Vascular Sphingosine Kinase Signaling in Cerebral Ischemic Tolerance

描述（由申请人提供）：中风是美国死亡和残疾的主要原因，但中风的有效治疗仍然有限。本文提出的研究旨在通过阐明脑血管鞘氨醇激酶（SphK）信号转导在低氧预处理（HPC）对卒中后神经血管单位提供缺血保护的机制中的作用，促进卒中治疗靶点的寻找。SphK会因缺氧或缺血而上调，在心脏和肾脏等组织中的研究已经将SphK通过多种信号通路与缺血保护联系起来。HPC对脑缺血引起的神经血管功能障碍具有保护作用，但其保护作用机制尚不清楚.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SphK 2亚型，这种SphK活性调节HPC诱导的缺血保护。因此，提出了以下具体目标：目的1：阐明脑血管缺血耐受对鞘氨醇激酶亚型的依赖性。使用SphK抑制剂和SphK 2基因敲除小鼠，我们打算证明，脑HPC是由血管内皮细胞内SphK 2的表达增加及其活性介导的。此外，我们将研究缺血后SphK对血管通透性的调节，并证明SphK介导的缺血耐受与血脑屏障的保护有关。此外，我们将检查粘附连接蛋白PECAM-1和VE-钙粘蛋白，以确定是否SphK的BBB的维护是通过粘附连接介导的。目标二：探讨SphK 2与HIF-1、HIF-2在脑血管内皮细胞诱导缺血耐受过程中的关系。在微血管内皮细胞培养物和HIF-2基因敲除小鼠中，使用慢病毒转导siRNA至特定的HIF亚型，我们打算表明血管HIF介导响应HPC的SphK信号传导的诱导。我们还将研究SphK在体外和体内HPC后调节HIF表达的可能作用。拟议的实验将提供更多的理解SphK在脑缺血保护的信号传导和保护诱导的途径。阐明神经血管单位保护机制将有助于卒中治疗药物的发展。

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.