Vascular Sphingosine Kinase Signaling in Cerebral Ischemic Tolerance

脑缺血耐受中的血管鞘氨醇激酶信号传导

• 批准号: 7918277
• 负责人: BRADLEY KENT WACKER
• 金额: $ 5.38万
• 依托单位: WASHINGTON UNIVERSITY
• 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 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7918277
• 关键词: Adherence; Adherens Junction; Adult; Biological Preservation; Blood - brain barrier anatomy; Blood Vessels; Brain; CD31 Antigens; Cause of Death; Cell Adhesion Molecules; Cell Death; Cerebral Ischemia; Cerebrum; Chemicals; Data; Dependency; Development; Endothelial Cells; Endothelium; Functional disorder; Goals; Heart; Hemorrhage; Hypoxia; In Vitro; Infiltration; Inflammatory; Intercellular Junctions; Ischemia; Ischemic Stroke; Kidney; Knockout Mice; Leukocytes; Link; Maintenance; Measures; Mediating; Mediation; Mediator of activation protein; Mus; Newborn Infant; Pathology; Pathway interactions; Permeability; Play; Protein Isoforms; Proteins; Regulation; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; Stroke; Therapeutic; Tissues; United States; Up-Regulation; Vascular Endothelial Cell; Vascular Permeabilities; cadherin 5; cerebrovascular; disability; effective therapy; hypoxia inducible factor 1; immunocytochemistry; in vivo; in vivo Model; inhibitor/antagonist; kinase inhibitor; neurovascular unit; preconditioning; prevent; receptor; relating to nervous system; research study; response; sphingosine 1-phosphate; sphingosine kinase; therapeutic target

DESCRIPTION (provided by applicant): Stroke is a leading cause of death and disability in the United States, yet effective treatments for stroke remain limited. The studies proposed within intend to facilitate the search for therapeutic targets for stroke by elucidating the role of cerebrovascular sphingosine kinase (SphK) signaling in the mechanism by which hypoxic preconditioning (HPC) affords ischemic protection to the neurovascular unit following stroke. SphK is upregulated by hypoxia or ischemia, and studies in tissues such as the heart and kidney have linked SphK to ischemic protection through multiple signaling pathways. HPC protects against cerebral ischemia- induced neurovascular dysfunction, however, the mechanisms of this protection are not well characterized. Recently, we obtained exciting preliminary data indicating that in cerebral microvessels, HPC upregulates the SphK2 isoform, and this SphK activity regulates HPC-induced ischemic protection. Thus, the following specific aims are proposed: Aim 1: To elucidate the dependency of cerebrovascular ischemic tolerance on the sphingosine kinase isoforms. Using a SphK inhibitor and SphK2 knockout mice, we intend to demonstrate that cerebral HPC is mediated by increased expression of SphK2 and its activity within vascular endothelial cells. Furthermore, we will study the regulation of vascular permeability by SphK following ischemia and demonstrate that ischemic tolerance mediated by SphK is associated with the preservation ofthe blood-brain barrier. Additionally, we will examine the adherens junction proteins PECAM-1 and VE-cadherin to determine if SphK maintenance of the BBB is mediated through adherens junctions. Aim 2: To elucidate the relationship between SphK2, and HIF-1 and HIF-2 during the induction of an Ischemic tolerant state in cerebrovascular endothelial cells. Using lentiviral transduction of siRNA to the specific HIF isoforms in a microvascular endothelial culture, and HIF-2 knockout mice, we intend to show that vascular HIF mediates the induction of SphK signaling in response to HPC. We will also examine the possible role of SphK in the regulation of HIF expression following HPC in vitro and in vivo. The proposed experiments will provide an increased understanding of the signaling of SphK in cerebral ischemic protection and the pathways by which that protection is induced. Elucidating the mechanisms of neurovascular unit protection will facilitate the development of stroke therapeutics.

描述（由申请人提供）：中风是美国死亡和残疾的主要原因，但是对中风的有效治疗仍然有限。在内部提出的研究打算通过阐明脑血管鞘氨醇激酶（SPHK）信号在proxic预处理（HPC）中提供脑血管鞘氨醇激酶（SPHK）信号的作用来促进中风的治疗靶标的，该机制可为神经血管造成的渗透率降低后的神经血管单位。 SPHK受到缺氧或缺血的上调，在心脏和肾脏等组织中的研究通过多种信号通路将SPHK与缺血保护联系在一起。 HPC可预防脑缺血引起的神经血管功能障碍，但是，这种保护的机制并未得到很好的特征。最近，我们获得了令人兴奋的初步数据，表明在脑微血管中，HPC上调SPHK2同工型，并且这种SPHK活性调节了HPC诱导的缺血性保护。因此，提出了以下特定目的：目标1：阐明脑血管缺血性耐受性对鞘氨酸激酶同工型的依赖性。使用SPHK抑制剂和SPHK2基因敲除小鼠，我们打算证明脑HPC是通过SPHK2表达增加及其在血管内皮细胞中的活性来介导的。此外，我们将研究缺血后SPHK对血管通透性的调节，并证明由SPHK介导的缺血性耐受性与保存血脑屏障的保存有关。此外，我们将检查粘附蛋白PECAM-1和VE-钙粘着蛋白，以确定BBB的SPHK维持是否是通过粘附连接介导的。目标2：阐明在脑血管内皮细胞中缺血性耐缺血状态时，SPHK2和HIF-1和HIF-2之间的关系。使用微血管内皮培养物中siRNA向特定的HIF同工型和HIF-2基因敲除小鼠的慢病毒转导，我们打算表明，血管HIF介导了响应于HPC的SPHK信号传导的诱导。我们还将检查SPHK在体外和体内HPC后SPHK在调节HIF表达中的可能作用。提出的实验将对脑缺血保护中SPHK的信号传导以及诱导该保护的途径有更多了解。阐明神经血管单位保护的机制将有助于中风治疗的发展。