Endothelial dysfunction in the cerebral circulation

描述(申请人提供)：在脑循环中，由一氧化氮(NO)生物活性降低和/或生物合成引起的内皮功能障碍是导致血管疾病发生和发展的主要机制。流行病学研究证实了血管疾病与阿尔茨海默病(AD)发病之间的密切联系。然而，脑血管疾病与阿尔茨海默病之间的分子机制尚不清楚。我们最近发表的研究结果(循环研究，107：1498-1502,2010)和初步实验证实，内皮NO在淀粉样前体蛋白(APP)、2-分泌酶(BACE1)和淀粉样蛋白-2肽(A2)的调节中发挥了先前未知的作用。内皮型一氧化氮合酶(ENOS)的药理或遗传失活导致APP和BACE1的上调，导致A2的产生增加。此外，我们有初步证据表明，激活过氧化物酶体增殖激活受体-4(PPAR4)通过促进NO的产生而对内皮功能起到有利的作用。此外，PPAR4的激活阻止了APP的淀粉样变过程。基于这些发现，我们提出了以下中心假说：脑循环内皮细胞功能受损在AD病理的发生和发展中起着关键作用。为了验证这一假说，我们提出了三个具体的目标：1)确定eNOS/cGMP信号对APP表达和加工的影响的分子机制；2)确定PPAR4在脑血管内皮细胞功能和APP加工中的作用；3)评估eNOS/cGMP信号在AD病理发生发展中的作用。培养的人脑微血管内皮细胞将被用于研究NO对A2生成的影响的信号转导途径。这些研究之后，将在转基因小鼠，包括eNOS、nNOS-和iNOS缺陷小鼠，11或21亚型可溶性鸟苷环化酶缺陷小鼠，以及内皮特异性PPAR4缺陷小鼠中对所提出的假设进行体内测试。为了确定eNOS在AD的发生和发展中的作用，我们建议将eNOS缺陷(eNOS-/-)小鼠与AD的小鼠模型杂交。所建立的缺乏eNOS的AD小鼠模型将为研究内皮功能障碍在淀粉样变发病中的作用提供新的、独特的模型。将对微血管和神经组织进行功能、生化和形态分析。我们预计，拟议研究的成功完成将确定eNOS/cGMP信号转导和激活PPAR4在A2产生中的作用。我们期望我们提出的研究结果将确立脑血管内皮细胞作为预防AD的关键靶点，从而指导和集中治疗重点在脑循环。 公共卫生相关性：内皮细胞功能障碍(脑动脉内层)被认为在阿尔茨海默病的发生和发展中起着重要作用。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变化的潜在机制。我们建议创建新的阿尔茨海默病实验模型，这将在旨在确定病变动脉对认知障碍发展的贡献的研究中非常有用。此外，这些模型还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.