REGULATION OF CELL MIGRATION IN VASCULAR REMODELING

血管的致病性重塑通常涉及平滑肌增殖和迁移。如在细胞运动的其他情况下，平滑肌细胞迁移到新生内膜中涉及由细胞表面受体和表面相关蛋白酶调节的一系列调节的粘附和去粘附事件。基质金属蛋白酶（MMP），明胶酶A，通过使细胞克服基质屏障，并在迁移过程中调节细胞-基质粘附，在这一过程中起着重要作用。它通过与其特异性激活剂MT 1-MMP的相互作用来实现这一点，MT 1-MMP是一种膜相关蛋白酶，其在涉及与细胞表面蛋白的多重相互作用的复杂过程中启动明胶酶A前肽的切割。一种这样的相互作用涉及MT 1-MMP本身，其通过与MMP抑制剂TIMP-2（明胶酶A活化的关键介质）的桥结合明胶酶A。TIMP-2结合MT 1-MMP的活性位点和明胶酶A的羧基结构域，形成三链体，其将明胶酶A定位为适于随后被另一MT 1-MMP分子活化。明胶酶A的羧基结构域还结合整合素α v β 3，其也在活化过程中起作用。最后，细胞将TIMP-2结合到其他未知位点，这些位点也可能是明胶酶A活化的关键因素。有趣的是，成纤维细胞和平滑肌细胞通过在独立于新蛋白质合成的过程中激活和结合明胶酶A来快速响应细胞形状的变化，这表明激活机制的组分存在于细胞上，但阻止适当的相互作用。在这项研究中，TIMP-2在细胞结合和激活明胶酶A的作用将被探索。首先，将探索TIMP-2在细胞结合和明胶酶A活化中的作用的生化基础。首先，TIMP-2对MT 1-MMP的特异性的生物化学基础将使用嵌合TIMP分子来推导，其中TIMP-2序列将被替换为TIMP-2内的位点，TIMP-2是一种高度同源的抑制剂，实际上对MT 1-MMP没有特异性。Net，将表征TIMP-2的非MMP细胞表面结合位点，并确定TIMP-2内赋予这种结合的关键氨基酸序列。最后，将模拟TIMP-2对明胶酶A结合和活化的贡献。了解这一机制将是重要的，在定义正常和疾病过程中血管重塑过程中发生的生化事件。

The pathogenic remodeling of blood vessels often involves smooth muscle proliferation and migration. As in other instances of cell movement, smooth muscle cell migration into the neointima involves a regulated series of adhesion and de-adhesion events regulated by cell-surface receptors and surface-associated proteases. The matrix metalloproteinase (MMP), gelatinase A, plays a significant role in this process by enabling cells to overcome matrix barriers, and also regulates cell-matrix adhesion during migration. It does this through its interaction with its specific activator, MT1-MMP, a membrane-associated proteinase that initiates cleavage of the gelatinase A pro-peptide in a complex process involving multiple interactions with cell-surface proteins. One such interaction involves MT1-MMP itself, which bindings gelatinase A through a bridge with the MMP inhibitor, TIMP-2, a critical mediator of gelatinase A activation. TIMP-2 binds the active site of MT1-MMP and the carboxyl domain of gelatinase A, forming a triplex which positions gelatinase A appropriate for subsequent activation by another MT1-MMP molecule. The carboxyl domain of gelatinase A also binds the integrin, alphavbeta3, which also plays a role in the activation process. Finally, cell bind TIMP-2 to other, as yet unknown sites which may also be key elements of gelatinase A activation. Interestingly, fibroblasts and smooth muscle cells rapidly respond to changes in cell shape by activating and binding gelatinase A in a process independent of new protein synthesis, suggesting that the components of the activation mechanism are present on cells but preventing from interacting appropriately. In this study, the role of TIMP-2 in both the cell binding and activation of gelatinase A will be explored. First, the biochemical basis for the role of TIMP-2 in both the cell binding and activation of gelatinase A will be explored. First, the biochemical basis for TIMP-2's specificity for MT1-MMP will be deduced using chimeric TIMP molecules in which TIMP-2 sequence will be exchanged for sites within TIMP-2, a highly homologous inhibitor with virtually no specificity for MT1-MMP. Net, the non-MMP cell-surface binding sites for TIMP-2 will be characterized, and the critical amino acid sequences within TIMP-2 that confer this binding will be determined. Finally, the contribution of TIMP-2 to gelatinase A binding and activation will be modeled. Understanding this mechanism will be important in defining the biochemical events that occur during vascular remodeling in both normal and disease processes.