Sphingosine 1-phosphate receptors in vascular homeostasis and pathology

描述（由申请人提供）：鞘氨醇1-磷酸（S1 P）是一种多功能脂质介质，通过S1 P受体（S1 PnR）发出信号以调节血管系统的发育、稳态和表型变化。我们最近的研究表明，Gi-，Rac-和Akt-偶联的S1 P1 R在血管生成血管中被诱导，是肿瘤血管生成所需的，协调细胞-细胞粘附分子功能，促进血管成熟/稳定并抑制血管通透性。相反，我们发现S1 P2 R，其激活肿瘤抑制磷酸酶PTEN的Rho GTP酶依赖性激活，抑制内皮细胞迁移，破坏细胞-细胞连接，诱导血管通透性，炎症基因表达和病理性血管生成。鉴于S1 P在血浆中是丰富的，S1 P受体在内皮细胞中的空间和时间调节的表达是S1 P在给定血管床中的生物反应的主要决定因素。这项研究计划的长期目标是更好地了解血管系统中S1 P信号的逻辑。由于第一代S1 P受体调节剂FTY 720目前正在进行多发性硬化症和移植排斥的临床试验（III期），因此该研究项目的结果可能提供可直接转化为临床的关键信息。该提议的中心假设是，S1 P通过S1 P1 R和S1 P2 R的时间和空间精确信号传导深刻地调节血管内皮的表型。在第一个具体的目标，我们将测试的假设，S1 P1 R是必不可少的生理血管生成。将使用遗传小鼠模型和药理学工具研究S1 P1 R调节的信号通路促进周细胞募集、抑制血管通透性和炎症的能力。其次，我们将解决的问题，如何在内皮细胞中的S1 P受体信号被衰减的事实，即配体是在血浆中高度丰富。我们将严格检验这一假设，即选择性亚细胞靶向S1 P1 R，即内体和质膜之间的再循环或基于泛素的靶向降解细胞器是调控的一个重要方面，并破译所涉及的分子机制。第三，我们将明确S1 P2 R信号在病理性血管生成中的意义。我们将测试S1 PR 2与Rho/ROCK/PTEN和p38通路的偶联是否分别诱导血管通透性和炎症基因表达。受体偶联的分子机制，调节内皮连接的完整性和转录调控将得到解决。最后，我们提出了S1 P1 R和S1 P2 R在血管稳态和发育中的协同信号转导的定义。将不内化的超形态S1 P1 R敲入小鼠基因组中，并表征所得的组织血管生成、血管稳定、血管渗透性和炎症。将通过在S1 P1 R超型体的背景下改变S1 P2 r的基因剂量来测试S1 P2 R调节S1 P1 R依赖性效应的能力。这些研究有望增强我们对S1 P在血管系统中功能的理解。这些信息可能有助于通过S1 P受体调节剂控制血管炎症和血管生成。 这项拟议的研究将调查鞘氨醇1-磷酸受体1和2（S1 P1 R和S1 P2 R）对血管内皮细胞生物学和病理生理学的作用。S1 P1 R和S1 P2 R介导相反的作用来调节生理和病理性血管生成的概念将使用体外细胞培养系统详细检查，以研究所涉及的机制。视网膜中血管生成的小鼠模型将用于获得体内相关物。最后，这两个受体的协调信号转导将被检查。一种新的小鼠模型，将使我们能够检查坐标信号将开发使用的内化缺陷突变体S1 P1 R。这些研究有望对S1 P受体的工作机制产生重要的见解，S1 P受体是控制血管和免疫系统的重要药物靶点。

DESCRIPTION (provided by applicant): Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, signals via the S1P receptors (S1PnR) to regulate the development, homeostasis and phenotypic changes of the vascular system. Our recent studies have shown that the Gi-, Rac- and Akt-coupled S1P1R is induced in angiogenic vessels, is required for tumor angiogenesis, orchestrates cell-cell adhesion molecule function, promotes vascular maturation/stabilization and inhibits vascular permeability. In contrast, we found that S1P2R, which activates the Rho GTPase- dependent activation of the tumor suppressor phosphatase PTEN, inhibits endothelial cell migration, disrupts cell-cell junctions, induces vascular permeability, inflammatory gene expression and pathological angiogenesis. Given that S1P is abundant in plasma, spatially- and temporally-regulated expression of S1P receptors in endothelial cells is a major determinant of the biological response of S1P in a given vascular bed. The long-term goal of this research program is to better understand the logic of S1P signaling in the vasculature. Since the first generation S1P receptor modulator called FTY720 is currently undergoing clinical trials (phase III) for multiple sclerosis and transplant rejection, the findings from this research program may provide critical information that is directly translatable to the clinic. The central hypothesis of this proposal is that S1P profoundly regulates the phenotype of vascular endothelium by temporally- and spatially-precise signaling of S1P1R and S1P2R. In the first specific aim, we will test the hypothesis that S1P1R is essential for physiological angiogenesis. The ability of S1P1R-regulated signaling pathways to promote pericyte recruitment, and inhibit vascular permeability, and inflammation will be investigated using genetic mouse models and pharmacological tools. Secondly, we will address the question of how the S1P receptor signaling in the endothelium is attenuated given the fact that the ligand is highly abundant in plasma. We will critically test the hypothesis that selective subcellular targeting of S1P1R, namely, recycling between endosomes and plasma membrane or ubiquitin-based targeting to degradative organelle is an important aspect of regulation and decipher the molecular mechanisms involved. Thirdly, we will define the significance of S1P2R signaling in pathological angiogenesis. We will test if S1PR2 coupling to Rho/ROCK/PTEN and p38 pathways induces vascular permeability and inflammatory gene expression, respectively. Molecular mechanisms of receptor coupling, regulation of endothelial junctional integrity and transcriptional regulation will be addressed. And finally, we propose to define the coordinate signaling of S1P1R and S1P2R in vascular homeostasis and development. A hypermorphic S1P1R that does not internalize will be knocked into the mouse genome and the resultant tissue angiogenesis, vascular stabilization, vascular permeability and inflammation will be characterized. The ability of S1P2R to modulate S1P1R-dependent effects will be tested by varying the gene dosage of S1p2r in the context of the S1P1R hypermorph. These studies are anticipated to enhance our understanding of S1P function in the vasculature. This information may be useful in the control of vascular inflammation and angiogenesis by S1P receptor modulators. PUBLIC HEALTH RELEVANCE This proposed research will investigate the role of sphingosine 1-phosphate receptors 1 and 2 (S1P1R and S1P2R) on vascular endothelial cell biology and pathophysiology. The concept that S1P1R and S1P2R mediate opposing actions to regulate physiological and pathological angiogenesis will be examined in detail using in vitro cell culture systems to investigate the mechanisms involved. Mouse models of angiogenesis in the retina will be used to obtain in vivo correlates. Finally coordinate signal transduction of these two receptors will be examined. A novel mouse model that will allow us to examine the coordinate signaling will be developed using an internalization deficient mutant of S1P1R. These studies are expected to derive critical insights into the workings of S1P receptors, which are important drug targets to control the vascular and immune systems.