Differential Expressions Of Lysyl Oxidase Family And Matrix Metalloproteinases In ACL And MCL Fibroblasts After Mechanical Injury

The anterior cruciate ligament （ACL） and medial collateral ligament （MCL） aretwo commonly injured areas of knee joints. In general, the MCL has the capacity toself-heal and restore the joint function of a completely ruptured mid-substance withinone month, and in most cases, without the need for surgery. On the other hand, the ACLdoes not heal satisfactorily, even if the surgical repair is attempted, and ACL injuriesoften lead to knee instability, pain, and even progressive degeneration of other jointtissues and Osteoarthritis （OA）.During lgament healing process, a fine balance between the synthesis ofextracellular matrix （ECM） and its degradation by a large family of enzymes, known asmatrix metalloproteinases （MMPs） of which is well known that the most important roleis that they could cleave one or more of the components of the extracellular matrix ofligaments, is required to maintain the structural integrity of healing tissue; Meanwhile,more and more evidences have inferred the relationship between lysyl oxidase family（LOXs） of which has the capability of oxidizing peptidyl lysine to peptidyl aldehyderesidues within collagen and elastin, thus initiating formation of the covalentcross-linkages that insolubilize these extracellular proteins, and facilitates the formationand repair of extracellular matrix （ECM）, and wound healing. The role of each familymember towards ECM cross-linkage has been consecutively identified. However, thevariations in expression of this LOX family in the normal and injured anterior cruciateligament （ACL） are not fully known.Inflammatory phase accompany with the ligament injury. In the inflammatoryphase, many factors such as transforming growth factor-beta1（TGF-β₁）, tumor necrosisfactor alpha （TNF-α） and interleukin-1beta （IL-1β） involved in mediating the healingprocess. We detected differential expressions of lysyl oxidases and matrixmetalloproteinases in injured ACL-MCL model in vitro. The main research and resultswere as follows:Firstly, we used equi-biaxial stretch system to mimic ligament injury in vivo anddetected the lysyl oxidases expressions in injured ACL and MCL （6%stretchrepresentative of physiological state and12%stretch representative of injurious state）.The results had shown that mechanical stretch up-regulated the expressions of LOXs,moreover,12%stretch-induced up-regulations of LOXs were much higher than6% stretch. In physiological state, LOX and LOXL-4were expressed higher in ACL thanthese in MCL fibroblasts, while LOXL-3in MCL was higher compared with in ACL,the other two members showed fluctuated. In injurious state, LOXs in MCL were moreprominent than those in ACL fibroblasts.Secondly, we detected the differential expressions of lysyl oxidases in normal ACLand MCL fibroblasts induced by TGF-β₁, TNF-α and IL-1β. The results had shown thatboth TGF-β₁and IL-1β could increase the expressions of lysyl oxidases in normal ACLand MCL fibroblasts, and the two factors enhaced LOXs in MCL were much higherthan in ACL. The TGF-β₁-induced-increases of LOXs showed persistent in both ACLand MCL, while IL-1β-induced-increases of LOXs showed a dose-dependent increase.TNF-α down-regulated expressions of lysyl oxidases, furthermore, decreases of LOXsin ACL were more prominent in ACL than in MCL fibroblasts.Thirdly, we detected the differential expressions of lysyl oxidases in ACL andMCL fibroblasts after12%stretch injury induced by TGF-β₁, TNF-α and IL-1β. Wefound that TGF-β₁and IL-1β enhaced different expressions of LOXs in both ACL andMCL after12%stretch injury, moreover, the effects of TGF-β₁on injured fibroblastswere much stronger than IL-1β. LOXL-1,3and4induced by TGF-β₁were much higherin injured fibroblasts than normal cells, LOX and LOXL-2relatively lower in injuredfibroblast than normal ones. IL-1β induced much higher expressions of LOXs in normalACL compared with the injured cells, while in MCL, IL-1β induced much higherexpressions of LOXs in injured MCL than normal cells. TNF-α induced value peaks inLOXs （except LOXL-2） at2hour after treatments, and then immediatelydown-regulated blew non-treated normal controls within24hours. The expression ofTNF-α-induced LOXL-2in injured fibroblasts was constantly down-regulated, but thedown-regulation was somewhat weaker than TNF-α-induced LOXL-2in normal cells.Fourthly, based on the previous data in our lab, we detected the expressions ofMMPs in ACL and MCL after12%stretch injury induced by TGF-β₁, TNF-α and IL-1β.We found these three factors all increased the expressions of MMP-1,2,3, and12inboth ACL and MCL fibroblasts. Inflammatory factors TNF-α and IL-1β bothup-regulated gene expressions of MMPs, moreover, the effect of TNF-α was strongerthan IL-1β. But protein expression of MMP-2by zymography showed the active formof MMP-2was much stronger induced by IL-1β than TNF-α. TGF-β₁also up-regulatedMMP-1,2,3, and12in both ACL and MCL, but TGF-β₁-induced-MMPs in ACL werehigher compared with in MCL. Fifthly, to mimic micro-envirenment, we established coculture system by transwellbwtween ACL fibroblasts and synovial cells. We found that coculture promoted MMP-2expressions. But coculture-induced increase of MMP-2was somewhat lower comparedwith high expression of MMP-2in injured ACL. We combined12%injury stretch andcoculture, found the expression of MMP-2was higher than single coculture or singleinjury.Finally, to resovle the high degradation induced by high expression of MMP-2ininjured ACL, we used signal pathway inhibitors aiming to decrease the high expressionof MMP-2induced by injury. Under coculutre system, we found all6inhibitors in theexperiments could reduce MMP-2expression, especially Bay11-7082, inhibitor ofNF-kappa B, and Curcumin, inhibitor of AP-1, which almostly inhibited the active formof MMP-2, and restore parts of the pro-form.In summary, we comfirmed that TGF-β₁, TNF-α, and IL-1β could inducedrelatively lower expressions of LOXs in normal and injured ACL fibroblasts comparedwith MCL, which mean to count against the repair of injured ligament. We inferred thatrelatively low expressions of LOXs might be on of the reseans why ACL could not selfheal. On the other hand, the three factors respectively promoted high MMPs in injuredACL compared with MCL fibroblasts, which mean to trend to degradation. We inferredthat high expressions of MMPs might be another reason why injured ACL had poorself-healing.The results of LOXs expressions in normal/injured ACL and MCL fibroblastsmight provide the basal data for cure of ligament injury in clinical trial. To enhance theexpressions of LOXs in injured ACL might provide a novel view for ACL healing;meanwhile, to reduce the expressions of MMPs in injured ACL by inhibitors undermimicked micro-envirenment might provide another therapeutial potential of injuredACL ligament.