We propose to continue our studies on the cellular and molecular mechanisms controlling new blood vessel formation (angiogenesis) during normal tissue development, using a concerted biochemical, pharmacological, and cell biological approach. Angiogenesis is a key step in the development of many normal and pathological tissues, and thus represents a potential control point for diseases such as cancer, rheumatoid arthritis, and retinopathies. Because adipocyte differentiation and angiogenesis are tightly coordinated during development, the interaction between cultured 3T3-adipocytes and vascular endothelial cells provides an excellent model system. Our previous work has shown that as 3T3-preadipocytes differentiate, they begin to secrete factors which stimulate angiogenesis in vivo, and three important components of the angiogenic response in vitro--protease activity, chemotaxis, and mitogenesis. Biochemical analysis indicates that: 1) multiple molecular signals are secreted by adipocytes, since at least one mitogen and two lipophilic chemotactic factors can be distinguished, 2) the mitogen is not angiogenic at its secreted concentration, whereas the two lipophilic factors are both angiogenic and chemotactic, and 3) one of the angiogenic factors is the E series prostaglandins; the other lipophilic component (designated TLC5) is very potent, strongly differentiation-dependent, and may be an unusual or novel prostanoid. We will purify and characterize the angiogenic TLC5 molecules and the endothelial mitogen, using chromatographic techniques, including high performance liquid chromatography. Our assays will include stimulation of angiogenesis on the chick chorioallantoic membrane together with growth, chemotaxis, plasminogen activator, and collagenase assays on cultured endothelial cells. The role of the endothelial mitogen in neovascularization will be examined by combining it with angiogenic molecules. We plan to identify the major influences regulating production of the angiogenic molecules by adipocytes, using agents known to affect adipocyte metabolism--hypoxia, insulin, cyclic AMP agents, and arachidonate derivatives. How the angiogenic factors modulate the biological response of endothelial cells will be investigated by examing receptors for these molecules. In particular, we will determine if the different angiogenic molecules compete for the same site, and if they alter cyclic nucleotide levels in endothelial cells. The mechanisms whereby heparin potentiates angiogenesis will be studied, using a series of chemically modified heparins.
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