Aging and Endothelial Cell Function

描述(申请人提供)：血管生成在衰老过程中受损。这损害了伤口的修复和缺血器官的血运重建。微血管内皮细胞(MECs)迁移受阻是增龄相关血管生成障碍的核心成分之一。在三维(3D)胶原中，MECs的运动受与细胞表面相关的基质金属蛋白酶(MMP2，MT1-MMPs)及其主要抑制物TIMP2的调节。此外，老年人胎盘上皮细胞迁移和小管生成减少与TIMP2增加和MMP2/MT1-MMPs活性降低有关。目前，介导TIMP2过度和MMP2/MT1-MMP2活性降低的机制尚不清楚，但初步数据表明一氧化氮(NO)可能是一种候选的调节因子。NO是一种血管活性物质，是TIMPs和MMPs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TIMP2过度和基质金属蛋白酶活性不足的原因，而这反过来又抑制了老年胎盘细胞的迁移和小管生成。因此，增加衰老的MECs中的NO水平将导致相应的MMPs活性的增加，从而改善迁移和小管形成。目标1和目标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)捐赠者的人骨髓间充质干细胞(HmECs)；所有细胞均在3D胶原凝胶中培养-体内间质ECM的模拟器。目的1研究NO对人脐静脉内皮细胞MMP2/MT1-MMP2/TIMP2合成和活性的影响。目的研究外源性和旁分泌(来自iNOS转导的成纤维细胞)对人脐静脉内皮细胞迁移和小管形成能力的影响。AIMS 3和AIMS 4将使用两个互补的模型定义在年轻和老年F1杂交小鼠体内和体外血管生成过程中对EC功能和MMP2/MT1-MMP1/TIMP2活性的影响：AIMS 3：体外培养的微血管在3D胶原中形成芽，AIMS 3b和4：血管长入聚乙烯醇(PVA)海绵体内。小鼠血管生成模型允许在除年龄以外的所有方面都匹配的动物中研究和操纵血管生长。 综上所述，本提案将使用NO来定义MMP2/MT1-MMP2/TIMP2的调节机制(S)以及随后在衰老过程中内皮细胞功能的损害。阐明衰老细胞和组织中基质金属蛋白酶活性的调节机制将有助于改善创伤修复和缺血器官血管重建的治疗方法的发展。

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.