AGING AND ENDOTHELIAL CELL FUNCTION

随着年龄的增长，伤口愈合受到损害，随之而来的是愈合延迟，裂开和感染的风险增加。这种与年龄相关的伤口修复损伤部分是由于新生血管的延迟。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。尽管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。该建议的长期目标是确定衰老内皮细胞迁移受损的机制，从而确定潜在的干预位点，以改善衰老过程中的伤口愈合。在体外实验中，衰老的人微血管内皮细胞（hmEC）在I型胶原（伤口修复血管生成阶段的主要细胞外基质（ECM）蛋白）的作用下迁移速度减慢。细胞的最佳迁移需要细胞-基质接触的动态断裂和形成。因此，运动是由基质降解蛋白酶（如间质胶原酶-基质金属蛋白酶I， MMP1）、整合素（如在与胶原I结合时诱导MMP1的alpha2beta1）及其与ECM的相互作用来调节的。与年轻hmEC相比，老年hmEC表达较少MMP1，分泌较多组织金属蛋白酶1抑制剂（TIMP1）。此外，在体内，老年小鼠聚乙烯醇海绵新生血管的延迟与MMP1的降低和TIMP1表达的增加有关。血管内皮生长因子（VEGF）刺激老年hmEC增加其运动，这可能是由诱导MMP1引起的。该应用的假设是，衰老的hmEC由于MMP1合成减少而导致迁移受损。我们认为，α - β -胶原I结合不能正确地诱导MMP1合成，而MMP1是迁移过程中选择性脱离所必需的。此外，由于需要MMP1来产生alpha2beta1的连接位点，因此选择性连接不能以协调的方式发生。以年轻hmEC和年轻小鼠为对照，提出以下具体目标：阐明MMP1/TIMP1在衰老hmEC对胶原i的迁移受损中的作用。确定alpha2beta1的表达和功能，以及它与MMP1在老年hmEC附着和迁移受损中的相互作用。3. 明确VEGF促进老年hmEC迁移的机制。4. 测定年龄和VEGF对体内血管生成过程中MMP1/TIMP1和alpha2beta1表达和功能以及微血管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.