Sphingolipid Signaling in Endothelial Responses to Injury

内皮损伤反应中的鞘脂信号传导

• 批准号: 8307783
• 负责人: TERESA SANCHEZ GARCIA-VAO
• 金额: $ 42.08万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8307783
• 关键词: 1-Phosphatidylinositol 3-Kinase; Accounting; Adherens Junction; Adhesions; Atherosclerosis; Blood; Blood Vessels; Cell Adhesion Molecules; Cell Line; Cell Migration Inhibition function; Cell Surface Receptors; Cell physiology; Cellular biology; Chromosomes, Human, Pair 10; Coupled; Coupling; Data; Diabetes Mellitus; Disease; Ear; Embryo; Endothelial Cells; Endothelium; Family; Fibroblasts; G-Protein-Coupled Receptors; Genetic; Heterotrimeric GTP-Binding Proteins; Human; Hydrogen Peroxide; Hypoxia; Immigration; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Injury; Ligands; Lipids; Lung; MAP3K5 gene; MAPK14 gene; Mediating; Metabolism; Modeling; Molecular; Molecular Biology; Molecular and Cellular Biology; Morphogenesis; Mus; PTEN gene; Pathway interactions; Pattern; Peritonitis; Permeability; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Plasma; Pulmonary Edema; Regulation; Renal Cell Carcinoma; Rho-associated kinase; Role; Sepsis; Signal Pathway; Signal Transduction; Small Interfering RNA; Sphingolipids; Sphingomyelins; Sphingosine-1-Phosphate Receptor; Stress; Subcutaneous Injections; Symptoms; System; Technology; Testing; Tumor Angiogenesis; Tumor Suppressor Proteins; Ubiquitination; Umbilical vein; Vascular Permeabilities; angiogenesis; cell motility; cell type; design; forkhead protein; gain of function; in vivo; insight; loss of function; matrigel; migration; monocyte; monolayer; public health relevance; receptor; response; response to injury; rho; sphingosine 1-phosphate; stress activated protein kinase; vascular bed; vascular inflammation

DESCRIPTION (provided by applicant): Sphingosine-1-phosphate (S1P), a blood-borne sphingolipid, regulates many cellular functions (e.g. migration, survival) through the interaction with its G protein coupled receptors S1P1-5R. At the cellular level, S1P1R and S1P2R mediate opposite effects due to the coupling of S1P1 to the Gi-phosphatidylinositol-3- kinase (PI3K) pathway and the coupling of S1P2R to the G12/13-Rho pathway, which activates the phosphatase PTEN. While S1P1R regulates vascular permeability, angiogenesis and the inflammatory response of the endothelium, the role of S1P2R in the regulation of endothelial responses to injury remains uncertain. The main hypothesis of this proposal is that S1P2R signaling in endothelial cells is a critical modulator of vascular permeability, angiogenesis and the inflammatory responses of the endothelium by counterbalancing S1P1R- Gi-PI3K signaling (via PTEN activation) and activating the pro-inflammatory ASK-1-SAPK pathway. To test this hypothesis we propose three specific aims: Specific Aim 1: To study the role of S1P2R-Rho-PTEN pathway in the regulation of endothelial cell barrier function and vascular permeability. Molecular and cellular biology approaches will be used to characterize the mechanisms of PTEN activation by S1P2R and the role of the S1P2R-Rho-PTEN pathway in the regulation of endothelial cell permeability using human lung microvascular endothelial cells (HLMVEC), and human aortic endothelial cells (HAEC). In addition, genetic and pharmacological approaches will be used to test the role of S1P2R-Rho-PTEN pathway in the regulation of vascular permeability in vivo. Specific aim 2: To study the role of S1P2R-Rho-PTEN pathway in angiogenesis. The regulation of FOXO-1 phosphorylation, subcellular localization, ubiquitination, stability and transcriptional activity by S1P will be determined. In addition, role of the S1P2R-Rho-PTEN pathway in angiogenesis will be determined i) in vitro using gain-of-function and loss-of-function approaches, and, ii) in vivo in the Matrigel model and in a model of tumor angiogenesis, involving subcutaneous injection of the murine renal cell carcinoma line, RENCA, into syngeneic wild type, S1p2r , S1p2r-/- and Pten mice. Specific Aim 3: To determine the role of S1P2R in the regulation of the inflammatory responses of the endothelium. The mechanisms of activation ASK-1 by S1P2R will be characterized by molecular and cell biology approaches. Secondly, the role of S1P2R-G12/13-Rho-PTEN and S1P2R-G12/13-ASK-1 pathways in the regulation of the inflammatory response of the endothelium, monocyte adhesion and transendothelial migration will be studied in vitro, by gain-of-function and loss-of-function approaches. Finally, genetic approaches will be used to study the role of S1P2R and PTEN in the regulation of endothelial inflammation in a mouse peritonitis model. These studies are anticipated to provide mechanistic insights into the role of S1P2R signaling in the regulation of endothelial responses to injury. PUBLIC HEALTH RELEVANCE: S1P is a lipid, very abundant in plasma, which regulates many cellular functions through the interaction with its cell surface receptors S1P1-5R. S1P2R is present in the endothelium, which overlays the blood vessels throughout the body, and it can be activated by S1P and by pharmacological modulators. This project aims to understand how S1P interaction with S1P2R regulates the responses of the endothelium to injury. Answering these questions will help us to design new drugs that inhibit or activate S1P2R, which could be used as new therapies to alleviate the symptoms of atherosclerosis, diabetes or sepsis.

