Endothelial-derived nitric oxide (NO) is an important regulator of blood flow and systemic blood pressure. However, little is known about the mechanisms by which NO acts to control endothelial cell (EC) function or how interference with the NO pathway may affect microvascular structure and function. The applicant's preliminary studies show that blockade of nitric oxide synthase (NOS) profoundly attenuates angiogenesis and in the face of NOS blockade, exogenous NO donors and cGMP analogues stimulate capillary tube formation. Thus, activation of soluble guanlyate cyclase and the attendant rise in cGMP are required for the pro-angiogenic actions of NO. To elucidate the mechanisms by which NO influences EC growth and blood flow, the following Specific Aims are proposed.
Aim 1. Elucidate the role of NO as a determinant of capillary organization in vitro and of microvascular structure in vivo. The applicant hypothesizes that (i) EC-derived NO regulates aspects of vasculogenesis, angiogenesis, microvascular structure and network remodeling; and (ii) Selective inhibition of NOS will impair vasculogenesis, angiogenesis and contribute to microvascular rarefaction. To test these hypotheses, embryos from ENOS and inducible NOS (iNOS) knockout mice and three dimensional (3D) cultures of capillary EC will be used to examine the NO dependency of capillary organization and remodeling in vitro, and the role of NO in vessel remodeling will be determined in the microcirculation of the cremaster muscle of eNOS knockout mice.
Aim 2. Define the NO dependent signal transduction pathways of angiogenesis, network modeling and microvascular hemodynamics. The applicant hypothesizes that (iii) Activation of cGMP-dependent pathway(s) will trigger pro-angiogenic pathways in EC; and (iv) Selective inhibition of NOS will reduce tissue blood flow and impair microvascular flow control thereby promoting network rarefaction. In 3D cultures of ECs, TGF-beta1 modulation of NOS, NO production and activation of the cGMP pathway will be studied in light of capillary morphogenesis. The influence of NO and cGMP on the expression and regulation of well described pro-angiogenic pathways will be determined. Short and long term roles for NO in control of pressure and flow will be investigated in the microcirculation of the cremaster muscle in eNOS knockout mice in vivo. The applicant's long term goals are to understand the interrelationships between NO as a molecular signal necessary for angiogenesis and vessel growth, and as a key determinant of pressure and flow control. Results derived from these studies will uniquely define the physiological role(s) of NO as a vasodilator that influences migration and organization of ECs; thereby defining microvascular geometry and tissue perfusion, in vivo.
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