AGING AND ENDOTHELIAL CELL FUNCTION

随着年龄的增长，伤口愈合受到损害，导致闭合延迟，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。虽然这些过程中的每一个都可能在衰老过程中受到损害，但我们的初步数据显示，微血管内皮细胞迁移减少导致血管生成延迟。这项建议的长期目标是确定老年内皮细胞迁移受损的机制(S)，从而确定潜在的干预部位，以促进衰老伤口的愈合。在体外，老化的人微血管内皮细胞(HmEC)在I型胶原上的迁移被减缓，I型胶原是伤口修复血管生成阶段存在的主要细胞外基质(ECM)蛋白。细胞的最佳迁移需要细胞-基质接触的动态断裂和形成。因此，运动受基质降解酶(如间质胶原酶-基质金属蛋白酶I，MMP1)、整合素(如α2beta1，在与I型胶原结合时诱导MMP1)以及它们与ECM的相互作用的调节。与青年HMEC相比，老年HMEC表达MMP1减少，分泌组织金属蛋白酶抑制因子1(TIMP1)增多。此外，在体内，衰老小鼠的聚乙烯醇海绵延迟新生血管与MMP1减少和TIMP1表达增加有关。用血管内皮生长因子(VEGF)刺激衰老的HMEC增加其运动，这一效应可能是通过诱导MMP1产生的。这一应用的假设是，老化的hmEC由于MMP1合成减少而损害了迁移。我们认为，α2beta1-I型胶原结合不能正确地诱导MMP1的合成，而MMP1是在迁移过程中选择性脱离所必需的。此外，由于MMP1需要为alpha2beta1生成连接位点，所以选择性连接不能以协调的方式发生。1.阐明MMP1/TIMP1在老年HMEC迁移受损的I型胶原中的作用；2.明确α2beta1的表达、功能及其与MMP1的相互作用在老年HMEC黏附和迁移受损中的作用。3.明确血管内皮生长因子促进老年人脐静脉内皮细胞迁移的机制。4.检测AGE和血管内皮生长因子对MMP1/TIMP1和α2beta1在体内血管生成过程中的表达和功能以及微血管内皮细胞迁移的影响。

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.