Reduction in disease progression by inhibition of transforming growth factor α-CCL2 signaling in experimental posttraumatic osteoarthritis.

OBJECTIVE: Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA. METHODS: Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C). RESULTS: Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage. CONCLUSION: TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo.

Transforming growth factor-alpha induces endothelin receptor A expression in osteoarthritis.

Previously, our lab identified transforming growth factor-alpha (TGFα) as a novel factor involved in osteoarthritis (OA) in a surgical model of the disease. In the same study, we also observed increased transcript levels for endothelin receptor A (ET(A)R), a known contributor to cartilage pathology. To investigate the connection between TGFα and endothelin signaling in OA, primary articular chondrocytes and osteochondral explants were isolated from Sprague-Dawley rats and treated with vehicle or TGFα. Expression of ET(A)R protein and its encoding gene Ednra was assessed. Chondrocytes and cartilage explants were also treated with the endothelin receptor A/B antagonist Bosentan, in order to determine whether TGFα effects could be blocked. TGFα induced expression of ET(A)R protein and its encoding gene Ednra. In primary chondrocyte cultures, Bosentan did not block TGFα responses of the anabolic genes Sox9, Agc1, and Col2a1, but reduced the induction of Mmp13 and Ednra transcripts by TGFα. In osteochondral explants, the inhibitor partially blocked TGFα reduction of type II collagen, as well as induction of MMP-13 and type II collagen neoepitopes. TGFα induces ET(A)R expression in articular chondrocytes and receptor antagonism appears to block some TGFα-induced catabolic effects in a three-dimensional organ culture system. Thus, TGFα may be a therapeutic target upstream of ET(A)R in OA.

Rho/ROCK and MEK/ERK activation by transforming growth factor-alpha induces articular cartilage degradation.

Identification and characterization of therapeutic targets for joint conditions, such as osteoarthritis (OA), is exceedingly important for addressing the increasing burden of disease. Transforming growth factor-alpha (TGFalpha) is upregulated by articular chondrocytes in experimentally induced and human OA. To test the potential involvement of TGFalpha, which is an activator of epidermal growth factor receptor (EGFR) signaling, in joint degeneration and to identify signaling mechanisms mediating articular chondrocyte responses to TGFalpha, rat chondrocytes and osteochondral explants were treated with TGFalpha and various inhibitors of intracellular signaling pathways. Stimulation of EGFR signaling in articular chondrocytes by TGFalpha resulted in the activation of RhoA/ROCK (Rho kinase), MEK (MAPK/ERK kinase)/ERK (extracellular-signal-regulated kinase), PI3K (phosphoinositide 3-kinase) and p38 MAPK (mitogen-activated protein kinase) pathways. Modification of the chondrocyte actin cytoskeleton was stimulated by TGFalpha, but inhibition of only Rho or ROCK activation prevented morphological changes. TGFalpha suppressed expression of anabolic genes including Sox9, type II collagen and aggrecan, which were rescued only by inhibiting MEK/ERK activation. Furthermore, catabolic factor upregulation by TGFalpha was prevented by ROCK and p38 MAPK inhibition, including matrix metalloproteinase-13 and tumor necrosis factor-alpha, which are well known to contribute to cartilage digestion in OA. To assess the ability of TGFalpha to stimulate degradation of mature articular cartilage, type II collagen and aggrecan cleavage fragments were analyzed in rat osteochondral explants exposed to exogenous TGFalpha. Normal articular cartilage contained low levels of both cleavage fragments, but high levels were observed in the cartilage treated with TGFalpha. Selective inhibition of MEK/ERK and Rho/ROCK activation greatly reduced or completely prevented excess type II collagen and aggrecan degradation in response to TGFalpha. These data suggest that TGFalpha is a strong stimulator of cartilage degradation and that Rho/ROCK and MEK/ERK signaling have critical roles in mediating these effects.

Vascular smooth muscle cells as a valvular interstitial cell surrogate in heart valve tissue engineering.

BACKGROUND: Vascular smooth muscle cells (VSMCs) are a potential autologous cell source for aortic valve tissue engineering, but have a phenotype that differs from that of valvular interstitial cells in vivo. We hypothesized that combining basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), or platelet-derived growth factor (PDGF) with transforming growth factor beta-1 (TGF-beta1) would achieve a valvular interstitial cell-like phenotype of VSMCs. METHODS: VSMC phenotype was assessed by immunofluorescence, proliferation was measured by the tetrazolium reduction (MTT) assay, and extracellular matrix gene expression was determined by real-time polymerase chain reaction. RESULTS: Combinations of growth factors that included PDGF showed the greatest increases in proliferation. Immunofluorescence for alpha-smooth muscle actin demonstrated an inverse correlation between proliferation and a myofibroblast-like phenotype, while combinations of TGF-beta1+ EGF+bFGF (TEF) and TGF-beta1+EGF+PDGF (TEP) induced the greatest change of alpha-smooth muscle actin expression compared to untreated controls. Finally, TEP treatment showed an increase in versican, fibronectin, and type I collagen mRNA expression, while decreasing matrix metalloproteinase 1 expression. CONCLUSIONS: Combination of TGF-beta1 with EGF and PDGF induces VSMC proliferation and expression of extracellular matrix constituents found in the aortic valve. In vitro preconditioning of VSMCs provides a potentially viable surrogate cell source for developing a valve graft.

