REGULATION OF MACROPHAGE DEPENDENT ANGIOGENIC ACTIVITY

描述(摘自《研究人员摘要》)：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的发展中发挥关键作用。血管内皮生长因子是MDAA的重要组成部分。巨噬细胞中血管内皮生长因子的生物活性受iNOS途径、低氧和乳酸的调节。申请人以前的研究表明，巨噬细胞对血管内皮生长因子的血管生成活性的调节部分是由单-ADP-核糖化过程控制的。基本的假设是，血管内皮生长因子是由巨噬细胞以未经修饰或ADP核糖化的形式产生的。未修饰的血管内皮生长因子是血管生成的；而ADP核糖基化的血管内皮生长因子是非血管生成的。ADP-核糖化受iNOS途径和缺氧的调节。本应用的具体目的是分析调节血管内皮生长因子的单-ADP-核糖化的机制，以及单-ADP-核糖化在巨噬细胞依赖的血管生成过程中的作用。INOS通路在控制VEGFADP-核糖化中的作用也将被检测。具体目标1是针对巨噬细胞中ADP-核糖掺入血管内皮生长因子的分子和细胞方面的特征。两个实验方案将用于监测血管内皮生长因子的共价修饰。首先，巨噬细胞将使用“Inflx”TM吞饮细胞加载系统在胞浆内负载32P-NAD+。第二，底物对外源ADP-核糖化的可用性将使用“反向ADP-核糖化”方法进行监测。此外，还将获得针对ADP-Ribose的特异性抗体，并利用SDS-PAGE和Western blotting分析ADP-Ribose掺入血管内皮生长因子的情况。具体目的2着重于确定ADP核糖化对血管内皮生长因子血管生成特性的影响。初步实验表明，rVEGF165可以被霍乱毒素或巨噬细胞胞浆提取物ADP核糖化，并且在大鼠角膜血管生成生物实验中，ADP核糖化的血管内皮生长因子是非血管生成的。进一步的研究将检验ADP核糖化血管内皮生长因子在体内和体外血管生成的其他检测中的血管生成活性。具体目标3将研究iNOS途径及其产物在巨噬细胞调节VEGFADP核糖化中的作用。目的是确定iNOS途径是否直接影响ADP-核糖转移酶的活性或表达，或者改变作为酶底物所需的NAD+的可用性。特异目的4研究ADP-核糖精氨酸水解酶在调节血管内皮生长因子核糖化中的作用。在不同条件下培养的巨噬细胞的酶活性和稳态mRNA表达水平将被测定。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。特异性目标5检测iNOS基因敲除小鼠巨噬细胞中ADP-核糖化及NO对其的调节。这将是早期结果的延续，该结果表明iNOS基因敲除小鼠的巨噬细胞显示MDAA的产生显著减少，而VEGF的产生没有减少。具体目的6集中在ADP核糖化抑制剂对正常和iNOS基因敲除小鼠伤口修复的影响。新诺贝菌素、维生素K1和维生素K3将接受测试。综上所述，这些研究应该阐明参与调节巨噬细胞依赖的血管生成活动的重要控制机制，并导致潜在的治疗慢性伤口的方式。

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.