ANGIOGENIC EFFECTS OF EXTRACELLULAR MATRIX PROTEINS

细胞外基质蛋白的血管生成作用

• 批准号: 6184262
• 负责人: Jose Martinez
• 金额: $ 27.29万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6184262
• 关键词: CHO cells; HIV envelope protein gp120; angiogenesis; cadherins; capillary; cell adhesion molecules; cell cell interaction; cell differentiation; cell membrane; chemical association; confocal scanning microscopy; extracellular matrix proteins; fibrin; fluorescence microscopy; gene deletion mutation; immunocytochemistry; immunoprecipitation; light microscopy; mutant; protein binding; protein protein interaction; protein structure function; scanning electron microscopy; transfection; vascular endothelium

The formation of new blood vessels plays a fundamental role in biological processes such as embryonic development, wound healing, and tumor growth. During these processes, the interactions of endothelial cells (EC) with proteins of the extracellular matrix are essential for the induction and maintenance of neovascularization. In vivo, following injury of a vessel, a fibrin clot is formed that interacts with the EC and serves as a provisional matrix for formation of new blood vessels. We have studied endothelial cell differentiation in a fibrin gel "sandwich" and observed that EC forms a network of capillary-like tubes, and the molecular determinants responsible for the morphogenesis in this system appears to be rather distinct. In fibrin II, the N-terminal region of the beta chain, a peptide 15-42, seems to express the biological activity which is mediated by associating VE-cadherin. We propose that endothelial cells respond in a fibrin sandwich by defined signals which originate from the direct association of apically fibrin, with the apical membrane via VE cadherin. In addition, the surface distribution of membrane cadherins, along with their association with specific catenins, may be crucial in the formation of capillary tubes. Based on these investigations, we propose to further characterize the domain of VE-cadherin that is responsible for its association with the N-terminal domain of fibrin, and to identify the VE-cadherin domain that intervenes in homophilic binding. Both interactions, heterophilic fibrin VE- cadherin and homophilic cell-cell play a fundamental role in the formation of capillaries. In these studies, we will construct mutants of VE-cadherin with specific deletions in the last two extracellular domains. CHO cells and endothelial cells devoid of VE-cadherin will be transfected with VE-cadherin mutants. Binding of the N-terminal fragment of fibrin, homophilic interactions and ability to form capillary-like structures in the fibrin assay will be analyzed and the results will be compared to those obtained with cells transfected with the wild type VE- cadherin. Pervious studies demonstrated the role of VE-cadherin- catenin complexes in EC homophilic binding and in this proposal, we will investigate the role of VE-cadherin association with beta-catenin, plakoglobin and p120 in hpmophilic binding and in the formation of capillary-like structures. VE-cadherin mutants with specific deletions of two cytoplasmic catenin binding domains will be prepared and functional studies will be done following transfection. Finally, we will also study the distribution of VE-cadherin and assembly of VE-cadherin-catenin complexes in cell monolayers and during the formation of capillaries. Immunocytochemical studies will also be done and analysis of the distribution of VE-cadherin and VE-cadherin- catenin complexes will be performed by using fluorescence microscopy, confocal microscopy and EM. These studies should elucidate the biochemical and biological responses that induce the assembly of EC into capillary-like structures as a response of the EC to the interaction with fibrin.

新血管的形成在诸如胚胎发育，伤口愈合和肿瘤生长等生物学过程中起着基本作用。 在这些过程中，内皮细胞（EC）与细胞外基质蛋白的相互作用对于诱导和维持新血管形成至关重要。 在体内，在血管受伤后，形成了与EC相互作用的纤维蛋白凝块，并用作形成新血管的临时基质。 我们已经研究了纤维蛋白凝胶“三明治”中的内皮细胞分化，并观察到EC形成了毛细管样管的网络，并且该系统中负责形态发生的分子决定因素似乎相当明显。 在纤维蛋白II中，β链的N末端区域（15-42）似乎表达了通过将VE-钙粘着蛋白关联而介导的生物学活性。 我们提出，内皮细胞通过定义的信号在纤维蛋白三明治中反应，该信号源自顶尖纤维蛋白的直接关联，通过VE钙粘着蛋白与顶端膜的直接关联。 另外，膜钙粘蛋白的表面分布以及它们与特定蛋白酶的关联对于毛细管的形成至关重要。 基于这些研究，我们建议进一步表征其与纤维蛋白的N末端结构域相关的VE-钙粘蛋白的结构域，并确定介入均匀结合的VE-钙粘蛋白结构域。 两种相互作用，异质纤维蛋白素 - 钙粘着蛋白和同质细胞 - 细胞在毛细血管的形成中都起着基本作用。 在这些研究中，我们将在最后两个细胞外结构域中构建具有特定缺失的VE-钙粘蛋白突变体。 没有VE-钙粘着蛋白的CHO细胞和内皮细胞将用VE-钙粘蛋白突变体转染。将分析纤维蛋白，同型相互作用的N末端片段的结合以及在纤维蛋白测定中形成毛细血管样结构的能力，并将分析结果，结果将与用野生型Ve-钙粘着蛋白转染的细胞获得的结果进行比较。 详尽的研究证明了Ve-钙粘蛋白 - 链氨蛋白络合物在EC均质结合中的作用，在该提案中，我们将研究VE-钙粘着蛋白与β-钙蛋白与β-蛋白质的作用结构。 将准备具有两个细胞质链氨酸结合结构域的特异性缺失的VE-钙粘蛋白突变体，转染后将进行功能研究。 最后，我们还将研究细胞单层中VE-钙粘蛋白 - 钙蛋白复合物的VE-钙粘蛋白和组装的分布以及毛细血管形成期间的分布。 还将进行免疫细胞化学研究，并通过使用荧光显微镜，共聚焦显微镜和EM来对VE-钙粘蛋白和Ve-钙粘蛋白 - 链氨酸蛋白酶复合物的分布进行分析。 这些研究应阐明生化和生物学反应，这些反应诱导EC组装成毛细管样结构，作为EC对与纤维蛋白相互作用的反应。