C. Thomas G. Appleton

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.

Rho/ROCK and MEK/ERK activation by transforming growth factor- α 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-α (TGFα) is upregulated by articular chondrocytes in experimentally induced and human OA. To test the potential involvement of TGFα, which is an activator of epidermal growth factor receptor (EGFR) signaling, in joint degeneration and to identify signaling mechanisms mediating articular chondrocyte responses to TGFα, rat chondrocytes and osteochondral explants were treated with TGFα and various inhibitors of intracellular signaling pathways. Stimulation of EGFR signaling in articular chondrocytes by TGFα 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 TGFα, but inhibition of only Rho or ROCK activation prevented morphological changes. TGFα 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 TGFα was prevented by ROCK and p38 MAPK inhibition, including matrix metalloproteinase-13 and tumor necrosis factor-α, which are well known to contribute to cartilage digestion in OA. To assess the ability of TGFα to stimulate degradation of mature articular cartilage, type II collagen and aggrecan cleavage fragments were analyzed in rat osteochondral explants exposed to exogenous TGFα. Normal articular cartilage contained low levels of both cleavage fragments, but high levels were observed in the cartilage treated with TGFα. Selective inhibition of MEK/ERK and Rho/ROCK activation greatly reduced or completely prevented excess type II collagen and aggrecan degradation in response to TGFα. These data suggest that TGFα is a strong stimulator of cartilage degradation and that Rho/ROCK and MEK/ERK signaling have critical roles in mediating these effects.

F-spondin, a neuroregulatory protein, is up-regulated in osteoarthritis and regulates cartilage metabolism via TGF-β 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 (‐7‐fold) expression of F‐spondin, a neuronal extracellular ma‐trix glycoprotein, in human OA cartilage (P<0.005). OA‐specific up‐regulation of F‐spondin was also dem‐onstrated in rat knee cartilage following surgical meni‐sectomy. F‐spondin treatment of OA cartilage explants caused a 2‐fold increase in levels of the active form of TGF‐β1(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‐β and the throm‐bospondin 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 (~20%;P<0.05), which were both TGF‐β 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 OAcartilage, where it may act in situ at lesional areas to activate latent TGF‐β and induce cartilage degradation via pathways that involve production of PGE2.—Attur, M. G., Palmer, G. D., Al‐Mussawir, H. E., Dave, M., Teixeira, C. C., Rifkin, D. B., Appleton, C. T. G., Beier, F., Abramson, S. B. F‐spondin, a neuroregulatory protein, is up‐regulated in osteoarthritis and regulates cartilage metabolism via TGF‐β activation. FASEB J. 23, 79‐89 (2009)

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-α 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-α in chondrocytes.

Molecular and Histological Analysis of a New Rat Model of Experimental Knee Osteoarthritis

Abstract:  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‐α signaling are discussed in the context of articular cartilage degeneration in early OA.

ADAMTS-7 forms a positive feedback loop with TNF-α in the pathogenesis of osteoarthritis

Objective To examine the expression of ADAMTS-7 during the progression of osteoarthritis (OA), defining its role in the pathogenesis of OA, and elucidating the molecular events involved. Methods ADAMTS-7 expression in cartilage of a rat OA model was assayed using immunohistochemistry. Cartilage-specific ADAMTS-7 transgenic mice and ADAMTS-7 small interfering (si)RNA knockdown mice were generated and used to analyse OA progression in both spontaneous and surgically induced OA models. Cartilage degradation and OA was evaluated using Safranin-O staining, immunohistochemistry, ELISA and western blotting. In addition, mRNA expression of tumour necrosis factor (TNF)-α and metalloproteinases known to be involved in cartilage degeneration in OA was analysed. Furthermore, the transactivation of ADAMTS-7 by TNF-α and its downstream NF-κB signalling was measured using reporter gene assay. Results ADAMTS-7 expression was elevated during disease progression in the surgically induced rat OA model. Targeted overexpression of ADAMTS-7 in chondrocytes led to chondrodysplasia characterised by short-limbed dwarfism and a delay in endochondral ossification in ‘young mice’ and a spontaneous OA-like phenotype in ‘aged’ mice. In addition, overexpression of ADAMTS-7 led to exaggerated breakdown of cartilage and accelerated OA progression, while knockdown of ADAMTS-7 attenuated degradation of cartilage matrix and protected against OA development, in surgically induced OA models. ADAMTS-7 upregulated TNF-α and metalloproteinases associated with OA; in addition, TNF-α induced ADAMTS-7 through NF-κB signalling. Conclusions ADAMTS-7 and TNF-α form a positive feedback loop in the regulation of cartilage degradation and OA progression, making them potential molecular targets for prevention and treatment of joint degenerative diseases, including OA.

