The overall aim of this revised proposal is to determine the signal transduction pathways that regulate normal neovascularization in the regenerating liver. The model of liver regeneration in the rat following 70% partial hepatectomy (PHx) provides a hitherto unexploited system by which to dissect the phenomenon of controlled angiogenesis. Following PHx, hepatocytes undergo peak mitoses at 24 hr, while sinusoidal endothelial cells (SEC) achiev peak mitoses at 96 hr. After hepatocyte division in the absence of SEC division, there exist a large percentage of non-vascularized hepatic islands within the liver. The SEC division, their subsequent migration into these islands, and formation of patent vessels within the liver is mediated by growth factor and extracellular matrix signals that have not been investigated and are relevant to elucidating mechanisms that control tissue vascularization in general. The presence of specific extracellular matrices, as well as endothelial mitogens and motogens including EGF, TGF-alpha, TGF-beta1, HGF, aFGF, bFGF and VEGF and the phosphorylation state of their receptors, will be examined during the revascularization process. All of these factors have been found in the liver following PHx and may mediate specific steps pertinent to angiogenesis. To investigate the signals that are critical for initiation and progression of angiogenesis within the time frame of liver regeneration, a novel in vivo membrane isolation technique will be utilized. SEC membranes of the liver will be non-covalently derivatized by perfusion of blood vessels with cationic colloids at specific times following PHx. This technology allows for the rapid, high yield isolation of SEC membranes from liver while maintaining the orientation of component proteins of the membrane at the exact time of derivitization. As a result, the signal transduction pathways acting at the membrane as well as cytosolic fractions of the SEC can be ascertained at any given time following PHx. The parallel in vitro studies will allow for the detailed examination of mechanisms involved in the mitogenic and motogenic signal transduction of SEC by growth factors and extracellular matrices under isolated conditions.
