REGULATION OF CELL MIGRATION IN VASCULAR REMODELING

血管的病理性重塑往往涉及到平滑肌的增殖和迁移。与细胞运动的其他情况一样，平滑肌细胞向新生内膜的迁移涉及一系列受细胞表面受体和表面相关蛋白酶调控的黏附和去黏附事件。基质金属蛋白酶，明胶酶A，在这一过程中起着重要的作用，使细胞能够克服基质屏障，并在迁移过程中调节细胞与基质的黏附。它通过与其特定的激活剂MT1-MMP相互作用来实现这一点，MT1-MMP是一种膜相关蛋白酶，在涉及与细胞表面蛋白多次相互作用的复杂过程中启动明胶酶A前肽的裂解。一种这样的相互作用涉及MT1-基质金属蛋白酶本身，它通过与基质金属蛋白酶抑制剂TIMP-2的桥梁结合明胶酶A，TIMP-2是明胶酶A激活的关键介质。TIMP-2结合MT1-MMP的活性部位和明胶酶A的羧基结构域，形成一个三链结构，使明胶酶A适合于随后被另一个MT1-MMP分子激活。明胶酶A的羧基结构域还与整合素αvbeta3结合，整合素在激活过程中也发挥作用。最后，细胞将TIMP-2结合到其他未知的位置，这些位置也可能是明胶酶A激活的关键因素。有趣的是，成纤维细胞和平滑肌细胞通过激活和结合明胶酶A来快速响应细胞形状的变化，这一过程独立于新的蛋白质合成，表明激活机制的组件存在于细胞上，但阻止了适当的相互作用。在本研究中，我们将探讨TIMP-2在明胶酶A的细胞结合和激活中的作用。首先，将探索TIMP-2在细胞结合和明胶酶A激活中的作用的生化基础。首先，将利用嵌合的TIMP分子来推断TIMP-2‘S对MT1-MMP特异性的生化基础，其中TIMP-2的序列将被TIMP-2中的位点交换，TIMP-2是一种高度同源的抑制物，实际上对Mt1-MMPs没有特异性。NET，将表征TIMP-2的非基质金属蛋白酶细胞表面结合位点，并将确定TIMP-2内实现这种结合的关键氨基酸序列。最后，我们将模拟TIMP-2在明胶酶A结合和激活中的作用。了解这一机制对于确定在正常和疾病过程中血管重塑过程中发生的生化事件将是重要的。

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.