THE ROLE OF A-BETA SPECIES IN VASCULAR SMOOTH MUSCLE CELL AND CEREBRAL ARTERIOLE

描述(由申请人提供)：A？种在血管平滑肌细胞和脑小动脉功能障碍中的作用。阿尔茨海默病(AD)是痴呆症的主要原因，但目前还没有治疗方法来减缓或阻止其发展。最近，脑血管(CV)病理被认为是AD的重要因素。两个关键的观察表明，淀粉样肽(A？)可能通过引起或增加对脑缺血的易感性而发挥作用。首先，在阿尔茨海默病的早期阶段，脑血流量(CBF)减少，脑血管的反应性受损--这两者都与A？的血管活性特性有关。其次，大多数阿尔茨海默病患者不仅在脑部，而且在血管中也会出现淀粉样蛋白沉积--这种情况被称为脑淀粉样血管病(CAA)。CAA是脑梗塞和痴呆的一个强有力的危险因素，并与严重的心血管功能障碍有关。A2以几种形式存在，包括可溶单体(如A？40和A？42)、可溶低聚物(有毒中间体)和不溶纤维(CAA的主要成分)。前者(主要是A？40)和后者(以CAA形式存在的纤维状A2)已被证明能有效地改变CV功能，而A2寡聚体的血管效应尚不清楚。我们的初步数据表明，单体A2和纤维A2导致CV功能障碍的方式和程度是不同的。我们发现，A？单体导致血管高度收缩表型是由于内皮细胞(EC)和血管平滑肌细胞(VSMC)功能障碍所致，而A？纤维以CAA的形式引起的血管低收缩表型主要是由于ROS介导的VSMC功能障碍。我们还发现了ROS对CAA形成的一个以前未被认识到的贡献。拟议项目的长期目标是验证中心假设，即A物种通过ROS介导的途径诱导VSMC介导的小动脉功能障碍，从而对脑循环产生强大和不利的影响。其具体目的是1)确定A物种是否引起不同的心血管效应(高收缩和低收缩损害)；2)确定A物种引起VSMC和脑小动脉功能障碍的ROS途径；3)确定ROS促进CAA形成的方式和程度，并评估通过抗ROS策略减少CAA的功能益处。使用的方法包括：a)使用外源性A？种后的VSMC功能的体外评估；b)在体内评估产生内源性A？种的转基因小鼠的脑小动脉功能；c)结合A？40、A？42、A？寡聚体和/或A？纤维的抗A？抗体的免疫治疗；d)对NADPH氧化酶的药理和遗传抑制；d)对A2结合的细胞表面受体LRP1和HSPGs的药理和遗传抑制；以及e)CAA、A？40、A？42、APP和ApoE的定量测定。如果成功，这些研究将有助于更好地理解A？诱导的CV缺陷和CAA形成的机制。这可能会促进针对A？及其下游效应物的治疗方法的开发，最终可能会改善AD和/或CAA患者的预后。

DESCRIPTION (provided by applicant): The role of A¿ species in vascular smooth muscle cell and cerebral arteriole dysfunction. Alzheimer's Disease (AD) is the leading cause of dementia; yet no therapy exists to slow or stop its progression. Recently, cerebrovascular (CV) pathology has been identified as a strong contributor to AD. Two key observations suggest that amyloid-¿ peptide (A¿) may play a role by either causing or increasing the susceptibility to cerebral ischemia. First, cerebral blood flow (CBF) is reduced in early stages of AD, and the reactivity of cerebral blood vessels is impaired - both of which have been linked to the vasoactive properties of A¿. Second, most AD patients develop A¿ deposits not only in brain but also in vessels - a condition known as cerebral amyloid angiopathy (CAA). CAA is a powerful risk factor for brain infarction and dementia, and is associated with severe CV dysfunction. A2 exists in several forms including soluble monomers (such as A¿40 and A¿42), soluble oligomers (toxic intermediate species), and insoluble fibrils (principle component of CAA). The former (primarily A¿40) and the latter (fibrillar A2 in the form of CAA) have been shown to powerfully alter CV function, while the vascular effects of A2 oligomers are not known. Our preliminary data suggest that the manner and extent to which monomeric A2 vs. fibrillar A2 cause CV dysfunction is different. We find that A¿ monomers cause a hyper-contractile vascular phenotype that is due to endothelial cell (EC) and vascular smooth muscle cell (VSMC) dysfunction that is mediated via reactive oxygen species (ROS), while A¿ fibrils in the form of CAA cause a hypo-contractile vascular phenotype that is primarily due to VSMC dysfunction that is mediated via ROS. We also identified a previously unrecognized contribution of ROS to CAA formation. The long-term objective of the proposed project is to test the central hypothesis that A¿ species powerfully and adversely affect the cerebral circulation by inducing VSMC-mediated arteriole dysfunction via an ROS- mediated pathway. The specific aims are 1) to determine whether A¿ species cause differential CV effects (hyper- vs. hypo-contractile impairment); 2) to determine the ROS pathways by which A¿ species cause VSMC and cerebral arteriole dysfunction; and 3) to determine the manner and extent to which ROS contribute to CAA formation, and to assess the functional benefits of reducing CAA via anti-ROS strategies. Methods used will include a) in vitro assessment of VSMC function after application of exogenous A¿ species; b) in vivo assessment of cerebral arteriole function in transgenic mice producing endogenous A¿ species; c) immunotherapy with anti-A¿ antibodies that bind A¿40, A¿42, A¿ oligomers, and/or A¿ fibrils; d) pharmacologic and genetic inhibition of NADPH oxidase; d) pharmacologic and genetic inhibition of the A2-binding cell surface receptors, LRP1 and HSPGs; and e) quantitation of CAA, A¿40, A¿42, APP, and ApoE. If successful, these studies will result in an improved understanding of the mechanisms underlying A¿-induced CV deficits and CAA formation. This will likely facilitate development of therapies targeting A¿ and its downstream effectors, which may ultimately improve the outcome of patients with AD, CAA, or both.