描述（由申请人提供）：血液传播的鞘脂鞘脂（S1P）通过与其G蛋白偶联受体S1P1-5R的相互作用来调节许多细胞功能（例如迁移，存活）。在细胞水平上，S1P1R和S1P2R介导了相反的作用，这是由于S1P1与Gi-phosphatidylyolinositol-3-激酶（PI3K）途径以及S1P2R偶联的偶联，而S1P2R的偶联是与G12/13-RHO途径与G12/13-RHO途径的偶联。尽管S1P1R调节内皮的血管通透性，血管生成和炎症反应，但S1P2R在调节内皮损伤的调节中的作用仍然不确定。该提议的主要假设是，内皮细胞中的S1P2R信号传导是血管通透性，血管生成和血管生成和内皮炎症反应的关键调节剂，通过平衡S1P1R-GI-PI-PI3K信号（通过PTEN激活）并激活亲侵袭性的Ask-1-Sapk途径。为了检验这一假设，我们提出了三个具体目标：具体目的1：研究S1P2R-RHO-PTEN途径在调节内皮细胞屏障功能和血管渗透性中的作用。分子和细胞生物学方法将用于表征S1P2R的PTEN激活机制，以及S1P2R-RHO-PTEN途径在使用人肺微血管内皮细胞（HLMVEC）和人类主意见内皮细胞（HALMVEC）的内皮细胞（HALMVEC）（HAEC）（HAEC）中的S1P2R-RHO-PTEN途径在调节内皮细胞通透性中的作用。此外，遗传和药理方法将用于测试S1P2R-RHO-PTEN途径在体内血管通透性调节中的作用。具体目标2：研究S1P2R-RHO-PTEN途径在血管生成中的作用。将确定S1P的FOXO-1磷酸化，亚细胞定位，泛素化，稳定性和转录活性的调节。 In addition, role of the S1P2R-Rho-PTEN pathway in angiogenesis will be determined i) in vitro using gain-of-function and loss-of-function approaches, and, ii) in vivo in the Matrigel model and in a model of tumor angiogenesis, involving subcutaneous injection of the murine renal cell carcinoma line, RENCA, into syngeneic wild type, S1p2r , S1P2R - / - 和PTEN小鼠。具体目的3：确定S1P2R在调节内皮炎症反应中的作用。 S1P2R激活ASK-1的机制将以分子和细胞生物学方法为特征。其次，S1P2R-G12/13-RHO-PTEN和S1P2R-G12/13-ASK-1途径在调节内皮，单核细胞粘附和跨内皮迁移的调节中的作用将在体外通过功能和官能功能丧失在体外进行研究。最后，遗传方法将用于研究小鼠腹膜炎模型中S1P2R和PTEN在调节内皮炎症中的作用。预计这些研究将提供有关S1P2R信号在调节内皮损伤反应中的作用的机械见解。 公共卫生相关性：S1P是一种脂质，在血浆中非常丰富，它通过与其细胞表面受体S1P1-5R的相互作用来调节许多细胞功能。 S1P2R存在于内皮中，该内皮覆盖了整个人体的血管，并且可以被S1P和药理调节剂激活。该项目旨在了解S1P与S1P2R的相互作用如何调节内皮对损伤的反应。回答这些问题将有助于我们设计抑制或激活S1P2R的新药物，这些药物可以用作减轻动脉粥样硬化，糖尿病或败血症症状的新疗法。