Dysregulation of MMP-9 in the neurovascular unit

在这个项目中，我们将研究神经血管单位(脑血管内皮细胞、星形胶质细胞、神经元)中基质金属蛋白酶-9(MMP9)异常调节的信号和后果。我们的总体假设是，中风后，氧化应激和黏附分子信号上调了降解神经血管基质的基质金属蛋白酶-9。这不仅会导致血脑屏障的渗漏，还会破坏细胞与基质的相互作用，从而引发内皮细胞、星形胶质细胞和神经元中的失巢凋亡样细胞死亡。血管紧张素II是一种炎症介质，它能放大基质金属蛋白酶-9。他汀类药物和抗粘连药物治疗下调了基质金属蛋白酶-9的表达。为了检验这一总体假设，我们将追求3个具体目标。在目标1中，我们将使用脑血管内皮细胞、星形胶质细胞和神经元的体外培养来检测上调基质金属蛋白酶-9的信号通路。对缺氧/复氧的反应将与细胞内黏附分子(ICAM-1)受体连接的反应进行比较。将评估MAP激酶、STAT1、AP-1和NFkB在基质金属蛋白酶-9调节中的作用。在目标2中，我们将测试基质金属蛋白酶-9上调触发内皮细胞、星形胶质细胞和神经元类失巢细胞死亡的想法。我们将表明，细胞外基质的蛋白降解破坏了整合素连接激酶(ILK)和Akt细胞生存信号，从而激活了caspase介导的细胞死亡。将检查药物抑制物、RNA干扰和缺乏基质金属蛋白酶-9或过表达内源性基质金属蛋白酶抑制物TIMP1的突变细胞。在目的3中，我们将在体外(细胞培养)和体内(小鼠局灶性脑缺血)研究修饰因素和治疗方法对基质金属蛋白酶-9水平的影响。血管紧张素II在内皮细胞培养中的暴露和高血压肾素/血管紧张素过度表达的小鼠将被用来测试基质金属蛋白酶-9的上调。辛伐他汀和抗ICAM-1治疗将被测试以降低基质金属蛋白酶-9。体内光学和荧光成像将得到科学核心的支持，以测量基质金属蛋白酶活性、血脑屏障通透性和脑血流灌注的变化。我们实验室和其他实验室的最新数据证实，基质金属蛋白酶-9是一种关键的蛋白酶，可以改变神经血管的动态平衡。这些拟议的研究应该为基质金属蛋白酶-9如何变得失调并有助于卒中的病理生理学提供新的见解。

In this project, we will investigate the signaling and consequences of matrix metalloproteinase-9 (MMP-9) dysregulation in cells of the neurovascular unit (cerebral endothelial cells, astrocytes, neurons). Our overall hypothesis is that after stroke, oxidative stress and adhesion molecule signaling upregulates MMP-9 that degrades neurovascular matrix. This leads to not only blood-brain barrier leakage, but also disrupts cell-matrix interactions, thus triggering anoikis-like cell death in endothelium, astrocytes, and neurons. Angiotensin II, an inflammatory mediator, amplifies MMP-9. Treatment with statins and anti-adhesion agents downregulate MMP-9. To test this overall hypothesis, we will pursue 3 specific aims. In Aim 1, we will examine signaling pathways that upregulate MMP-9 using in vitro cultures of cerebral endothelial cells, astrocytes, and neurons. Responses to hypoxia/reoxygenation will be compared with responses to intracellular adhesion molecule (ICAM-1) receptor ligation. The roles of MAP kinase, STAT1, AP-1 and NFkB in MMP-9 regulaton will be assessed. In Aim 2, we will test the idea that MMP-9 upregulation triggers anoikis-like death in endothelial cells, astrocytes, and neurons. We will show that proteolytic degradation of extracellular matrix disrupts integrin linked kinase (ILK) and Akt cell survival signaling, thus activating caspase-mediated cell death. Pharmacologic inhibitors, RNA interference, and mutant ceils lacking MMP-9 or overexpressing the endogenous MMP inhibitor TIMP1 will be examined. In Aim 3, we will investigate the effects of modifying factors and therapy on MMP-9 levels in vitro (cell culture) and in vivo (mouse focal cerebral ischemia). Angiotensin II exposure in endothelial cultures and hypertensive renin/angiotensin overexpressing mice will be used to test for MMP-9 upregulation. Simvastatin and anti-ICAM-1 treatments will be tested to reduce MMP-9. In vivo optical and fluorescence imaging will be supported by the Scientific Core to measure changes in MMP activity, BBB permeability, and cerebral perfusion. Recent data from our lab and others established MMP-9 as a key protease that modifies neurovascular homeostasis. These proposed studies should provide novel insight into how MMP-9 becomes dysregulated and contributes to stroke pathophysiology.