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.
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