The tissue macrophage plays a central role in the regulation of acute wound repair. The macrophage participates in the clearance of debris, and modulates epidermal and fibrous tissue regrowth through the production of soluble factors. In addition, the macrophage is a central mediator of new capillary growth, or angiogenesis. More than 20 macrophage derived factors have been described to influence angiogenesis, yet the specific factors that regulate capillary growth in wound repair have not been clearly characterized. Previous in vivo studies have shown that wound macrophages produce the positive angiogenic regulators TNF-alpha and TGF-alpha, as well as the negative angiogenic regulator thrombospondin (TSP). This proposal utilizes two murine models of acute wound repair (the dorsal incision and the dermal punch wound) to investigate the regulation of the angiogenesis by macrophages. The goal of this proposal is 1) to determine the time course of production of the angiogenic modulators TNF-alpha, TGF- alpha and TSP1 by macrophages in acute wounds, and 2) to assess the importance of these factors in directing wound angiogenesis. The first series of experiments will utilize RNA analysis to determine if TNF-alpha, TGF-alpha, and TSP1 mRNA levels increase during the course of acute wound repair. The cellular source of the transcripts will then be determined by in situ hybridization. In the second series of experiments, the production of each of these angiogenic modulators will be blocked by topical administration of specific anti sense oligonucleotides to healing wounds. To assess the angiogenic role of each modulator, capillary growth in control and antisense treated wounds will be quantitated by specific immunohistochemical staining and compared. Preliminary results indicate that the production of TSP1 in dermal wounds can be down-regulated by the administration of antisense TSP1 oligonucleotides; this down-regulation of TSP1 production causes a significantly delayed pattern of repair. These results suggest that (1) TSP1 plays an important role in the reparative process, and (2) the application of antisense oligonucleotides can be a useful strategy for dissecting the wound repair process in vivo. The continuation of these studies will allow for a direct assessment of the role of TSP1, TNF-alpha, and TGF-alpha in regulating angiogenesis in acute wounds. These investigations should lead to a better understanding of the key elements of the response of macrophages in healing wounds, and might ultimately lead to new therapeutic strategies for modulating repair in chronic, traumatic, or surgical wounds.
