Vascular Sphingosine Kinase Signaling in Cerebral Ischemic Tolerance

描述（由申请人提供）：在美国，中风是导致死亡和残疾的主要原因，但有效的治疗方法仍然有限。本文提出的研究旨在通过阐明脑血管鞘氨酸激酶（SphK）信号在缺氧预处理（HPC）对脑卒中后神经血管单位提供缺血保护的机制中的作用，促进寻找脑卒中的治疗靶点。SphK在缺氧或缺血时上调，在心脏和肾脏等组织中的研究已经通过多种信号通路将SphK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，但其保护机制尚不明确。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亚型，而这种SphK活性调节HPC诱导的缺血保护。因此，提出了以下具体目的：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SphK2敲除小鼠，我们打算证明脑HPC是由血管内皮细胞中SphK2表达及其活性的增加介导的。此外，我们将研究SphK对缺血后血管通透性的调节，并证明SphK介导的缺血耐受与血脑屏障的保存有关。此外，我们将检查粘附体连接蛋白PECAM-1和VE-cadherin，以确定SphK是否通过粘附体连接介导血脑屏障的维持。目的2：阐明SphK2与HIF-1和HIF-2在诱导脑血管内皮细胞缺血耐受状态中的关系。在微血管内皮培养和HIF-2敲除小鼠中，通过慢病毒将siRNA转导到特定的HIF亚型，我们打算证明血管HIF在HPC反应中介导SphK信号的诱导。我们还将在体外和体内研究SphK在HPC后调控HIF表达中的可能作用。所提出的实验将增加对SphK在脑缺血保护中的信号传导和诱导这种保护的途径的理解。阐明神经血管单位保护的机制将促进脑卒中治疗的发展。

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.