We propose to study the cellular and molecular mechanisms controlling new blood vessel formation (angiogenesis) during embryonic development, using a concerted biochemical, pharmacological, and 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 embryogenesis, the interaction between cultured 3T3-adipocytes and vascular endothelial cells provides an excellent model system. We have previously shown that as 3T3-preadipocytes differentiate, they begin to secrete factor(s) into the medium which stimulate angiogenesis in vivo, and three important components of the angiogenic response in vitro--protease activity, chemotaxis, and mitogenesis. Preliminary results suggest that prostaglandins or other arachidonic acid derivatives may be involved. We plan to identify the major influences modulating production of the angiogenic activities by adipocytes, using agents known to affect adipocyte metabolism--insulin, cyclic AMP agents, hypoxia, and arachidonate derivatives. We plan to examine factors which may modulate the response of blood vessels to various angiogenic stimuli, including arachidonate derivatives and heparin. We will fractionate adipocyte-conditioned medium using various chromatographic techniques (including high-pressure liquid chromatography) to determine if a single factor stimulates angiogenesis, or if several molecular signals are required. If factors stimulating growth, chemotaxis, protease activity, and angiogenesis are separable, the effects of combining individual factors will be studied. Subsequently, we will continue purification of the angiogenesis factor(s). A key aspect of this proposal is that we intend to follow angiogenesis in vivo as well as endothelial cell protease activity, chemotaxis, and proliferation in vitro after each of the biochemical or pharmacological manipulations. Techniques employed will include: the chick chorioallantoic membrane assay for angiogenesis; plasminogen activator and collagenase assays; modified Boyden chamber assays for chemotaxis; and growth curves of endothelial cell number to assay mitogenesis.
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