Aging and Endothelial Cell Function

描述（由申请人提供）：血管生成在衰老中受损。这损害了伤口的修复和缺血器官的血管再生。年龄相关的血管生成损伤的中心组成部分之一是微血管内皮细胞（mECs）的迁移抑制。在三维（3D）胶原蛋白中，mEC的运动受与细胞表面相关的基质金属蛋白酶（MMP）（MMP 2，MT 1-MMP）及其主要抑制剂TIMP 2的调节。此外，老年人mECs迁移和小管形成减少与TIMP 2增加和MMP 2/MT 1-MMP活性降低相关。目前，在老年人mECs中介导过量TIMP 2和缺陷MMP 2/MT 1-MMP活性的机制尚不清楚;然而，初步数据暗示一氧化氮（NO）作为候选调节因子。NO是血管活性的，TIMP和MMP的“上游”调节剂，在老化的细胞和组织中减少。我们的假设是，NO水平的降低是导致TIMP 2过量和MMP活性不足的原因，而TIMP 2和MMP活性不足反过来又抑制了老年mECs的迁移和小管形成。因此，增加老化mEC中的NO水平将导致MMP活性的相应增加，从而改善迁移和小管形成。目标1和2 将利用来自7名年轻（平均年龄=26 ± 6岁）和8名老年（平均年龄=67 ± 11岁）供体的人mEC（hmEC）;所有细胞都在3D胶原凝胶中培养-体内间质ECM的模拟物。AIM 1将确定hmECs对NO的反应中MMP 2/MT 1-MMP/TIMP 2的合成和活性的变化。AIM 2将检查外源性和旁分泌（来自iNOS转导的成纤维细胞）诱导的变化对hmECs迁移和进行小管形成能力的功能后果。AIM 3和4将使用两种互补模型定义在年轻和老年F1杂交小鼠中离体和体内血管生成过程中NO对EC功能和MMP 2/MT 1-MMP/TIMP 2活性的影响：AIM 3：在3D胶原中离体培养的微血管形成芽，以及AIM 3b和4：体内皮下植入的聚乙烯醇（PVA）海绵中的血管向内生长。鼠血管生成模型允许在除了年龄之外的所有方面都匹配的动物中研究和操纵血管生长。 总之，该建议将使用NO来定义MMP 2/MT 1-MMP/TIMP 2调节和随后的衰老中内皮细胞功能损伤的机制。阐明在老化细胞和组织中调节MMP活性的机制将有助于开发改善缺血器官的伤口修复和血管重建的疗法。

DESCRIPTION (provided by applicant): Angiogenesis is impaired in aging. This compromises the repair of wounds and revascularization of ischemic organs. One of the central components of age-associated impairment of angiogenesis is inhibited migration of microvascular endothelial cells (mECs). In 3-dimensional (3D) collagen, movement of mECs is regulated by matrix metalloproteinases (MMPs) associated with the cell surface (MMP2, MT1-MMP) and their primary inhibitor, TIMP2. Moreover, diminished migration and tubulogenesis by aged human mECs is associated with increased TIMP2 and decreased MMP2/MT1-MMP activity. Currently, the mechanism that mediates excess TIMP2 and deficient MMP2/MT1-MMP activity in aged human mECs is not understood; however, preliminary data implicate nitric oxide (NO) as a candidate regulatory factor. NO, a vasoactive, "upstream" modulator of TIMPs and MMPs, is decreased in aged cells and tissues. Our hypothesis is that diminished levels of NO are responsible for excess TIMP2 and deficient MMP activity that, in turn, inhibits the migration and tubulogenesis of aged mECs. Consequently, increasing NO levels in aged mECs will lead to corresponding increases in MMP activity, thereby improving migration and tubulogenesis. AIMs 1 and 2 will utilize human mECs (hmECs) from 7 young (mean age=26+6yrs) and 8 aged (mean age=67+11 yrs) donors; all cells are cultured in 3D collagen gels- a simulator of interstitial ECM in vivo. AIM 1 will define changes in synthesis and activity of MMP2/MT1-MMP/TIMP2 by hmECs in response to NO. AIM 2 will examine the functional consequences of exogenous and paracrine (from iNOS transduced fibroblasts) N)-induced changes on the ability of hmECs to migrate and undergo tubulogenesis. AIMs 3 and 4 will define NO effects on EC function and MMP2/MT1-MMP/TIMP2 activity during angiogenesis ex vivo and in vivo in young and aged F1 hybrid mice using two complementary models: AIM 3: formation of sprouts from explanted microvessels cultured ex vivo in 3D collagen, and AIMs 3b and 4: vascular ingrowth into polyvinyl alcohol (PVA) sponges implanted subcutaneously in vivo. The models of murine angiogenesis permit vascular growth to be studied and manipulated in animals that are matched in all respects except for age. In summary, this proposal will use NO to define mechanism(s) of MMP2/MT1-MMP/TIMP2 regulation and subsequent impairment of endothelial cell function in aging. The elucidation of mechanisms that regulate MMP activity in aged cells and tissues will assist the development of therapies to improve wound repair and revascularization of ischemic organs.