Mechanisms of Cerebral Vascular Remodeling

描述（由申请人提供）：高水平的同型半胱氨酸（Hcy）称为高同型半胱氨酸血症（HHcy），与脑血管疾病、痴呆、中风和阿尔茨海默病有关。什么？-氨基丁酸（GABA）刺激抑制性神经递质GABA-A受体并减少血管性痴呆和中风。该提议的新奇在于，Hcy特异性地与GABA-A受体竞争，并作为兴奋毒性神经递质。同型半胱氨酸通过诱导氧化还原应激和活性氧（ROS）激活脑血管基质金属蛋白酶（MMPs）：该提案的长期目标是了解HHcy脑血管重塑的机制。这一建议的假设是，同型半胱氨酸诱导MMPs和抑制金属蛋白酶组织抑制剂（TIMPs），部分，通过抑制GABA-A受体。这导致基质的降解和血脑屏障的破坏。我们将通过三个具体目的来测试这一假设：具体目的#1：确定Hcy是否通过减弱GABA-A受体来增加NADH氧化酶和ROS的水平，并降低硫氧还蛋白和过氧化物氧还蛋白的水平。将测量用蝇蕈醇（GABA-A受体激动剂）和不用蝇蕈醇（GABA-A受体激动剂）处理的HHcy（胱硫醚合成酶，CBS -/+）转基因小鼠模型和GABA-A受体缺失小鼠的脑皮质中的NADH氧化酶活性和硫氧还蛋白水平。将通过Q-RT-PCR测量mRNA水平。具体目标#2：确定Hcy是否通过拮抗GABA-A受体增加金属蛋白酶活性并降低TIMP活性。MMP-2、-9、-13和TIMP-1、-2、-3和-4的水平将通过创新的2-D酶谱法、功能蛋白质组学、蛋白质印迹和Q-RT-PCR分析来测量。具体目标3：目的探讨同型半胱氨酸是否通过增强γ-氨基丁酸A受体而改变脑微血管反应性和增加脑微血管通透性。将使用荧光标记的白蛋白，通过体内视频荧光照相术测量脑微血管通透性。将通过局部应用血管活性剂来测量血管反应性。这些研究将证明一种新的机制，其中脑微血管通透性的变化在高同型半胱氨酸和血管性痴呆，并将有治疗阿尔茨海默氏症患者的微血管疾病的分支。

DESCRIPTION (provided by applicant): High levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with cerebral- vascular disease, dementia, stroke, and Alzheimer's disease. The ?-amino butyric acid (GABA) stimulates the inhibitory neurotransmitter GABA-A receptor and decreases vascular dementia and stroke. The novelty of this proposal is that Hcy specifically competes with the GABA-A receptors and acts as an excitotoxic neurotransmitter. Hcy activates cerebral vascular matrix metalloproteinases (MMPs) by inducing redox stress and reactive oxygen species (ROS): The long-term goal of this proposal is to understand the mechanisms of cerebral vascular remodeling in HHcy. The hypothesis of this proposal is that Hcy induces MMPs and suppresses tissue inhibitors of metalloproteinase (TIMPs), in part, by inhibiting the GABA-A receptor. This leads to degradation of the matrix and disruption of the blood brain barrier We will test this hypothesis by three specific aims: Specific aim #1: To determine whether Hcy increases levels of NADH oxidase and ROS, and decreases levels of thioredoxin and peroxiredoxin by attenuating the GABA-A receptor. Levels of NADH oxidase activity and thioredoxin in brain cortex of transgenic mouse model of HHcy (cystathionine ¿ synthetase, CBS -/+) and GABA-A receptor null mice treated with and without muscimol (GABA-A receptor agonist) will be measured. The mRNA levels will be measured by Q-RT-PCR. Specific aim #2: To determine whether Hcy increases metalloproteinase activity and decreases TIMP activity by antagonizing the GABA-A receptor. Levels of MMP-2, -9, -13, and TIMP-1, -2, -3, and -4 will be measured by innovative 2-D zymography, functional proteome, Western blots and Q-RT-PCR analyses. Specific aim #3: To determine whether Hcy alters brain microvascular reactivity and increases permeability of brain microvessels by augmenting GABA-A receptor. Brain microvascular permeability will be measured by in vivo video fluorography, using fluorescence-labeled albumin. The vascular reactivity will be measured by a topical application of vasoactive agents. These studies will demonstrate a novel mechanism in which brain microvascular permeability changes during HHcy and vascular dementias, and will have therapeutic ramifications for microvascular disease in Alzheimer's patients.