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 Gα 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.

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

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.

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.

‘Weighing in' on Framingham OA: Measuring biomechanical and metabolic contributions to osteoarthritis

Thirty years ago, Altman et al told us that osteoarthritis (OA) is not a single disease (1). That 1986 description of OA as “a heterogeneous group of conditions that lead to joint symptoms and signs. . .” remains true today. But the simple recognition of OA as a group of related but distinct joint disorders among clinicians and researchers is hampered by the lack of a clearly accepted set of criteria to distinguish independent clinical OA phenotypes. Moreover, the description of these clinical OA phenotypes in molecular, anatomic, and physiologic domains remains a formidable, yet fundamental task before us in the field of OA research. Notwithstanding, the blanket term “OA” should no longer be used in isolation to describe the typical joint pathology and symptoms of the most common form of arthritis in humans. An effort should be made in all OA cases to apply accompanying adjectives to at least describe the context in which the joint disease arose. Candidate clinical phenotypes include OA related to joint trauma (posttraumatic OA), advanced age at disease onset (agerelated/senescent OA), strong family history (inherited/ genetic OA), pain sensitization, inflammatory features, and metabolic syndrome (metabolic OA) (2). Given that ;25% of the world’s adult population develops metabolic syndrome (3), the association of metabolic syndrome with OA is especially alarming. Metabolic syndrome consists of 4 core features, variably defined, including hypertension, atherogenic dyslipidemias, visceral obesity, and insulin resistance. The most recent metabolic syndrome definitions from the US National Cholesterol Education Program Adult Treatment Panel III and the International Diabetes Federation were presented in 2005. Regardless of the definition of metabolic syndrome, a clear link between metabolic syndrome and OA has been established in many different studies. Analyses of Third National Health and Nutrition Examination Survey (NHANES-III) data show that metabolic syndrome prevalence is higher among people with OA than those without OA (59% versus 23%, respectively) and that this form of OA occurs in younger age groups (ages 45–65 years) than age-related OA (4). The individual components of metabolic syndrome are also associated with excess OA risk. For example, in the Japanese Research on Osteoarthritis Against Disability (ROAD) study, the risk of OA increased with each additional component of metabolic syndrome (5), although that was a cross-sectional analysis without adjustment for body mass index (BMI). The nature of the interaction between metabolic syndrome and OA remains unresolved. It is unclear whether the most important link is due to an influence of OA on metabolic syndrome (e.g., decreased mobility due to OA leads to obesity and therefore metabolic syndrome), vice versa (abnormal joint loading—with or without metabolic derangement—fuels OA pathophysiology), or if a common set of risk factors exist which drive both conditions in parallel. A shared etiology in the latter case would suggest that metabolic OA is an underrecognized fifth (or sixth) feature of metabolic syndrome rather than a separate condition per se, as some have suggested (6). As is often the case, the answer may lie in a combination of these possibilities. But the reliance on prevalence data and crosssectional analyses in most OA/metabolic syndrome studies C. Thomas Appleton, MD, PhD, FRCPC, Janet E. Pope, MD, MPH, FRCPC: Western University and St. Joseph’s Health Care, London, Ontario, Canada; Gillian A. Hawker, MD, MSc, FRCPC: University of Toronto, Women’s College Research Institute, Women’s College Hospital, and Institute for Clinical Evaluative Sciences, Toronto, Ontario, Canada; Catherine L. Hill, MBBS, MD, MSc, FRACP: The Queen Elizabeth Hospital, Woodville, South Australia, Australia, and the Health Observatory, University of Adelaide, Adelaide, South Australia, Australia. Address correspondence to C. Thomas Appleton, MD, PhD, FRCPC, St. Joseph’s Health Care London, 268 Grosvenor Street, London, Ontario N6A 4V2, Canada. E-mail: tom.appleton@sjhc.london.on.ca. Submitted for publication January 14, 2017; accepted in revised form March 2, 2017.