our third hypothesis, that widening of the pelvis was simply due to periosteal appositional bone formation, because we observed no significant change in the pelvic inlet width whereas appositional formation would presumably result in a reduction in the width of the inlet. If periosteal apposition is due to the previously described bone enlargement in response to osteoporosis, one would expect that there would be more marked differences between the enlargement of male and female subjects as well as a significant change in the slope of the line (as represented by the triangular decade means) after age 45 in women, which we did not observe in this data. The significant increases in inter-femoral head distance also cannot be explained by simple periosteal apposition and would appear to require some conformational change in the pelvis."<-Bone formation by means other than periosteal deposition is very promising for longitudinal height growth as of course periosteum is not on the longitudinal ends of the bones.  If this mechanism for width increase can be found than it probably can be used for height growth.

"Our first thought was that the enlargement was due to calcification of the deeper layers of the cartilage (apparent enlargement only). It may be, but 3.3 mm of enlargement of the diameter would represent 1.6 mm of calcification of the cartilage on each side of the head over the years from age 20 to 80 which seems to be a great deal of calcification as the total thickness of the cartilage is only 2–3 mm (joint space 4–5 mm)."<-now endochondral ossification could take a smaller part of cartilage and turn it into a larger total width due to chondrocyte hypertrophy.

This study provides strong evidence for non-periosteal bone growth either occurring through endochondral ossification within the synovium or within the bone.



"mice in which the Spry2 gene has been inactivated have smaller and undermineralized bones"

"Sprouty genes encode molecules that antagonize signaling by FGF receptors as well as other receptor-tyrosine kinases (RTKs). "

Spry2 may inhibit FGFR3 making you taller.

Monday, August 8, 2011

Grow taller with Matrilin's?

LSJL upregulates Matrilin's 2 & 4 by approximately 3.5 fold.  It also upregulates Matrilin 3

Expression of matrilin-1, -2 and -3 in developing mouse limbs and heart.

"Throughout development matrilin-3 expression was strictly limited to cartilage, while matrilin-1 was also found in some other forms of connective tissue. Matrilin-2, albeit present around hypertrophic chondrocytes in the growth plate, was mainly expressed in non-skeletal structures."

"matrilin-1 associates with the aggrecan core protein"  It may also be involved in collagen assembly.

"In the skeleton matrilin-2 occurs only in the periosteum, perichondrium and the hypertrophic zone of the growth plate"

Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state.

"The two aggrecanases ADAMTS-4 and ADAMTS-5 play roles in the breakdown of cartilage extracellular matrix in osteoarthritis [and] mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail.  ADAMTS-5 and a matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. In the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of matrilins and potentially of other extracellular matrix proteins."

"Aggrecan has a modular structure and ADAMTSs cleave its core protein at distinct sites either in the interglobular domain, located between the N-terminal globular domain G1 and domain G2, or in the long region of the core protein substituted with chondroitin sulfate chains"

"Matrilin-4 is expressed in all cartilaginous regions of the maturing mouse joint, including proliferating and hypertrophic cells of the growth plate"<-maybe the increase in matrilin-4 expression in LSJL could indicate new growth plate formation.

According to Molecular structure, processing, and tissue distribution of matrilin-4., osteoblasts showed low levels of matrilin 4 whereas the joint surface, where there's chondrocytes and cartilage, expressed matrilin 4 at much higher levels.  Thus, indicating that such a large increase of MATN4 levels could be due to new chondrogenesis.

Although according to Normal skeletal development of mice lacking matrilin 1: redundant function of matrilins in cartilage?, MATN1 and MATN3 are only expressed in cartilage which makes it disturbing that MATN1 is not expressed in LSJL at over 2-fold.

Increased expression of matrilin-3 not only in osteoarthritic articular cartilage but also in cartilage-forming tumors, and down-regulation of SOX9 via epidermal growth factor domain 1-dependent signaling.

