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、中风和阿尔茨海默病相关。 γ-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。该提议的新颖之处在于，Hcy 专门与 GABA-A 受体竞争，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（ROS）激活脑血管基质金属蛋白酶（MMP）：该提案的长期目标是了解HHcy脑血管重塑的机制。该提议的假设是，Hcy 部分通过抑制 GABA-A 受体来诱导 MMP 并抑制金属蛋白酶组织抑制剂 (TIMP)。这会导致基质降解和血脑屏障破坏。我们将通过三个具体目标来检验这一假设：具体目标#1：确定 Hcy 是否会增加 NADH 氧化酶和 ROS 的水平，并通过减弱 GABA-A 受体来降低硫氧还蛋白和过氧化还原蛋白的水平。将测量 HHcy（胱硫醚 ¿ 合成酶，CBS -/ ）转基因小鼠模型和用或不用蝇蕈醇（GABA-A 受体激动剂）治疗的 GABA-A 受体缺失小鼠的脑皮质中 NADH 氧化酶活性和硫氧还蛋白的水平。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 受体来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、功能蛋白质组、蛋白质印迹和 Q-RT-PCR 分析进行测量。具体目标#3：确定 Hcy 是否通过增强 GABA-A 受体来改变脑微血管反应性并增加脑微血管的通透性。将使用荧光标记的白蛋白通过体内视频荧光检查来测量脑微血管通透性。血管反应性将通过局部应用血管活性剂来测量。这些研究将证明 HHcy 和血管性痴呆期间大脑微血管通透性发生变化的新机制，并将对阿尔茨海默病患者的微血管疾病产生治疗影响。

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.