The pattern recognition receptor CD36 is a chondrocyte hypertrophy marker associated with suppression of catabolic responses and promotion of repair responses to inflammatory stimuli.

Multiple inflammatory mediators in osteoarthritis (OA) cartilage, including S100/calgranulin ligands of receptor for advanced glycation end products (RAGE), promote chondrocyte hypertrophy, a differentiation state associated with matrix catabolism. In this study, we observed that RAGE knockout was not chondroprotective in instability-induced knee OA in 8-wk-old mice. Hence, we tested the hypothesis that expression of the alternative S100/calgranulin and patterning receptor CD36, identified here as a marker of growth plate chondrocyte hypertrophy, mediates chondrocyte inflammatory and differentiation responses that promote OA. In rat knee joint destabilization-induced OA, RAGE expression was initially sparse throughout cartilage but increased diffusely by 4 wk after surgery. In contrast, CD36 expression focally increased at sites of cartilage injury and colocalized with developing chondrocyte hypertrophy and aggrecan cleavage NITEGE neoepitope formation. However, CD36 transfection in normal human knee-immortalized chondrocytes (CH-8 cells) was associated with decreased capacity of S100A11 and TNF-alpha to induce chondrocyte hypertrophy and ADAMTS-4 and matrix metalloproteinase 13 expression. S100A11 lost the capacity to inhibit proteoglycans synthesis and gained the capacity to induce proteoglycan synthesis in CD36-transfected CH-8 cells. Moreover, S100A11 required the p38 MAPK pathway kinase MKK3 to induce NITEGE development in mouse articular cartilage explants. However, CH-8 cells transfected with CD36 demonstrated decreased S100A11-induced MKK3 and p38 phosphorylation. Therefore, RAGE and CD36 patterning receptor expression were linked with opposing effects on inflammatory, procatabolic responses to S100A11 and TNF-alpha in chondrocytes.

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-beta activation.

In osteoarthritis (OA) articular chondrocytes undergo phenotypic changes culminating in the progressive loss of cartilage from the joint surface. The molecular mechanisms underlying these changes are poorly understood. Here we report enhanced (approximately 7-fold) expression of F-spondin, a neuronal extracellular matrix glycoprotein, in human OA cartilage (P<0.005). OA-specific up-regulation of F-spondin was also demonstrated in rat knee cartilage following surgical menisectomy. F-spondin treatment of OA cartilage explants caused a 2-fold increase in levels of the active form of TGF-beta1 (P<0.01) and a 10-fold induction of PGE2 (P<0.005) in culture supernatants. PGE2 induction was found to be dependent on TGF-beta and the thrombospondin domain of the F-spondin molecule. F-spondin addition to cartilage explant cultures also caused a 4-fold increase in collagen degradation (P<0.05) and a modest reduction in proteoglycan synthesis (approximately 20%; P<0.05), which were both TGF-beta and PGE2 dependent. F-spondin treatment also led to increased secretion and activation of MMP-13 (P<0.05). Together these studies identify F-spondin as a novel protein in OA cartilage, where it may act in situ at lesional areas to activate latent TGF-beta and induce cartilage degradation via pathways that involve production of PGE2.

Transforming growth factor alpha suppression of articular chondrocyte phenotype and Sox9 expression in a rat model of osteoarthritis.

OBJECTIVE: To define the roles of transforming growth factor alpha (TGFalpha) in cartilage degradation. METHODS: Primary rat articular chondrocytes and articular osteochondral explants were cultured with TGFalpha to assess the effects of TGFalpha on chondrocyte physiology and phenotype. RESULTS: TGFalpha altered chondrocyte morphology through reorganization of the actin cytoskeleton and formation of stress fibers. Expression of anabolic genes, including aggrecan, type II collagen, and cartilage link protein, was reduced in response to TGFalpha. Proliferation of chondrocytes and formation of articular chondrocyte clusters was stimulated by TGFalpha. Expression of matrix metalloproteinase 13 and cathepsin C was increased by TGFalpha. We demonstrated the down-regulation of Sox9 messenger RNA and protein levels by TGFalpha. This was associated with reduced levels of phosphorylated and total SOX9 in cartilage explants upon TGFalpha treatment. In contrast, another growth factor identified in our microarrays, Kitl, had no effects on the chondrocyte parameters tested. To examine correlations between the increased levels of TGFalpha in experimental knee osteoarthritis (OA) with the levels of TGFalpha in humans with knee OA, a microarray analysis of mRNA from 13 normal and 12 late-stage OA cartilage samples was performed. Seven OA samples showed TGFA mRNA levels similar to those in the normal controls, but expression was markedly increased in the other 5 OA samples. These data confirm that TGFA transcript levels are increased in a subset of patients with OA. CONCLUSION: This study adds TGFalpha to the list of dysregulated cytokines present in degrading cartilage in OA. Since TGFalpha inhibits articular chondrocyte anabolic capacity, increases catabolic factors, and contributes to the development of chondrocyte clusters, TGFalpha may be a potential target for therapeutic strategies in the treatment of OA.

