PARACRINE REGULATION OF ANGIOGENESIS BY FIBROBLASTS

成纤维细胞对血管生成的旁分泌调节

• 批准号: 2230040
• 负责人: ROBERTO NICOSIA
• 金额: $ 23.34万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2230040
• 关键词: RNase protection assay; angiogenesis; antisense nucleic acid; basement membrane; cell growth regulation; collagen; fibroblast growth factor; fibroblasts; fibronectins; immunocytochemistry; immunoelectron microscopy; immunoprecipitation; laboratory rat; laminin; muscle cells; neutralizing antibody; northern blottings; paracrine; platelet derived growth factor; polymerase chain reaction; tissue /cell culture; vascular endothelium; western blottings

Angiogenesis, i.e. the formation of blood vessels by endothelial cells, plays a critical role in wound healing and in pathologic processes such as neoplastic growth, rheumatoid arthritis, diabetic retinopathy, and atherosclerosis. The long term goal of these studies is to elucidate the molecular mechanisms of angiogenesis and to facilitate the treatment of angiogenesis-dependent disorders. We have developed a serum-free in vitro model of angiogenesis which is based on the capacity of rat aorta rings to generate microvessels in collagen gels. Angiogenesis in this model is regulated by endogenous basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF). We also postulate a role for vascular endothelial growth factor (VEGF) based on its potent stimulatory effect on the system. Our hypothesis is that aortic fibroblasts stimulated by bFGF and PDGF promote the formation of microvessels by producing heparin-binding growth factors including VEGF. We also hypothesize that fibroblasts stabilize microvessels by promoting the perivascular deposition of basement membrane. Using a serum-free co-culture model recently developed in our laboratory we will evaluate the effect of fibroblasts on the proliferative and vasoformative response of endothelial cells to bFGF and PDGF. We will also investigate the inhibitory effects on rat aortic angiogenesis of neutralizing antibodies against these growth factors. Immunoprecipitation studies together with light and electron immunohistochemical analysis will evaluate how fibroblasts influence the perivascular production, deposition, and distribution of fibronectin, laminin, and type IV collagen, which are components of the basement membrane. The stabilizing effects of these molecules on the microvessels will be also evaluated. The production of VEGF by fibroblasts and by aortic cultures at different stages of angiogenesis will be studied by Western blotting, Northern blotting, reverse transcriptase-polymerase chain reaction (RT-PCR), and RNAase protection assay. The expression of the endothelial VEGF receptor during angiogenesis will be investigated by affinity labeling studies, RT-PCR, and RNAase protection assay. The function of endogenous VEGF in the rat aorta model will be inhibited with anti-VEGF antibodies and antisense oligonucleotides. By studying how fibroblasts regulate angiogenesis we will gain insight into the mechanisms of angiogenesis-dependent disorders which are often characterized by a combination of fibroblastic and vascular proliferation.

血管生成，即由内皮细胞形成血管， 在伤口愈合和病理过程中起关键作用 肿瘤生长，类风湿关节炎，糖尿病性视网膜病和 动脉粥样硬化。这些研究的长期目标是阐明 血管生成的分子机制，并促进 血管生成依赖性疾病。我们已经开发了一种无血清体外 血管生成模型基于大鼠主动脉环的能力 在胶原蛋白凝胶中产生微丝。该模型中的血管生成是 由内源性基本成纤维细胞生长因子（BFGF）和 血小板衍生的生长因子（PDGF）。我们还假设了一个角色 基于其有效刺激的血管内皮生长因子（VEGF） 对系统的影响。我们的假设是刺激主动脉成纤维细胞 通过BFGF和PDGF通过产生生产来促进微血管的形成 包括VEGF在内的肝素结合增长因素。 我们还假设 成纤维细胞通过促进血管来稳定微血管 地下膜的沉积。使用无血清共培养模型 最近在我们的实验室开发的，我们将评估 内皮的增生和血管生成反应的成纤维细胞 细胞到BFGF和PDGF。我们还将研究对 对这些生长的中和抗体的大鼠主动脉血管生成 因素。免疫沉淀研究与光和电子 免疫组织化学分析将评估成纤维细胞如何影响 纤连蛋白的血管周围产生，沉积和分布， 层粘连蛋白和IV型胶原蛋白，它们是地下室的组成部分 膜。这些分子对微血管的稳定作用 还将评估。通过成纤维细胞生产VEGF和 在不同阶段的血管生成阶段的主动脉培养会通过 蛋白质印迹，北印迹，逆转录酶 - 聚合酶 链反应（RT-PCR）和RNAase保护测定法。表达 血管生成过程中的内皮VEGF受体将通过 亲和力标记研究，RT-PCR和RNAase保护分析。这 内源性VEGF在大鼠主动脉模型中的功能将被抑制 抗VEGF抗体和反义寡核苷酸。通过研究如何 成纤维细胞调节血管生成我们将深入了解机制 血管生成依赖性疾病的特征是 成纤维细胞和血管增殖的组合。