Mechanisms of Cerebral Vascular Remodeling

描述(由申请人提供)：高水平的同型半胱氨酸(Hcy)被称为高同型半胱氨酸血症(HHcy)，与脑血管疾病、痴呆症、中风和阿尔茨海默病有关。氨基丁酸(GABA)刺激抑制性神经递质GABA-A受体，减少血管性痴呆和中风。这项提议的新奇之处在于，同型半胱氨酸与GABA-A受体特异性竞争，并充当兴奋性毒性神经递质。Hcy通过诱导氧化还原应激和活性氧自由基(ROS)激活脑血管基质金属蛋白酶(MMPs)：本研究的长期目标是了解HHcy脑血管重构的机制。这一建议的假设是，Hcy诱导MMPs并抑制金属蛋白酶组织抑制物(TIMPs)，部分是通过抑制GABA-A受体。这将导致基质的降解和血脑屏障的破坏我们将通过三个特定的目标来检验这一假说：特定的目标1：确定Hcy是否通过减弱GABA-A受体而增加NADH氧化酶和ROS的水平，并降低硫氧还蛋白和过氧化还蛋白的水平。检测转基因HHcy(CBS-/)小鼠和GABA-A受体缺陷小鼠(GABA-A受体激动剂)处理前后大脑皮层NADH氧化酶活性和硫氧还蛋白的水平。采用Q-RT-PCR法检测基因表达水平。特定目的#2：确定同型半胱氨酸是否通过拮抗GABA-A受体而增加金属蛋白酶活性和降低TIMP活性。基质金属蛋白酶-2、-9、-13和TIMP-1、-2、-3和-4的水平将通过创新的二维酶谱、功能蛋白质组、Western blotts和Q-RT-PCR分析来测量。具体目的#3：确定同型半胱氨酸是否通过增强GABA-A受体改变脑微血管反应性和增加脑微血管通透性。脑微血管通透性将通过体内视频荧光照相，使用荧光标记的白蛋白来测量。血管反应性将通过局部应用血管活性物质来测量。这些研究将展示HHcy和血管性痴呆期间脑微血管通透性变化的新机制，并将对阿尔茨海默病患者的微血管疾病具有治疗意义。

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.