Endothelial dysfunction in the cerebral circulation

描述（由申请人提供）：在脑循环中，由一氧化氮（NO）生物活性和/或生物合成降低引起的内皮功能障碍是导致血管疾病发生和进展的主要机制。流行病学研究证实血管疾病与阿尔茨海默病（AD）之间存在密切联系。然而，脑血管疾病和AD之间的分子机制尚不清楚。我们最近发表的发现（Circulation Research，107：1498-1502，2010）和初步实验鉴定了内皮NO在淀粉样前体蛋白（APP）、β 2-分泌酶（BACE 1）和淀粉样蛋白-2肽（A2）的调节中的先前未被认识的作用。内皮型一氧化氮合酶（eNOS）的药理学或遗传失活导致APP和BACE 1的上调，导致A2的产生增加。此外，我们有初步证据表明，过氧化物酶体增殖激活受体4（PPAR 4）的激活通过增加NO的产生对内皮功能产生有益的影响。此外，PPAR 4的激活阻止APP的淀粉样蛋白形成过程。基于这些发现，我们提出了以下中心假设：脑循环中内皮功能的损伤在AD病理的开始和发展中起关键作用。为了验证这一假设，我们提出了三个具体目标：1）确定强调eNOS/cGMP信号传导对APP表达和加工的影响的分子机制，2）确定PPAR 4在脑血管内皮功能和APP加工中的作用，3）评估eNOS/cGMP信号传导在AD病理学的起始和进展中的作用。培养的人脑微血管内皮细胞将被用来研究信号转导通路负责NO的影响产生的A2。这些研究之后将在转基因小鼠中对所提出的假设进行体内测试，包括eNOS、nNOS和iNOS缺陷小鼠、可溶性鸟苷酸环化酶缺陷小鼠的11或21亚型和内皮特异性PPAR 4缺陷小鼠。为了确定eNOS在AD发病和进展中的作用，我们建议将eNOS缺陷型（eNOS-/-）小鼠与AD小鼠模型交叉。创建缺乏eNOS的AD小鼠将提供与研究内皮功能障碍对淀粉样蛋白病理学发展的贡献相关的新的和独特的AD模型。将对微血管和神经元组织进行功能、生化和形态学分析。我们预计，成功完成拟议的研究将确定eNOS/cGMP信号传导和激活PPAR 4在A2产生中的作用。我们希望我们提出的研究结果将建立脑血管内皮作为预防AD的关键目标，从而将治疗重点集中在脑循环上。 公共卫生相关性：内皮细胞（脑动脉的内层）的功能障碍被认为在阿尔茨海默病的发生和发展中起重要作用。本申请中提出的研究的长期目标是确定可能导致阿尔茨海默病发展的脑血管功能改变的潜在机制。我们建议建立新的阿尔茨海默病实验模型，这将是非常有用的研究，旨在确定患病动脉的发展的认知障碍的贡献。此外，这些模型将用于测试预防和治疗阿尔茨海默病的新疗法。

DESCRIPTION (provided by applicant): In the cerebral circulation, endothelial dysfunction caused by decreased biological activity and/or biosynthesis of nitric oxide (NO) is principal mechanism responsible for initiation and progression of vascular disease. Epidemiological studies established strong association between vascular disorders and incident Alzheimer's disease (AD). However, the molecular mechanisms linking cerebrovascular disease and AD are unknown. Our recently published findings (Circulation Research, 107:1498-1502, 2010) and preliminary experiments identified previously unrecognized role of endothelial NO in modulation of amyloid precursor protein (APP), 2- secretase (BACE1) and amyloid-2 peptide (A2). Pharmacological or genetic inactivation of endothelial nitric oxide synthase (eNOS) causes up-regulation of APP and BACE1, leading to increased production of A2. Moreover, we have preliminary evidence suggesting that activation of peroxisome proliferation-activated receptor-4 (PPAR4) exerts beneficial effect on endothelial function by enhancing production of NO. Moreover, activation of PPAR4 prevents amyloidogenic processing of APP. Based on these findings we formulated following central hypothesis: impairment of endothelial function in the cerebral circulation plays a critical role in initiation and progression of AD pathology. To test this hypothesis we propose three specific aims: 1) Determine the molecular mechanisms that underline the effects of eNOS/cGMP signaling on expression and processing of APP, 2) Define the role of PPAR4 in cerebrovascular endothelial function and processing of APP and 3) Assess the role of eNOS/cGMP signaling in initiation and progression of AD pathology. Cultured human brain microvascular endothelial cells will be used to study signal transduction pathways responsible for NO effects on generation of A2. These studies will be followed by in vivo testing of the proposed hypothesis in genetically modified mice including eNOS, nNOS-, and iNOS-deficient mice, 11 or 21 isoform of soluble guanylate cyclase-deficient mice, and endothelial specific PPAR4-defcient mice. To determine the role of eNOS in onset and progression of AD we propose to cross eNOS-deficient (eNOS-/-) mice with murine models of AD. Created AD mice lacking eNOS will provide new and unique models of AD relevant to study contribution of endothelial dysfunction to development of amyloid pathology. Functional, biochemical and morphological analyses will be performed on microvessels and neuronal tissue. We anticipate that successful completion of the proposed studies will define the role of eNOS/cGMP signaling and activation of PPAR4 in production of A2. We expect the results of our proposed studies will establish cerebrovascular endothelium as a critical target in the prevention of AD thereby directing and concentrating therapeutic focus on the cerebral circulation. PUBLIC HEALTH RELEVANCE: Dysfunction of endothelial cells (inner lining of the brain arteries) is believed to play an important role in initiation and progression of Alzheimer's disease. The long-term goal of the studies proposed in this application is to determine the mechanisms underlying alterations in function of brain blood vessels that could contribute to development of Alzheimer's disease. We propose to create new experimental models of Alzheimer's disease which will be very useful in studies designed to determine contribution of diseased arteries to development of the cognitive impairment. In addition, these models will be employed in testing novel therapies for prevention and treatment of Alzheimer's disease.