Dysregulation of MMP-9 in the neurovascular unit

在这个项目中，我们将研究神经血管单元细胞（脑内皮细胞、星形胶质细胞、神经元）中基质金属蛋白酶-9 （MMP-9）失调的信号传导和后果。我们的总体假设是，中风后，氧化应激和粘附分子信号上调MMP-9，从而降解神经血管基质。这不仅会导致血脑屏障渗漏，还会破坏细胞-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的嗜酸样细胞死亡。血管紧张素II是一种炎症介质，可扩增MMP-9。他汀类药物和抗黏附剂治疗可下调MMP-9。为了验证这一总体假设，我们将追求3个具体目标。在目的1中，我们将通过体外培养的脑内皮细胞、星形胶质细胞和神经元来研究上调MMP-9的信号通路。对缺氧/再氧化的反应将与细胞内粘附分子（ICAM-1）受体连接的反应进行比较。我们将评估MAP激酶、STAT1、AP-1和NFkB在MMP-9调控中的作用。在Aim 2中，我们将测试MMP-9上调引发内皮细胞、星形胶质细胞和神经元的嗜酸样死亡的观点。我们将证明细胞外基质的蛋白水解降解会破坏整合素连接激酶（ILK）和Akt细胞存活信号，从而激活caspase介导的细胞死亡。将检测药物抑制剂、RNA干扰和缺乏MMP-9或过表达内源性MMP抑制剂TIMP1的突变细胞。在Aim 3中，我们将研究修饰因子和治疗对体外（细胞培养）和体内（小鼠局灶性脑缺血）MMP-9水平的影响。血管紧张素II暴露于内皮培养和高血压肾素/血管紧张素过表达小鼠中，将用于检测MMP-9的上调。将测试辛伐他汀和抗icam -1治疗是否能降低MMP-9。体内光学和荧光成像将由Scientific Core提供支持，以测量MMP活性、血脑屏障通透性和脑灌注的变化。我们实验室和其他实验室最近的数据证实MMP-9是一种关键的蛋白酶，可以改变神经血管的内稳态。这些拟议的研究应该为MMP-9如何变得失调并有助于中风病理生理提供新的见解。

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.