We have demonstrated recently that Macrophage-derived angiogenic activity (MDAA) is either identical or closely related immunologically to Tumor Necrosis Factor-alpha (TNF-alpha). Recombinant (r) TNF-alpha is potently angiogenic in vivo in the rat cornea and chick chorioallantoic membrane, is a chemoattractant for bovine capillary endothelial cells (BCE's) in vitro, and induces confluent monolayers of BCE's cultured on collagen gels to invade the gels and from capillary-like tubular structures. MDAA in conditioned media of macrophage cultures is neutralized by a rabbit anti-murine TNF-alpha polyclonal antibody. We have also shown that Transforming Growth Factor-beta (TGF-beta), a product of both normal (platelets and activated lymphocytes) as well as transformed cells, is a potent chemoattractant for human monocytes in vitro, with maximal activity in the femtomolar. At picomolar concentrations, TGF-beta induces expression of angiogenic activity by monocytes. The projects in this application are designed to study: (a) Events involved in the activation of monocyte/macrophage expression of angiogenic activity (MDAA/TNF- alpha). TGF-beta and oxygen/lactate concentrations will be studied. C-DNA clones obtained from Genentech, Inc. will be used to probe for TNF-alpha, TGF-alpha and TGF-beta mRNA levels in monocytes/macrophages. Levels of expressed protein will also be determined. (b) Structural features of TNF-alpha that relate to its angiogenic activity. Five monoclonal antibodies to TNF-alpha, two that neutralize cytotoxic activity, three that do not, will b compared for anti-angiogenic activity. Specific proteolytic and chemical cleavage of TNF-alpha will be carried out, and the peptides tested for angiogenic and cytotoxic activity. Synthetic peptide analogs of active sequences will ultimately be examined in relation to potential inhibitors. (c) The mechanism of action of TNF-alpha as an angiogenic agent, by studying its effects on capillary endothelial cells in culture. Endothelial cell receptors for TNF-alpha will be studied using 125I-labelled TNF-alpha. The effects of TNF-alpha on basic Fibroblast Growth Factor (bFGF) mRNA levels will be examined. Secretion of metalloproteinases, such as collagenase, and TIMP (Tissue Inhibitor of Metalloproteinases) will be studied. (d) The expression of TNF-alpha in vivo will be examined in wounds, inflammation and tumors, by in situ hybridization. Either a labelled oligonucleotide probe for TNF- alpha, or an RNA probe (""""""""Riboprobe""""""""), will be used. (e) The effects of TNF-alpha on wound repair in vivo will be studied, using the implanted polyvinyl (lvalon) sponge and the implanted Gortex micropermeable tubing models.
