REGULATION OF MACROPHAGE DEPENDENT ANGIOGENIC ACTIVITY

描述（改编自研究者摘要）：巨噬细胞通过产生巨噬细胞衍生的血管生成活性（MDAA），在伤口修复、纤维增生性疾病和实体肿瘤发展中介导血管生成的诱导中发挥关键作用。VEGF是MDAA的重要组成部分。巨噬细胞中VEGF的生物活性受iNOS途径、缺氧和乳酸盐的调节。申请人之前的研究表明，巨噬细胞对VEGF血管生成活性的调节部分受单adp核糖基化过程控制。主要假设是巨噬细胞以未修饰或adp核糖基化形式产生VEGF。未修饰的VEGF是血管生成的；而ADP-核糖化VEGF则无血管生成作用。adp核糖基化受iNOS途径和缺氧调节。本应用的具体目的是分析VEGF单adp -核糖基化的调节机制，以及单adp -核糖基化在巨噬细胞依赖性血管生成过程中的作用。iNOS途径在控制VEGF adp核糖基化中的作用也将被研究。特异性Aim 1旨在表征巨噬细胞中adp -核糖掺入VEGF的分子和细胞方面。两种实验方案将用于监测VEGF的共价修饰。首先，巨噬细胞将使用“内流”TM胞饮细胞装载系统细胞质装载32P-NAD+。其次，将使用“反向ADP-核糖基化”方法监测外源性ADP-核糖基化的底物有效性。此外，我们将获得针对ADP-核糖的特异性抗体，并使用SDS-PAGE和Western blotting分析ADP-核糖进入VEGF的情况。特异性目标2侧重于确定adp核糖基化对VEGF血管生成特性的影响。初步实验表明，霍乱毒素或巨噬细胞细胞质提取物可以使rVEGF165被adp核糖化，并且在大鼠角膜血管生成生物实验中，adp核糖化的VEGF是非血管生成的。进一步的研究将在体内和体外血管生成的附加实验中检验adp -核糖基化VEGF的血管生成活性。特异性目的3将研究iNOS途径及其产物在巨噬细胞调节VEGF adp核糖基化中的作用。目的是确定iNOS途径是否直接影响ADP-核糖基转移酶的活性或表达，或改变作为酶底物所需的NAD+的可用性。特异性目的4探讨adp -核糖精氨酸水解酶在VEGF核糖基化调节中的作用。在不同条件下培养的巨噬细胞中测定酶活性和稳态mRNA表达水平。然后分析inos衍生产物对ADP核糖精氨酸水解酶活性和表达的影响。特异性目的5检测iNOS敲除小鼠巨噬细胞中ADP-核糖基化及其受NO的调节。这将是早期结果的延续，表明来自iNOS敲除小鼠的巨噬细胞明显减少MDAA的产生，而VEGF的产生没有减少。特异性目的6侧重于ADP-核糖基化抑制剂对正常小鼠和iNOS敲除小鼠伤口修复的影响。将测试新生物素，维生素k1和维生素k3。综上所述，这些研究应阐明巨噬细胞依赖性血管生成活性调节的重要控制机制，并为慢性伤口的治疗提供潜在的治疗方式。

DESCRIPTION (Adapted from Investigator's Abstract): 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.