Chondromodulin-1 ameliorates osteoarthritis progression in vitro and in vivo

Abstract

Purpose: Articular cartilage is an avascular tissue by nature, in which resident chondrocytes maintain a stable phenotype that is resistant to hypertrophy and angiogenesis throughout life. Hypoxia is known to stabilize hypoxia-inducible factor-α (mainly HIF-1α and HIF-2α) and initiate an angiogenic signalling cascade. However, normal cartilage maintains avascularity under hypoxic conditions; and the abrogation of avascularity in cartilage is related to several joint diseases such as osteoarthritis (OA), indicating the important role of anti-angiogenic factors in normal cartilage homeostasis. Chondromodulin-1 (ChM-1) is an anti-angiogenic protein endogenously expressed in cartilage and has recently been shown to stabilize the chondrocyte phenotype during cartilage tissue repair via an unknown mechanism. These findings suggested that anti-angiogenic factors had a critical role in maintaining the physiological functions of chondrocytes and prevent hypertrophy. Our aims in the present study were to clarify the role of ChM-1 during chondrocyte maturation, OA development and therapy, in an animal model, as well as to explore the mechanistic pathways of ChM-1 with a focus of the regulation of the HIF pathways. Methods: ChM-1 expression during chondrocyte maturation was investigated in chondrogenic culture conditions in cells derived from cartilage and bone marrow. Lentivirus vectors carrying ChM-1 complementary DNA (LV-ChM-1) and ChM-1 siRNA were constructed to respectively over-express and down-regulate ChM-1. Human OA cartilage samples were collected and subjected to gene profiling of angiogenic and anti-angiogenic cytokines using Human Angiogenesis PCR Arrays (Qiagen, Australia). Tumor necrosis factor alpha (TNFα) was applied to mimic the inflammatory environment in OA. Articular cartilage cells (ACCs) over-expressing or under-expressing ChM-1 were exposed to TNFα, and the chondrogenic and hypertrophic markers (RUNX2, COL10, MMP13, ALP and VEGF) were assayed by qRT-PCR and western blot. To evaluate the effect of ChM-1 in OA development, an OA model was created in rats by surgical sectioning of the meniscus. For the mechanistic study, intracellular protein levels, nuclear accumulation and transcriptional activity of HIF-2α with ChM overexpression were evaluated by western blot and Chromatin-Immunoprecipitation (ChIP) assay, respectively. Results: Compared with healthy cartilage, the expression of angiogenic factors was significantly upregulated and anti-angiogenic factors were suppressed in OA cartilage (Figure 1A). ChM-1 expression was strongly correlated with chondrogenesis in cells derived from both cartilage and bone marrow. In OA cartilage, the Angiogenesis PCR Array revealed decreased gene expression in the majority of anti-angiogenic factors, whereas the gene expression of angiogenic-related cytokines increased (Figure 1B). ACCs over-expressing ChM-1 appeared to protect chondrocytes from TNFα induced hypertrophy, and was commensurate with decreased gene and protein level of MMP13, COL10 and VEGF; whereas siRNA-decreased ChM-1 levels resulted in the loss of chondrogenic marker expression and chondrocytes undergoing hypertrophy and becoming more sensitive to inflammatory cytokines. LV-ChM-1 induced over-expression of ChM-1 in the rat model resulted in delayed OA development. It was noted that ChM-1 delayed HIF-2α nuclear translocation at an early time-point and decreased the transcriptional activity of HIF-2α on VEGF and MMP13. Conclusions: These findings demonstrate that ChM-1 is a potent anti-angiogenic factor that is essential to maintain the chondrocyte phenotype and prevent these cells from undergoing hypertrophy during OA development by inhibition of the HIF-2α mediated angiogenic pathway. Thus, this study shows that anti-angiogenic factors such as ChM-1 are essential for the maintenance of chondrocyte homeostasis, prevent hypoxia induced angiogenesis in cartilage, and protect chondrocyte from inflammatory cytokines induced hypotrophy.