Molecular and histological analysis of a new rat model of experimental knee osteoarthritis.

Articular cartilage degeneration is the most consistently observed feature of osteoarthritis (OA). Animal and human studies have shown that various forms of exercise influence the course of the disease in different ways. In addition, early changes in articular cartilage that influence the progression of OA, such as the expression of cytokines, require further investigation. We have used a surgically induced experimental model of knee OA to address these questions. Here, we discuss our recent studies investigating the effects of an exercise paradigm in surgically induced OA, which determined that the destabilized knee joint is susceptible to enhanced degeneration when subjected to low-intensity, low-impact exercise. Further, we investigated early global changes in gene expression in articular chondrocytes from degenerating cartilage. Identified candidate genes including genes involved in chemokine, endothelin, and transforming growth factor-alpha signaling are discussed in the context of articular cartilage degeneration in early OA.

Forced mobilization accelerates pathogenesis: characterization of a preclinical surgical model of osteoarthritis.

Preclinical osteoarthritis (OA) models are often employed in studies investigating disease-modifying OA drugs (DMOADs). In this study we present a comprehensive, longitudinal evaluation of OA pathogenesis in a rat model of OA, including histologic and biochemical analyses of articular cartilage degradation and assessment of subchondral bone sclerosis. Male Sprague-Dawley rats underwent joint destabilization surgery by anterior cruciate ligament transection and partial medial meniscectomy. The contralateral joint was evaluated as a secondary treatment, and sham surgery was performed in a separate group of animals (controls). Furthermore, the effects of walking on a rotating cylinder (to force mobilization of the joint) on OA pathogenesis were assessed. Destabilization-induced OA was investigated at several time points up to 20 weeks after surgery using Osteoarthritis Research Society International histopathology scores, in vivo micro-computed tomography (CT) volumetric bone mineral density analysis, and biochemical analysis of type II collagen breakdown using the CTX II biomarker. Expression of hypertrophic chondrocyte markers was also assessed in articular cartilage. Cartilage degradation, subchondral changes, and subchondral bone loss were observed as early as 2 weeks after surgery, with considerable correlation to that seen in human OA. We found excellent correlation between histologic changes and micro-CT analysis of underlying bone, which reflected properties of human OA, and identified additional molecular changes that enhance our understanding of OA pathogenesis. Interestingly, forced mobilization exercise accelerated OA progression. Minor OA activity was also observed in the contralateral joint, including proteoglycan loss. Finally, we observed increased chondrocyte hypertrophy during pathogenesis. We conclude that forced mobilization accelerates OA damage in the destabilized joint. This surgical model of OA with forced mobilization is suitable for longitudinal preclinical studies, and it is well adapted for investigation of both early and late stages of OA. The time course of OA progression can be modulated through the use of forced mobilization.

Regulator of G-protein signaling (RGS) proteins differentially control chondrocyte differentiation.

Control of chondrocyte differentiation is attained, in part, through G-protein signaling, but the functions of the RGS family of genes, well known to control G-protein signaling at the Galpha subunit, have not been studied extensively in chondrogenesis. Recently, we have identified the Rgs2 gene as a regulator of chondrocyte differentiation. Here we extend these studies to additional Rgs genes. We demonstrate that the Rgs4, Rgs5, Rgs7, and Rgs10 genes are differentially regulated during chondrogenic differentiation in vitro and in vivo. To investigate the roles of RGS proteins during cartilage development, we overexpressed RGS4, RGS5, RGS7, and RGS10 in the chondrogenic cell line ATDC5. We found unique and overlapping effects of individual Rgs genes on numerous parameters of chondrocyte differentiation. In particular, RGS5, RGS7, and RGS10 promote and RGS4 inhibits chondrogenic differentiation. The identification of Rgs genes as novel regulators of chondrogenesis will contribute to a better understanding of both normal cartilage development and the etiology of chondrodysplasias and osteoarthritis.

Microarray analyses of gene expression during chondrocyte differentiation identifies novel regulators of hypertrophy.

Ordered chondrocyte differentiation and maturation is required for normal skeletal development, but the intracellular pathways regulating this process remain largely unclear. We used Affymetrix microarrays to examine temporal gene expression patterns during chondrogenic differentiation in a mouse micromass culture system. Robust normalization of the data identified 3300 differentially expressed probe sets, which corresponds to 1772, 481, and 249 probe sets exhibiting minimum 2-, 5-, and 10-fold changes over the time period, respectively. GeneOntology annotations for molecular function show changes in the expression of molecules involved in transcriptional regulation and signal transduction among others. The expression of identified markers was confirmed by RT-PCR, and cluster analysis revealed groups of coexpressed transcripts. One gene that was up-regulated at later stages of chondrocyte differentiation was Rgs2. Overexpression of Rgs2 in the chondrogenic cell line ATDC5 resulted in accelerated hypertrophic differentiation, thus providing functional validation of microarray data. Collectively, these analyses provide novel information on the temporal expression of molecules regulating endochondral bone development.