Expressions Of Lysyl Oxidases And Matrix Metalloproteinases In Synovial Fibroblasts After Cruciate Ligament Injury

Injured knee joint cruciate ligament does not self-heal satisfactorily making it as one of the most challenging and intractable clinical problem today. This raises the necessity to detail the reason why the injured cruciate ligament hardly repair.The damage of cruciate ligament is always accompanied by the injury of synovium. Previous ex vivo studies showed that the synovium has the greatest ability for converting the72kDa pro-MMP-2to the62kDa active-MMP-2and releasing MMP-2into the synovial fluids compared with other intraarticular tissues (such as posterior cruciate ligament (PCL), meniscus and cartilage). Therefore the synovium is considered to be the major regulator of the microenvironment in the joint cavity after cruciate ligament injury. In order to further explore the role of synovium in regulating the microenvironment of joint cavity after cruciate ligament injury, we mimicked the microenvironment of joint cavity after cruciate ligament injury and study the effects of mechanical-chemical factors on expressions of LOXs (Lysyl oxidases, LOXs) and MMPs and MMP-2activity in synovial fibroblasts at the cellular level. The main research and results are as follows:①Combined effects of TNF-a and IL-1β on LOXs and MMPs expressions in synovial fibroblastsProinflammatory factors are considered important chemical mediators in the acute inflammatory phase of wound healing. The results showed that lOng/ml TNF-α alone down-regulated the expressions of LOXs. lOng/ml IL-1β up-regulated the expressions of LOX、LOXL-1and LOXL-3, down-regulated the expressions of LOXL-2and LOXL-4. Both TNF-a and IL-1β increased the expressions of MMP-1,2,3. When the two inflammatory factors were mixed, expressions of LOXs were inhibited differently. However, the combination of TNF-a plus IL-1β caused a greater increase in the expressions of MMP-1,-2, and-3relative to TNF-a and IL-1β alone. Zymography assays showed that the combination of TNF-a and IL-1β increased MMP-2activity in a dose and time dependent manner and the MMP-2activity was much higher than MMP-2activity induced by TNF-α and IL-1β alone. In the presence of proinflammatory factors, the LOXs and MMPs showing a state of imbalanced expressions in synovial fibroblasts were released into synovial fluids and alter the balance of cruciate ligament healing, which may be one of the important reasons why cruciate ligament cannot be repaired. These results suggest that besides self-factors of cruciate ligament, microenvironment in knee joint cavity also affect the cruciate ligament’s healing ability and synovium plays an important role in regulating the microenvironment in the joint cavity and cannot be ignored in the healing process of cruciate ligament.②Effects of TNF-a on expressions of LOXs and MMPs in synovial fibroblasts under injurious mechanical stretchIn the real physiological situation, the stretching force may exert its effect together with other factors to induce the injurious cascade. The results showed that12%injurious stretch down-regulated the expressions of LOXs (except LOXL-2) and up-regulated the expressions of MMP-1,2,3. As above lOng/ml TNF-α,5ng/ml TNF-α also down-regulated the expressions of LOXs and up-regulated the expressions of MMP-1,2,3. In the presence of TNF-a, the12%injurious stretch-induced up-regulation of LOXs at1,2and3h was significantly inhibited and decreased further below control after6h. In contrast, injurious stretch had a synergistic effect with TNF-a on increasing expressions of MMP-1,2,3and MMP-2activity. In addition, in vitro wound healing assay revealed that TNF-a inhibited the migration of synovial fibroblasts. Under the stimulation of injurious stretch and TNF-a, the LOXs and MMPs of imbalance in synovial fibroblasts released into synovial fluids and caused the change of microenvironment in knee joint cavity which would promoted the ECM degradation of cruciate ligament and inhibit the ligament repair. These results suggest that besides self-factors of cruciate ligament, microenvironment in knee joint cavity also affect the cruciate ligament’s healing ability. In addition, these results further indicate that synovial fibroblasts are very sensitive to mechanical injury and proinflammatory factors and plays an important role in regulating the microenvironment in the joint cavity and cannot be ignored in the healing process of cruciate ligament.③Combined effects of mechanical stretch and TGF-β1on LOXs and MMPs expressions in synovial fibroblastsIn the inflammatory phase of ligament healing, not only proinflammatory factors (such as TNF-a and IL-1β) but also growth factors (such as TGF-β1) participate in the ligament repair. The results showed that injurious stretch down-regulated the expressions of LOXs (except LOXL-2), however, TGF-β1up-regulated the expressions of LOXs. Both injurious stretch and TGF-β1alone increased the expressions of MMP-1, 2,3. When injurious stretch and TGF-β1were mixed, expressions of LOXs and MMP-1,2,3were up-regulated and the elevated level of MMP-1,2,3was higher than LOXs. In addition, the combination of injurious stretch and TGF-β1increased MMP-2activity in a time dependent manner and the MMP-2activity was much higher than MMP-2activity induced by injurious stretch alone. Under the stimulation of injurious stretch and TGF-β1the much higher expressions of MMPs in synovial fibroblasts compared to LOXs promoted the ECM degradation of cruciate ligament, which would inhibit the ligament repair. These results suggest that change of microenvironment in knee joint cavity after cruciate ligament injury affects the cruciate ligament’s healing ability. The synovial fibroblasts are very sensitive to mechanical injury and growth factors, and further indicate that synovium plays an important role in regulating the microenvironment in the joint cavity after cruciate ligament injury must be considered seriously.④NF-κB pathway is involved in the injurious stretch and TGF-β1induction of MMP-2in human synovial fibroblastsThe signal pathways underlining the injurious stretch and TGF-β1-induced MMP-2activity in synovial fibroblasts were investigated through signal pathway inhibitor. These results suggest that the induced MMP-2by injurious stretch and TGF-β1was inhibited significantly due to the addition of NF-κB pathway inhibitor Bay11-7082and Bay11-7085.⑤In order to mimicked the microenvironment of joint cavity more really, we established coculture system and investigated the direct regulation of synovial fibroblasts on PCL fibroblasts. The results showed that coculture promoted the expressions of LOXs in PCL fibroblasts compared mono-culture.These results suggest that synovial fibroblasts and PCL fibroblasts do exist interaction and crosstalk, and further indicate that synovium participates in the healing process of cruciate ligament as microenvironment regulator in the joint cavity.In summary, these results suggest that the change of microenvironment in knee joint cavity after cruciate ligament injury may be one of the important reasons why cruciate ligament cannot be repaired and synovial fibroblasts participate in the healing process of cruciate ligament as microenvironment regulator. Therefore, improving the microenvironment in the joint cavity after cruciate ligament injury through regulating LOXs and MMPs expressions in synovial fibroblasts would has important significance for cruciate ligament repair.