MECHANISMS OF ENDOTHELIAL CELL-COLLAGEN INTERACTIONS

描述（由申请人提供）：血管生成依赖于适当的胶原生物合成和交联，而且，I型胶原是体外血管生成的理想支架。尽管如此，I型胶原介导的血管生成机制仍然知之甚少。我们建议定义类型/胶原原纤维、内皮细胞表面和细胞内信号通路的特征，这些信号通路共同作用介导I型胶原诱导的血管生成。我们建立了一个独特的模型来研究内皮管的体外形态发生。我们使用我们的系统的初步数据支持这样的假设：在内皮管形态发生过程中，α 2 β 1整合素受体和I型胶原整合素结合序列之间的结合导致D38MAPK激活，而Focal Adhesion Kinase失活。我们将在以下目标中验证这一假设：1)通过测试整合素或纤维连接蛋白功能阻断抗体和GAG功能抑制剂的活性，确定内皮细胞表面alpha1beta1、alpha2beta1和alphaVbeta3整合素受体、硫酸化蛋白聚糖和纤维连接蛋白在血管生成中的作用；2)合成含有整合素结合位点的三螺旋I型胶原模拟肽（THPs），并研究其抑制细胞胶原附着、结合整合素受体和影响血管生成的能力；3)研究整合素功能阻断抗体、整合素结合THPs以及信号通路功能的化学和显性负构建抑制剂对p38MAPK和FAK激活和血管生成的影响；4)检测α 2 β 1整合素连接离散胶原序列的作用，以及p38MAPK和FAK途径，a)在鸡CAM体内，b)在其他聚合物（包括异型胶原原纤维和纤维蛋白）诱导血管生成中的作用。我们的工作将定义I型胶原的结构特征，对其形态发生活性至关重要，探索α 2 β 1整合素-胶原结扎、p38 MAPK和FAK激活与血管生成之间的功能联系，并有助于理解和治疗涉及血管不足的人类疾病，如缺血或异常血管生成，如肿瘤生长。

DESCRIPTION (provided by applicant): Angiogenesis depends upon proper collagen biosynthesis and crosslinking, moreover, type I collagen is an ideal scaffold for angiogenesis in vitro. Despite this, mechanisms of type I collagen-mediated angiogenesis remain poorly understood. We propose to define the features of the type / collagen fibril, endothelial cell surface, and intracellular signaling pathways that act together to mediate type I collagen-induced angiogenesis. We have developed a unique model for studying endothelial tube morphogenesis in vitro. Our preliminary data using our system support the hypothesis that engagement between the alpha2beta1 integrin receptor and integrin-binding sequences of type I collagen result in D38MAPK activation, and inactivation of Focal Adhesion Kinase during endothelial tube morphogenesis. We will test this hypothesis in the following aims:1) Define the roles of endothelial cell surface alpha1beta1, alpha2beta1, and alphaVbeta3 integrin receptors, sulfated proteoglycans, and fibronectin by testing the activities of integrin or fibronectin function-blocking antibodies and inhibitors of GAG function on angiogenesis; 2) Synthesize triple helical, type I collagen mimetic peptides (THPs) including putative integrin-binding sites, and study their capacities to inhibit cell collagen attachment, bind integrin receptors, and influence angiogenesis; 3) Study the consequences of integrin function-blocking antibodies, integrin-binding THPs, and chemical and dominant-negative construct inhibitors of signaling pathway function on p38MAPK and FAK activation and angiogenesis; and 4) Examine the role of alpha2beta1 integrin ligation of discrete collagen sequences, and the p38MAPK and FAK pathways, a) in vivo in the chick CAM, and b) in angiogenesis induction by other polymers including heterotypic collagen fibrils and fibrin. Our work will define the type I collagen structural features critical for its morphogenic activity, probe the functional link between alpha2beta1 integrin-collagen ligation, p38 MAPK and FAK activation, and angiogenesis, and contribute to the understanding and treatment of human diseases involving vascular insufficiencies, such as ischemia, or abnormal angiogenesis, as in tumor growth.