This proposal is based on the working hypothesis t hat the degeneration of capillary blood vessels or """"""""vascular rarefaction"""""""" observed in patients with hypertension and diabetes may result from alterations in extracellular matrix (ECM) structure that induce a cellular suicide program (apoptosis) within the endothelium. These changes in ECM structure may be induced by altering the proteolytic balance between ECM-degrading enzymes (metalloproteinases; MMPs) and their inhibitors (TIMPs) or by mechanically deforming endothelial cells as a results of changes in hemodynamic parameters. In this proposal, we propose to t est this hypothesis by determining whether changes in cell-ECM binding and associated deformation of endothelial cells alters the proteolytic balance that is responsible for ECM remodeling. We also will carry out studies to analyze how changes in ECM structure and cell shape induce apoptosis.
In Aim 1, immunohistochemistry, in situ zymography, and biochemical techniques will be used to characterize athe relationship between ECM turnover, MMP activity, TIMP distribution, and changes in capillary cell shape and structure during both capillary growth and regression in the chick chorioallantoic membrane. Structural changes that mediate regression induced by interfering with ECM deposition will be compared with those induced by inhibiting ECM degradation or interfering with binding of cell surface integrin receptors to identify common mechanisms of capillary involution.
In Aim 2, we will use biochemical and immunocytochemical techniques to determine whether changes in ECM binding and endothelial cell shape feed back to produce capillary basement membrane breakdown by altering expression of MMPs and TIMPs. Endothelial cell shape and integrin binding will be varied independently by culturing cells on adhesive islands of defined shape and size that are coated with ECM molecules or anti- integrin antibodies in the presence or absence of different angiogenic simulators and inhibitors. Finally, in Aim 3, we will use similar micropatterned surfaces in conjunction with immunocytochemical, biochemical, and molecular biological techniques to map out the signaling pathway that begins with changes in integrin binding or cell deformation and results in activation of the gene program that culminates in endothelial cell death. In this manner, we hope to identify critical regulatory mechanisms that underly capillary regression as well as specific molecular mediators that may serve as future targets for therapeutic intervention in patients with hypertensive diseases.
