Dysregulation of MMP-9 in the neurovascular unit

神经血管单元中 MMP-9 的失调

• 批准号: 7072645
• 负责人: Eng H. Lo
• 金额: $ 28.87万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7072645
• 关键词: RNA interference; angiotensin II; apoptosis; astrocytes; biological signal transduction; blood brain barrier; cell adhesion molecules; cerebral ischemia /hypoxia; enzyme activity; enzyme inhibitors; enzyme mechanism; extracellular matrix; gene expression; inflammation; laboratory mouse; metalloendopeptidases; mutant; neurons; neuropathology; oxidative stress; proteolysis; simvastatin; stroke; tissue /cell culture; vascular endothelium

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.

在这个项目中，我们将研究基质金属蛋白酶-9（MMP-9）在神经血管单位（脑内皮细胞，星形胶质细胞，神经元）细胞中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降解神经血管基质。这不仅导致血脑屏障渗漏，而且破坏细胞-基质相互作用，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，一种炎症介质，放大MMP-9。他汀类药物和抗粘连剂治疗下调MMP-9。为了验证这个假设，我们将追求三个具体目标。在目标1中，我们将研究在体外培养的脑内皮细胞，星形胶质细胞和神经元的信号通路，上调MMP-9。将对缺氧/复氧的反应与对细胞内粘附分子（ICAM-1）受体连接的反应进行比较。本研究将评估MAP激酶、STAT 1、AP-1和NF-κ B在MMP-9调节中的作用。在目标2中，我们将测试MMP-9上调触发内皮细胞、星形胶质细胞和神经元失巢凋亡样死亡的想法。我们将表明，细胞外基质的蛋白水解降解破坏整合素连接激酶（ILK）和Akt细胞生存信号，从而激活半胱天冬酶介导的细胞死亡。将检查药理学抑制剂、RNA干扰和缺乏MMP-9或过表达内源性MMP抑制剂TIMP 1的突变细胞。在目的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。将使用内皮培养物和高血压肾素/血管紧张素过度表达小鼠中的血管紧张素II暴露来测试MMP-9上调。将测试辛伐他汀和抗ICAM-1治疗以降低MMP-9。Scientific Core将支持体内光学和荧光成像，以测量MMP活性、BBB渗透性和脑灌注的变化。我们实验室和其他实验室的最新数据确定MMP-9是一种改变神经血管稳态的关键蛋白酶。这些拟议的研究应该提供新的见解MMP-9如何变得失调，并有助于中风的病理生理。