Articular Chondrocyte MTORC1 Promotes Cartilage Angiogenesis And Osteoarthritis

BackgroundOsteoarthritis(OA)is a common joint disease,mainly afflicting the weight-bearing joints.Particularly,OA is characterized by a progressive degradation of articular cartilage,joint pain and functional impairment.But,more and more data showed that cartilage angiogenesis played an important role in OA development.However,the exact mechanism underlying the potential contributions of cartilage angiogenesis to articular cartilage degeneration during OA progression is largely unknown..ObjectiveWe investigate the changes of H-vessel,and the relationship between the H-vessel and OA development,by chondrocyte-specific Tscl CKO mice.Further,we aim to clarify the type,source and function of blood vessels during OA development.Materials and Methods2-month-old male C57BL/6 mice(wild type;WT),and Tsc1KO mice DMM-operated,treated with saline,or DMM-operated,treated with bevacizumab.Immunostaining and western blot analyses were conducted to detect relative protein.Primary chondrocytes and ADTC5 cells were investigated the role of VEGF-A in chondrocyte changes and were performed to induce vascular formation.Results1.The H-vesselexpression increases in subchondral bone during.The H-type vessel in OA mice dramatically changed relative to no surgery controls by performing double immunofluorescence staining for CD31 and endomucin(Emcn).H-type vessels were obviously increased in the subchondral bone of OA mice.Moreover,proteoglycan loss and cartilage degeneration were observed in OA mice.Interestingly,the abundance of H-type vessels was strongly increased in subchondral bone from aged animals,which were nore prone to osteoarthritis.2.Enhanced articular chondrocyte VEGF-A production stimulates vascular formation during OA.Strong VEGF-A immunostaining was seen in OA chondrocytes.In vitro,VEGF was increased in supernatant of OA primary chondrocyte by ELISA assay.Moreover,the supernatant of OA chondrocyte promoted angiogenesis in HUVEC tube forlation.3.Articular chondrocyte mTORC1 stimulates cartilage angiogenesis and promotes O A development.H-type vessels were obviously increased in the subchondral bone of Tsc1 CKO mice,and were obviously decreased in contro1 mice and Tsc1 CKO Rapa mice.Proteoglycan loss and cartilage degeneration was observed in Tsc1 CKO mice,but was no change in control mice and Tsc1 CKO Rapa mice.Then,we construct a DMM model of OA for 8-week-old male control and Tsc1 CKO mice and Raptor CKOER mice,and the mTORCl expression(indicated by p-S6(S235/236))in articular chondrocytes was analyzed by iununohistochemistry.Similarly,immunofluorescence analysis at 5 weeks post-OA surgery,H-type vessels were obviously increased in the subchondral bone of Tsc1 CKO mice,and were obviously deereased in control mice and Raptor CKOER mice.Tsc1 CKO mice showed obviously loss of proteoglycans and destruction in the articular cartilage at 5 weeks post-OA surgery,and Raptor CKOER mice showed less destruction and greater proteoglycans.4.mTORC1 activation enhances articular chondrocyte VEGF production and stimulate angiogenesis in vitro.Strong VEGF-Aimmunostaining was seen in Tsc1 CKO mice chondrocytes at 8 weeks old,and weak VEGF-A immunostaining was seen in control mice and Tsc1 CKO Rapa mice chondrocytes.In vitro,the supernatant of primary chondrocyte from Tsc1 CKO mice showed nrore egression of VEGF-A than control mice,and whereas no difference was noted in Tsc1 CKO Rapa mice versus control mice.Further,Tsc1 CKO mice increased significantly angiogenesis and Tsc1 CKO Rapa mice restored angiogenesis similar to control mice.Similarly,treatment with Tsc1 siRNA showed more expression of VEGF-A,but not any statistically significant difference in control,Tscl siRNA and treatment with rapamycin.5.Articular chondrocyte mTORC1-stimulated cartilage angiogenesis is important for OA development.Bevacizumab reduced H-type vessel number relative to saline treatment in Tsc1 CKO mice,retaining vessel number similar to control mice in post-surgery 5 weeks.Moreover,Bevacizumab reduced proteoglycan loss and cartilage degeneration in Tsc1 CKO mice.Similarly,some Tsc1 CKO mice after induction of OA were treated with local intra-articular injections of bevacizumab for 5 weeks and some were treated with local intra-articular injections of saline for 5 weeks.Bevacizumab reduced H-type vessel number relative to saline treatment in Tscl CKO mice after induction of OA,retaining vessel number similar to control mice after induction of OA.(fig5e,f)And bevacizumab reduced proteoglycan loss and cartilage degeneration in Tscl CKO mice after induction of OA.6.Increased the nutrition upregulates mTORC1 activation in articular cartilage.Whether ATDC5 or primary chondrocytes,the expression of p-S6(S235/236)increased with the enhanced time of nutrient treatment in lh.Western blotting analysis of articular cartilage showed that p-S6 reduced significantly in OA mice treated with bevacizumab,and weak p-S6 immunostaining was seen in OA mice treated with bevacizumab..7.Inhibition of cartilage angiogenesis delays OA development.Bevacizumab reduced proteoglycan loss and cartilage degeneration in OA mice and reduced H-type vessel number.Indeed,bevacizumanb increased autophagy(immunofluorescence staining of LC3B)in OA mice.As expected,weak immunofluorescence staining of MMP13,ColX,Runx2 and less apoptosis were seen in OA mice treated with bevacizumab.These results showed that enhanced angiogenesis can active mTORC1 signaling and promote OA development.Conclusions1.The H-vessel expression increases in subchondral bone during OA development,and H-vessel shows an increasing trend in subchondral bone during aging.2.Up-regulated articular chondrocyte mTORC1 stimulates H-vessel in subchondral bone and aggravates OA development,wheras down-regulated articular chondrocyte mTORC1 reduces H-vessel in subchondral bone and alleviate OA development.3.mTORCl positively regulaties the expression of VEGF in articular chondrocytes,and plays an imortant role in the regulation of H-vessel in subchondral bone.4.Inhibition the H-vessel in subchondral bone,reducing nutrition,down-regulates the articular chondrocyte mTORC1 activity,and delays OA development.Articular chondrocyteTo sum up,mTORC1 can regulate H-type of blood vessels in subchondral bone through VEGF-A.Moreover,the abundance of H-type vessels may promote OA progression by activating of articular chondrocyte mTORC1 signalling.