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

描述（由申请人提供）：本提案的目标是了解血管生成（新血管生长）由微血管内皮细胞表面表达的称为CD36的蛋白质受体调节的机制。这些细胞构成了毛细血管的内膜。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，了解身体正常情况下如何开启和关闭血管生成，有可能直接导致许多疾病的新治疗和预后方法。CD36的功能是，当它暴露于含有称为血栓反应蛋白I型重复（TSR）结构域的任何一组其他蛋白质时，向微血管内皮细胞发送信号以停止血管生成。我们的实验室最近发现，血液中循环的一种叫做HRGP的蛋白质与CD36具有结构相似性，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设，即血管生成是通过对组织中CD36、TSR和HRGP表达的精细控制来调节的。为了解决这一假设，我们提出了三个具体目标。首先是确定参与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。该方法将包括生成重组肽，研究它们的相互作用，并使用核磁共振波谱在原子水平上定义相互作用的机制。目的2将表征人微血管内皮细胞中CD36的表达，重点关注CD36的表达或功能通过蛋白激酶C激活和外显域磷酸化被修饰的磷脂下调的机制。在mRNA和蛋白水平上测量表达。这些调节途径在体内的生物学重要性将在目标3中确定，其中血管生成与UVB皮肤辐射和肿瘤生长相关，将在靶向缺失cd36和hrgp基因的小鼠和皮肤中过度表达hrgp的转基因小鼠中进行研究。实现这些目标可能会导致通过cd36介导的抗血管生成开关来调节血管生成的新治疗方法。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，了解身体正常情况下如何开启和关闭血管生成，有可能直接导致许多疾病的新治疗和预后方法。该项目旨在了解血管内膜细胞（内皮细胞）上的抗血管生成开关被打开和关闭的细胞机制。这种开关是由一种叫做CD36的细胞受体介导的，当内皮细胞暴露于一组含有称为血栓反应蛋白I型重复（TSR）结构域的其他蛋白质中时，CD36的功能是向内皮细胞发送信号，阻止血管生成。本项目将明确TSR- CD36相互作用的结构基础，确定该系统关键组分的表达是如何被调节的，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.