Angiogenesis, or neovascularization, is essential to successful wound healing. Cell-free fluid drawn from the wound site, wound fluid, contains a potent angiogenesis factor that is chemotactic but not mitogenic for capillary endothelial cells. In a new wound, macrophages are established as the dominant inflammotary cell within 3 days. Macrophages remain dominant until the wound is healed. Removal of macrophages from the wound site by antimacrophage serum prevents angiogenesis and wound healing. Our work has shown that, when grown under the hypoxic conditions that mimic the tissue oxygen found in the central deal space of a wound, macrophages are stimulated to secrete a stable angiogenesis factor.
The specific aim of this proposal is to investigate in detail the angiogenesis factor secreted by rabbit macrophages and compare it to the angiogenesis factor that has been purified from rabbit wound fluid. This study on macrophage angiogenesis factor will include an HPLC purification of the factor to homogeneity and the production of a monoclonal antibody to it. The characterization of macrophage angiogenesis factor will include a determination of its chemical identity and structure as well as functional studies. Angiogenesis will always be determined by the corneal implant assay. The mode of action of purified macrophage angiogenesis factor on capillary endothelial cells will be monitored by the stimulation of capillary endothelial cell mitogenesis and chemotaxis. The chemotaxis will be carried out in a modified Boyden blind-well chamber that is sensitive to less than 1 ng of wound fluid angiogenesis factor. The monoclonal antibody to macrophage angiogenesis factor will be used to compare it to wound fluid angiogenesis factor and to angiogenesis factors from other sources. The antibody will also facilitate the study of capillary endothelial cell receptors for macrophage angiogenesis factor and the cellular specificity of the binding of macrophage angiogenesis factor.
