Sphingosine 1-phosphate receptors in vascular homeostasis and pathology

描述(申请人提供)：1-磷酸鞘氨醇(S1P)，一种多功能的脂质介体，通过S1P受体(S1PnR)发出信号，调节血管系统的发育、动态平衡和表型变化。我们最近的研究表明，Gi-、Rac-和Akt偶联的S1P1R在血管生成中被诱导，是肿瘤血管生成所必需的，协调细胞-细胞黏附分子功能，促进血管成熟/稳定，抑制血管通透性。相反，我们发现S1P2R激活肿瘤抑制基因PTEN依赖的Rho GTP酶激活，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S1P在血浆中含量丰富，血管内皮细胞中S1P受体的时空调控表达是S1P在特定血管床上生物学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S1P信号在血管系统中的逻辑。由于名为FTY720的第一代S1P受体调节剂目前正在进行多发性硬化症和移植排斥反应的临床试验(第三阶段)，该研究计划的结果可能提供可直接翻译到临床的关键信息。这一假设的中心假设是，S1P通过S1P1R和S1P2R在时间和空间上的精确信号深刻地调节血管内皮细胞的表型。在第一个特定的目标中，我们将检验S1P1R对于生理性血管生成是必不可少的假设。S1P1R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将使用遗传小鼠模型和药理学工具进行研究。其次，鉴于S1P受体在血浆中的含量非常丰富，我们将解决血管内皮细胞中S1P受体信号如何减弱的问题。我们将严格检验选择性亚细胞靶向S1P1R的假说，即内体和质膜之间的循环或基于泛素的降解细胞器靶向是调控的一个重要方面，并破译其中的分子机制。再次，我们将明确S1P2R信号在病理性血管生成中的意义。我们将测试S1PR2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的表达。受体偶联的分子机制，内皮细胞连接完整性的调节和转录调节将被解决。最后，我们建议定义S1P1R和S1P2R在血管内稳态和发育中的协调信号。一种不内化的超形态S1P1R将被敲入小鼠基因组，由此产生的组织血管生成、血管稳定、血管通透性和炎症将被表征。S1P2R调节S1P1R依赖效应的能力将在S1P1R超晶型的背景下通过改变S1p2r的基因剂量来测试。这些研究有望加强我们对S1P在血管系统中的功能的理解。这一信息可能有助于通过S1P受体调节剂控制血管炎症和血管生成。 公共卫生相关性这项拟议的研究将调查鞘氨醇1-磷酸受体1和2(S1P1R和S1P2R)在血管内皮细胞生物学和病理生理学中的作用。关于S1P1R和S1P2R调节生理和病理血管生成的相反作用的概念，将利用体外细胞培养系统进行详细的研究，以探讨涉及的机制。小鼠视网膜血管生成的模型将被用来获得体内的相关信息。最后，我们将研究这两种受体的协同信号转导。将使用S1P1R的内化缺陷突变体开发一种新的小鼠模型，该模型将允许我们检查坐标信号。这些研究有望获得对S1P受体工作原理的关键见解，S1P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.