Regulation Of Osteochondral Remodeling By Hypertrophic Chondrocyte-Derived Factors During Osteoarthritis

Purpose: Osteochondral angiogenesis is an important step in the pathological remodeling of the cartilage-bone interface in osteoarthritis (OA) and could be a crucial driving factor of OA progression. The identification of factors responsible for osteochondral angiogenesis in OA is challenging, as it could be a potential therapeutic target. We hypothesize that the hypertrophic differentiation of chondrocytes within OA cartilage drives osteochondral angiogenesis, as it occurs in the endochondral ossification process during development and growth. Thus, chondrocyte phenotypic changes in the deep layer of cartilage, among which the differentiation of prehypertrophic chondrocytes into hypertrophic chondrocytes could be associated with a switch of the angiogenic balance, allowing vascular channels to penetrate the articular cartilage. The aim of this study was to investigate the biological activity of hypertrophic chondrocytes in the pathological remodeling of the osteochondral junction. Methods: After destabilization of medial meniscus (DMM), 10 weeks-old Lewis rats were injected intraperitoneally with an antiangiogenic molecule (1/week during 8 weeks). The effect of the molecule on cartilage integrity and osteophyte formation was assessed by histology (Safranin-O/Fast Green staining and OARSI score).The expression of angiogenic and angiostatic factors by articular, prehypertrophic and hypertrophic chondrocytes was determined by RT-qPCR, ELISA, proteome array and immunoblotting in a model of progressive hypertrophic differentiation of murine immature articular chondrocytes (iMACs), we recently developed (van Eegher et al., Osteoarthritis & Cartilage, In press).Angiogenesis functional assays were set up in order to evaluate chondrocyte activity. Proliferation assay of human endothelial cells (HUVECs: Human Umbilical Vein Endothelial Cells) in response to prehypertrophic and hypertrophic chondrocyte conditioned media was determined by EdU incorporation. Tube formation assay on Matrigel was performed and total network length and junction number were analysed.Adherence capacity of fluorescent HUVECs on prehypertrophic and hypertrophic extracellular matrices (ECM) was assessed by spectrophotometry. Results: A high OARSI score (20/24) was observed in control DMM rats 8-weeks post-surgery. Cartilage degradation was associated with osteophyte formation and subchondral bone remodeling. The IP injected angiostatic molecule limited OA development (OARSI score on tibial plateau 12/24, p= 0.0144, n=7). The integrity of articular cartilage was preserved, osteophytes were less developed and no subchondral bone remodeling events were observed.The mRNA expression of chondrocyte markers (Sox9, Aggrecan, Col2A1) drastically decreased with hypertrophic differentiation, whereas that of hypertrophic markers (RunX2, Osteocalcin, Col10) was significantly increased. In addition, hypertrophic chondrocytes showed strong matrix calcifications. Prehypertrophic chondrocytes expressed intermediate levels of iMAC and hypertrophic chondrocyte markers and also expressed markers of prehypertrophic chondrocytes, including Snorc and Osteomodulin. Differentiation of prehypertrophic into hypertrophic chondrocytes was associated with an increased mRNA and protein expression of angiogenic factors, including bFGF, VEGF-A, VEGF-C and CXCL-12. In contrast, a decreased mRNA expression of the angiostatic factors CHM-1, ANGPTL4 and TSP-1 was observed.As cartilage is an avascular tissue, it is known to have angiostatic properties. Interestingly, prehypertrophic chondrocytes displayed more angiostatic activity than hypertrophic ones (p=0.0311, n=7) on HUVECs. In addition, HUVECs were more prone to adhere to calcified ECM of hypertrophic chondrocytes than to ECM of prehypertrophic chondrocytes (p=0.0434, n=8). Hypertrophic chondrocyte conditioned media displayed a stronger cell proliferation activity on HUVECs than prehypertrophic chondrocyte conditioned media (p=0.0195, n=10). Consequently, conditioned media from hypertrophic chondrocytes induced more capillary-like structures of HUVECs on Matrigel than prehypertrophic chondrocyte conditioned media. Total network length and total junction number were 2-fold higher after hypertrophic chondrocyte conditioned media stimulation (p=0.0313, n=5) than prehypertrophic stimulation. This effect was already observed at 2h of experiment (p=0.0313, n=5). Conclusions: These findings suggest that the differentiation of prehypertrophic to hypertrophic chondrocytes in the deeper layer of OA cartilage, close to the vascularized subchondral bone, is a crucial step in the osteochondral angiogenesis in OA. Hypertrophic chondrocytes have angiogenic properties, promoting endothelial adherence to their matrix, endothelial cell proliferation and tube formation. Targeting osteochondral angiogenesis could be a promising therapeutical strategy to prevent OA progression.