AGING AND ENDOTHELIAL CELL FUNCTION

随着年龄的增长，伤口愈合受损，从而延迟闭合，并增加裂开和感染的风险。 这种与年龄相关的伤口修复损伤部分是由于延迟的新血管形成。 新血管的成功生长需要协调的细胞功能，包括迁移、增殖和基质蛋白的生物合成/分解。 虽然这些过程中的每一个都可能在衰老中受损，但我们的初步数据表明，微血管内皮细胞迁移的减少有助于延迟血管生成。 该提案的长期目标是确定老年内皮细胞迁移受损的机制，从而确定干预的潜在部位，以改善衰老中的伤口愈合。在体外，老年人微血管内皮细胞（hmEC）的迁移在胶原蛋白I上减慢，胶原蛋白I是伤口修复的血管生成阶段期间存在的主要细胞外基质（ECM）蛋白。 细胞的最佳迁移需要细胞-基质接触的动态破坏和形成。 因此，运动受基质降解蛋白酶（如间质胶原酶-基质金属蛋白酶I，MMP 1）、整联蛋白（如α 2 β 1，其在与胶原I结合时诱导MMP 1）及其与ECM的相互作用调节。 相对于年轻hmEC，老年hmEC表达较少的MMP 1，并分泌增加量的金属蛋白酶组织抑制剂1（TIMP 1）。 此外，在体内，老年小鼠中聚乙烯醇海绵的延迟新生血管形成与MMP 1降低和TIMP 1表达增加相关。用血管内皮生长因子（VEGF）刺激老化的hmEC增加它们的运动，这可能是由MMP 1的诱导引起的效果。本申请的假设是，由于MMP 1的合成减少，老化的hmEC具有受损的迁移。 我们认为，α 2 β 1-胶原蛋白I结合不能正常发挥作用，诱导MMP 1的合成，这是必要的迁移过程中的选择性脱离。 此外，由于需要MMP 1来产生α 2 β 1的连接位点，因此选择性连接不能以协调的方式发生。 以幼龄hmEC和幼龄小鼠为对照，提出以下具体目的：1.阐明MMP 1/TIMP 1在老化hmEC迁移受损中的作用。 2.明确alpha 2 β 1的表达和功能及其与MMP 1的相互作用，在老化hmEC的附着和迁移受损中。 3.确定VEGF增强老化hmEC迁移的机制。 4.确定年龄和VEGF对MMP 1/TIMP 1和α 2 β 1的表达和功能以及体内血管生成过程中微血管EC迁移的影响。

Wound healing is impaired in aging with consequent delay in closure and increased risk for dehiscence and infection. This age-associated impairment in wound repair is due, in part, to delayed neovascularization. The successful growth of new blood vessels requires coordinated cellular functions including migration, proliferation, and biosynthesis/breakdown of matrix proteins. Although each of these processes may be impaired in aging, our preliminary data show that decreased migration of microvascular endothelial cells contributes to delayed angiogenesis. The long-term objective of this proposal is to define the mechanism(s) of impaired migration in aged endothelial cells, and thus, identify potential sites for interventions to improve wound healing in aging. In vitro, migration of aged human microvascular endothelial cells (hmEC) is slowed on collagen I, the major extracellular matrix (ECM) protein present during the angiogenic phase of wound repair. Optimal migration of cells requires the dynamic breaking and forming of cell-matrix contacts. Thus, movement is regulated by matrix degrading proteases (such as interstitial collagenase- matrix metalloprotease I, MMP1), integrins (such as alpha2beta1, which induces MMP1 upon binding to collagen I), and their interaction with the ECM. Aged hmEC express less MMP1 and secrete increased amounts of tissue inhibitor of metalloprotease 1 (TIMP1) relative to young hmEC. Moreover, in vivo, delayed neovascularization of polyvinyl alcohol sponges in aged mice is associated with decreased MMP1 and increased TIMP1 expression. Stimulation of aged hmEC with vascular endothelial growth factor (VEGF) increases their movement, an effect which may result from induction of MMP1. The hypothesis of this application is that aged hmEC have impaired migration due to decreased synthesis of MMP1. We propose that alpha2beta1-collagen I binding does not properly function to induce MMP1 synthesis which is necessary for selective detachment during migration. Moreover, as MMP1 is required to generate ligation sites for alpha2beta1, selective attachment cannot occur in a coordinated fashion. Using young hmEC and young mice as controls, the following Specific Aims are proposed: 1. Elucidate the role of MMP1/TIMP1 in the impaired migration of aged hmEC on collagen I. 2. Define the expression and function of alpha2beta1, and its interaction with MMP1, in impaired attachment and migration of aged hmEC. 3. Identify the mechanism by which VEGF enhances the migration of aged hmEC. 4. Determine the effects of age and VEGF on: expression and function of MMP1/TIMP1 and alpha2beta1, and the migration of microvascular EC during angiogenesis in vivo.