An angiogenic role for the alpha 5 beta 1 and alpha V beta 3 integrins during cer

描述(申请人提供)：在西方世界，缺血性中风是发病率和死亡率的主要原因。尽管在这一领域进行了密集的研究，但目前的治疗选择仍然有限。预防中风或促进中风后恢复的一种方法是鼓励新血管的生长(血管生成)，恢复血管对缺血区的输送。血管生成受生长因子、细胞因子和细胞外基质(ECM)蛋白的调节，如纤维连接蛋白。这一建议要检验的假设是，脑缺血诱导脑内皮细胞表达a5？1和av？3整合素，纤维连接蛋白通过这些整合素促进脑血管生成。中枢神经系统(CNS)外的研究表明，a5？1和av？3整合素在新生血管中被强烈诱导，在肿瘤形成和糖尿病新生血管的动物模型中，对这些整合素的功能阻断抑制了血管生成。我们实验室的证据表明，这些整合素还可能促进中枢神经系统的血管生成：(I)发育中的血管生成与脑内皮细胞a5？1整合素的表达密切相关，(Ii)成人中枢神经系统缺氧时血管生成内皮细胞也强烈诱导a5？1整合素的表达，(Iii)脑缺血后，缺血半影区的a1整合素mRNA水平升高，出现新的血管萌发，(Iv)脑缺血后血管生成内皮细胞迅速诱导av？3整合素，(V)脑内皮细胞的体外研究表明，纤维连接蛋白的存活和促有丝分裂作用是由a5？1和av？3整合素介导的。这一假说将采用体内和体外相结合的方法进行验证。在局灶性脑缺血和脑缺氧的小鼠模型中，将检测a5？1和av？3整合素的内皮表达。Av？3整合素在脑血管生成中的作用将直接在两种动物模型中使用3整合素缺失的小鼠进行检测。A5？1和av？3整合素在调节小鼠脑内皮细胞行为中的潜在作用将通过使用缺乏这些整合素的内皮细胞来解决。最后，将在这些系统中确定促血管生成因子--转化生长因子-1的时间进程和细胞来源。本研究有三个具体目的：1)证实a5？1和av？3整合素在脑缺血或缺氧后对脑内皮细胞的诱导作用；2)证实a5？1和av？3整合素在促进脑内皮细胞存活、增殖、迁移和毛细血管形成方面的指导作用；3)明确脑缺血和脑缺氧后促血管生成因子转化生长因子-1的时程和细胞来源。这些研究将直接检验a5？1和av？3整合素在促进脑缺血反应中的血管生成方面发挥重要作用的观点。基于这项工作，操纵这些整合素的表达水平或信号很可能提供一种有希望的方法来增强脑血管生成，无论是在缺血性卒中之前或之后。公共卫生相关性：该项目的总体目标是研究脑缺血诱导内皮细胞表达a5？1和av？3整合素，以及纤维连接蛋白通过这些整合素促进大脑中新血管的形成。在这项工作的基础上，操纵这些整合素的表达水平或信号通路可能提供一种有希望的治疗方法，以促进脑血管的生长，无论是在缺血性中风之前还是之后。如果成功，这项工作可能会对缺血性中风的发病率和死亡率产生重大影响。

DESCRIPTION (provided by applicant): Ischemic stroke is a major cause of morbidity and mortality in the western world. Despite intensive research in this field, current options for treatment are still limited. One approach to preventing stroke or promoting recovery after stroke is to encourage the growth of new blood vessels (angiogenesis), to restore vascular delivery to the ischemic area. Angiogenesis is regulated by growth factors, cytokines, and extracellular matrix (ECM) proteins like fibronectin. The hypothesis to be tested by this proposal is that cerebral ischemia induces brain endothelial cell expression of the a5¿1 and av¿3 integrins, and that fibronectin promotes cerebral angiogenesis, via these integrins. Studies outside the central nervous system (CNS) show that the a5¿1 and av¿3 integrins are strongly induced on sprouting blood vessels, and functional blockade of these integrins inhibits angiogenesis in animal models of tumor formation and diabetic neovascularization. Evidence from our laboratory suggests that these integrins may also promote angiogenesis in the CNS: (i) developmental angiogenesis is strongly associated with brain endothelial expression of the a5¿1 integrin, (ii) the a5¿1 integrin is also strongly induced on angiogenic endothelial cells during hypoxia in the adult CNS, (iii) following cerebral ischemia, ¿1 integrin mRNA levels are increased in the ischemic penumbra, where new vessel sprouting occurs, (iv) the av¿3 integrin is induced on angiogenic endothelial cells rapidly following cerebral ischemia, and (v) in vitro studies of brain endothelial cells demonstrate that the survival and mitogenic effects of fibronectin are mediated by both the a5¿1 and av¿3 integrins. The hypothesis will be tested using a combination of in vivo and in vitro approaches. Endothelial expression of the a5¿1 and av¿3 integrins will be examined in mouse models of focal cerebral ischemia, and cerebral hypoxia. The role of the av¿3 integrin in cerebral angiogenesis will be directly examined in both animal models using ¿3 integrin null mice. Potential roles for the a5¿1 and av¿3 integrins in regulating mouse brain endothelial cell behavior will be addressed by using endothelial cells deficient in these integrins. Finally, the time-course and cellular source of the pro-angiogenic factor, TGF-¿1 will be defined in these systems. This study has three Specific Aims: 1) demonstrate induction of the a5¿1 and av¿3 integrins on brain endothelial cells following ischemia or hypoxia in cerebral tissue; 2) demonstrate an instructive role for the a5¿1 and av¿3 integrins in promoting brain endothelial cell survival, proliferation, migration, and capillary formation; and 3) define the time-course and cellular origin of the pro-angiogenic factor, TGF- ¿1 following cerebral ischemia and cerebral hypoxia. These studies will directly test the idea that the a5¿1 and av¿3 integrins play an important role in promoting cerebral angiogenesis in response to ischemia. Based on this work, manipulation of the expression level or signaling of these integrins are likely to provide a promising approach to enhance cerebral angiogenesis, either before or immediately after ischemic stroke. PUBLIC HEALTH RELEVANCE: The overall aim of this Project is to examine the notion that cerebral ischemia induces endothelial cell expression of the a5¿1 and av¿3 integrins, and that fibronectin promotes new blood vessel formation in the brain via these integrins. Based on this work, manipulation of the expression levels or signaling pathways of these integrins may provide a promising therapeutic approach to enhance cerebral blood vessel growth, either before or immediately after ischemic stroke. If successful, this work could have major impact on the incidence and mortality of ischemic stroke.