Regulation of the anti-antiangiogenic switch by CD36, Thrombospondin, and HRGP

描述(申请人提供)：这项提案的目标是了解微血管内皮细胞表面表达的一种名为CD36的蛋白质受体调节血管生成(新血管生长)的机制。这些细胞构成了毛细血管的内层。血管生成在许多人类疾病的发展中是一个至关重要的过程，包括心脏病发作、中风、糖尿病和癌症。因此，了解人体如何正常开启和关闭血管生成，直接有可能导致许多疾病的新的治疗和预后方法。CD36的功能是当它接触到一组其他蛋白质中的任何一种时，向微血管内皮细胞发送信号，阻止血管生成，这些蛋白质包含一种称为凝血酶敏感蛋白I重复序列(TSR)的结构域。我们的实验室最近发现了一种在血液中循环的蛋白质，称为HRGP，它在结构上与CD36具有相似的结构，并可以作为“诱饵”，阻断含有TSR的抗血管生成蛋白的活性。这些发现导致了一种假说，即血管生成受到组织中CD36、TSR和HRGP表达的精细调控。为了解决这一假设，已经制定了三个具体目标。首先是确定CD36识别TSR结构域的结构决定因素，并表征氧化磷脂对TSR结合的调节作用。该方法将包括产生重组多肽，研究它们的相互作用，并使用核磁共振光谱在原子水平上定义这些相互作用的机制。目的2研究CD36在人微血管内皮细胞中的表达，重点阐述修饰磷脂通过蛋白激酶C活化和胞外结构域磷酸化下调CD36表达或功能的机制。表达水平将在信使核糖核酸和蛋白质水平进行测量。这些调节通路在体内的生物学重要性将在目标3中确定，其中将在定向缺失CD36和hrgp基因的小鼠和在皮肤中过度表达HRGP的转基因小鼠中研究与UVB皮肤辐射和肿瘤生长相关的血管生成。实现这些目标可能导致新的治疗方法，通过CD36介导的抗血管生成开关来调节血管生成。公共卫生相关性：血管生成(新血管生长)在许多人类疾病的发展过程中是一个至关重要的过程，包括心脏病发作、中风、糖尿病视网膜病变和癌症。因此，了解人体如何正常开启和关闭血管生成，直接有可能导致许多疾病的新的治疗和预后方法。这个项目试图了解血管衬里细胞(内皮细胞)上的抗血管生成开关开启和关闭的细胞机制。这种转换是由一种名为CD36的细胞受体介导的，当它接触到一组其他蛋白质中的任何一种时，CD36都会向内皮细胞发送信号，阻止血管生成，这些蛋白质含有一种称为凝血酶敏感蛋白I重复序列(TSR)的结构域。该项目将确定TSR-CD36相互作用的结构基础，确定系统关键组件的表达是如何调节的，并确定该系统在体内炎症和肿瘤生长过程中调节血管生成的功能作用。

DESCRIPTION (provided by applicant): The goal of this proposal is to understand the mechanisms by which angiogenesis (new blood vessel growth) is regulated by a protein receptor called CD36 that is expressed on the surface of microvascular endothelial cells. These are the cells that form the lining of blood capillaries. Angiogenesis is a critically important process in the development of many human diseases, including heart attack stroke, diabetes and cancer. Therefore, understanding how the body normally turns angiogenesis on and off has direct potential to lead to new therapeutic and prognostic approaches to many diseases. CD36 functions by sending a signal to microvascular endothelial cells to halt angiogensesis when it is exposed to any of a group of other proteins that contain a structural domain called the thrombospondin type I repeat (TSR). Our laboratory has recently disovered that a protein circulating in blood, called HRGP, bears structural similarity to CD36 and can act as a "decoy", blocking the activity of TSR-containing anti-angiogenic proteins. These discoveries have led to the hypothesis that angiogenesis is modulated by the fine control of CD36, TSR and HRGP expression in tissues. To address the hypothesis three specific aims have been developed. The first is to identify the structural determinants involved in recognition of TSR domains by CD36 and to characterize modulation of TSR binding by oxidized phospholipids. The approach will involve generating recombinant peptides, studying their interaction, and using NMR spectroscopy to define the mechanisms of the interactions at the atomic level. Aim 2 will characterize expression of CD36 in human microvascular endothelial cells, focusing on the mechanisms by which CD36 expression or function is down-regulated by modified phospholipids via protein kinase C activation and by ecto-domain phosphorylation. Expression will be measured at the mRNA and protein levels. The biological importance of these regulatory pathways in vivo will be determined in aim 3, in which angiogenesis associated with UVB skin radiation and tumor growth will be studied in mice with targeted deletion of the cd36 and hrgp genes and transgenic mice that over-express HRGP in skin. Accomplishing these aims could lead to new therapeutic approaches to modulate angiogenesis through the CD36-mediated anti-angiogenic switch. PUBLIC HEALTH RELEVANCE: Angiogenesis (new blood vessel growth) is a critically important process in the development of many human diseases, including heart attack, stroke, diabetic retinopathy, and cancer. Therefore, understanding how the body normally turns angiogenesis on and off has direct potential to lead to new therapeutic and prognostic approaches to many diseases. This project seeks to understand the cellular mechanisms by which an anti-angiogenic switch on blood vessel lining cells (endothelial cells) is turned on and off. The switch is mediated by a cellular receptor called CD36 which functions by sending a signal to endothelial cells to halt angiogenesis when it is exposed to any of a group of other proteins that contain a structural domain called the thrombospondin type I repeat (TSR). This project will define the structural basis of TSR- CD36 interactions, determine how expression of the key components of the system are regulated, and determine the functional role of the system in regulating angiogenesis during inflammation and tumor growth in vivo.