"Differential proteomics analysis revealed matrilin-3 (MATN3) as a candidate regulator of the cartilaginous phenotype. Its capacity to modulate gene expression was investigated in human HCS-2/8 chondrosarcoma cells and transfected chondrocytes.
Increased expression of the cartilage-specific matrix protein MATN3 was specifically observed in enchondromas and conventional chondrosarcomas. A substantial fraction of MATN3 was found in cytoplasmic structures of tumor cells, as demonstrated by immunohistochemistry. Analyses of intracellular MATN3 revealed that it corresponded to an imperfectly maturated MATN3 polypeptide, both in HCS-2/8 human chondrosarcoma cells and in transfected human chondrocytes. Moderately increased expression of MATN3 resulted in its intracellular retention. Antibody-mediated blockade of soluble, extracellular MATN3 in HCS-2/8 cell cultures resulted in increased expression of MATN3 and the chondrogenic transcription factor SOX9. Conversely, increased ectopic expression of MATN3 resulted in decreased expression of MATN3 and SOX9 in primary chondrocytes, while a mutant MATN3 lacking its first epidermal growth factor (EGF)-like domain failed to down-regulate SOX9.
Aberrant expression and processing of MATN3 are hallmarks of conventional cartilaginous neoplasms. A particular step in the maturation of MATN3 limits its processing through the secretion machinery, resulting in its intracellular accumulation upon increased expression. Soluble, secreted MATN3, however, down-regulates SOX9 at the messenger RNA and protein levels. The first EGF-like domain of MATN3 is a critical determinant of its regulatory activity toward SOX9."

"Most chondrogenic tumors are benign (enchondromas) and are comparable with differentiated chondrocytes with respect to their gene expression profile and matrix composition"

"extracellular MATN3 can down-regulate the expression of both SOX9 mRNA and its own mRNA."<-Sox9 was still upregulated in LSJL despite upregulation of MATN3.

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix.

"Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. [MATN3's] concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation[Akt is phosphorylated with LSJL] and favored survival and ECM synthesis.  MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. The integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. MATN3 plays either beneficial or detrimental functions in cartilage."

"MATN3A, but not MATN3B, exerted up-regulation of catabolism and down-regulation of anabolism within 48 h"

"MATN3 does not arise from outside the cartilage but from the pericellular matrix of chondrocytes"

Matrilin-3 Induction of IL-1 receptor antagonist Is required for up-regulating collagen II and aggrecan and down-regulating ADAMTS-5 gene expression.

"The effects of recombinant human (rh) MATN3 protein were examined in C28/I2 immortalized human chondrocytes, primary human chondrocytes (PHCs), and primary mouse chondrocytes (PMCs)."

"rhMATN3 protein induced gene expression of IL-1Ra in C28/I2 cells, PHCs, and PMCs in a dose- and time-dependent manner. Treatment of C28/I2 cells and PHCs with MATN3 protein stimulated gene expression of COL2A1 and ACAN. Conversely, mRNA levels of COL2A1 and ACAN were decreased in MATN3 KO mice. MATN3 protein treatment inhibited IL-1β-induced MMP-13, ADAMTS-4 and ADAMTS-5 in C28/I2 cells and PHCs. Knocking down IL-1Ra abolished the MATN3-mediated stimulation of COL2A1 and ACAN and inhibition of ADAMTS-5, but had no effect on MATN3 inhibition of MMP-13 mRNA."

"MATN3 KO mice have relatively normal skeletal development"

Matrilin-3 activates the expression of osteoarthritis-associated genes in primary human chondrocytes.

"we tested the matrilin-3-dependent induction of osteoarthritis-associated genes in primary human chondrocytes. Matrilin stimulation leads to the induction of MMP1, MMP3, MMP13, COX-2, iNOS, IL-1beta, TNFalpha, IL-6 and IL-8. ADAMTS4 and ADAMTS5 [participate] in the in vitro degradation of matrilin-3. [There's] a matrilin-3-dependent feed-forward mechanism of matrix degradation, whereby proteolytically-released matrilin-3 induces pro-inflammatory cytokines as well as ADAMTS4 and -5 indirectly via IL-1beta. ADAMTS4 and ADAMTS5, in turn, cleave matrilin-3 and may release more matrilin-3 from the matrix, which could lead to further release of pro-inflammatory cytokines and proteases in cartilage."

It seems as though Matrilin-3 is involved in endochondral ossification.

" IL-6 release can be induced by collagen II, but not by collagen I [in chondrocytes]"  Type II collagen induced the release of the other interleukin's IL8 and IL1B in relatively similar quantities to MATN3.  However MATN3 increased TNFa levels more than Type II Collagen.

"ADAMTS4 and ADAMTS5 cleave matrilin-3 and may release more matrilin-3 from the matrix"