Macrophages play a key role in mediating the induction of angiogenesis in wound repair, fibroproliferative diseases and solid tumor development, by producing macrophage-derived angiogenic activity (MDAA). VEGF is an important component of MDAA. The bio-activity of VEGF is regulated in macrophages by the iNOS pathway, by hypoxia and by lactate. The applicant's previous studies indicate that regulation of the angiogenic activity of VEGF by macrophages is controlled in part by the process of mono-ADP-ribosylation. The primary hypothesis is that VEGF is produced by macrophages in either the unmodified or the ADP-ribosylated form. Unmodified VEGF is angiogenic; while ADP- ribosylated VEGF is non-angiogenic. ADP-ribosylation is regulated by the iNOS pathway and by hypoxia.
The Specific Aims of this application are designed to analyze the mechanisms that regulate the mono-ADP-ribosylation of VEGF, and the role of the mono-ADP- ribosylation in the process of macrophage-dependent angiogenesis. The role of the iNOS pathway in controlling VEGF ADP-ribosylation will also be examined.
Specific Aim 1 is directed at characterizing molecular and cellular aspects of ADP-ribose incorporation into VEGF in macrophages. Two experimental protocols will be used to monitor covalent modification of VEGF. First, macrophages will cytoplasmically loaded with 32P-NAD+ using the """"""""Influx""""""""TM pinocytic cell loading system. Second, the substrate availability to exogenous ADP-ribosylation will be monitored using a """"""""Back ADP- ribosylation"""""""" approach. In addition, specific antibodies to ADP- Ribose will be obtained and used to analyze the incorporation of ADP-Ribose into VEGF, using SDS-PAGE and Western blotting.
Specific Aim 2 focuses on determining the effects of ADP-ribosylation on the angiogenic properties of VEGF. Initial experiments have shown that rVEGF165 can be ADP-ribosylated with cholera toxin or macrophage cytoplasmic extracts, and that the ADP-ribosylated VEGF is non- angiogenic in the rat corneal bio-assay of angiogenesis. Further studies will examine the angiogenic activity of ADP-ribosylated VEGF in additional assays of angiogenesis in vivo and in vitro.
Specific Aim 3 will examine the role of the iNOS pathway and its products in the regulation of VEGF ADP-ribosylation by macrophages. The goal will be to determine whether the iNOS pathway effects ADP- ribosyl transferase activity or expression directly, or alters the availability of the NAD+ required as a substrate for the enzyme.
Specific Aim 4 examines the role of ADP-ribosylarginine hydrolases in the regulation of VEGF ribosylation. Enzyme activity and steady state mRNA expression levels will be determined in macrophages cultured under various conditions. The effects of iNOS-derived products on the activity and expression of ADP ribosylarginine hydrolase will then be analyzed.
Specific Aim 5 examines ADP- ribosylation and its regulation by NO in macrophages from iNOS knockout mice. This will be a continuation of earlier results indicating that macrophages from iNOS knockout mice show markedly reduced production of MDAA, with no reduction in VEGF production.
Specific Aim 6 focuses on the effect of inhibitors of ADP- ribosylation on wound repair in normal and iNOS knockout mice. Novobiocin, Vitamin-K1, and Vitamin-K3 will be tested. Taken together, these studies should elucidate an important control mechanism involved in the regulation of macrophage-dependent angiogenic activity, and lead to potential therapeutic modalities for the treatment of chronic wounds.
