MECHANISMS OF ENDOTHELIAL CELL-COLLAGEN INTERACTIONS

说明(申请人提供)：血管生成依赖于适当的胶原生物合成和交联，此外，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于介导型胶原诱导的血管生成。我们开发了一种独特的模型来研究体外内皮管的形态发生。我们使用我们的系统的初步数据支持这样的假设，即在内皮细胞形态形成过程中，α2beta1整合素受体与I型胶原整合素结合序列之间的结合导致D38MAPK激活，并导致粘着斑激酶失活。我们将在以下方面验证这一假说：1)通过检测整合素或纤维连接蛋白功能阻断抗体和GAG功能抑制剂对血管生成的作用，确定内皮细胞表面α1beta1、α2beta1和alphaVbeta3整合素受体、硫化蛋白多糖和纤维连接蛋白的作用；2)合成包括可能的整合素结合位点在内的三螺旋I型胶原模拟肽，并研究它们抑制细胞胶原附着、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以及化学和显性信号通路抑制物对p38MAPK和FAK激活和血管生成的影响；4)研究α2beta1整合素连接离散的胶原序列的作用，以及p38MAPK和FAK通路，a)在鸡CAM体内，b)在包括异型胶原纤维和纤维蛋白在内的其他聚合物诱导血管生成中。我们的工作将确定对I型胶原的形态生成活性至关重要的I型胶原结构特征，探索α2beta1整合素-胶原连接、p38MAPK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.