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

描述（申请人提供）：A¿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；然而，目前还没有任何治疗方法可以减缓或阻止其发展。最近，脑血管（CV）病理已被确定为AD的一个重要因素。两个关键的观察结果表明，淀粉样蛋白-肽（A¿）可能通过引起或增加对脑缺血的易感性发挥作用。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——这两种情况都与A¿的血管活性有关。其次，大多数AD患者不仅在大脑中而且在血管中都有A -沉积物，这种情况被称为脑淀粉样血管病（CAA）。CAA是脑梗死和痴呆的重要危险因素，并与严重的心血管功能障碍相关。A2以多种形式存在，包括可溶性单体（如A¿40和A¿42）、可溶性低聚物（有毒的中间物质）和不溶性原纤维（CAA的主要成分）。前者（主要是A¿40）和后者（以CAA形式存在的原纤维A2）已被证明能有力地改变心血管功能，而A2低聚物对血管的影响尚不清楚。我们的初步数据表明，单体A2与纤维A2引起心血管功能障碍的方式和程度是不同的。我们发现A¿单体引起血管过度收缩的表型，这是由于内皮细胞（EC）和血管平滑肌细胞（VSMC）功能障碍是通过活性氧（ROS）介导的，而CAA形式的A¿原纤维引起血管低收缩的表型，主要是由于VSMC功能障碍是通过ROS介导的。我们还发现了以前未被认识到的ROS对CAA形成的贡献。该项目的长期目标是验证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烈的不利影响的中心假设。具体目的是：1)确定A¿物种是否会引起不同的CV效应（超收缩损伤与低收缩损伤）；2)确定A¿物种引起VSMC和脑小动脉功能障碍的ROS通路；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)外源性a¿物种应用后体外评估VSMC功能；b)内源性A¿转基因小鼠脑小动脉功能的体内评价；c)使用结合A¿40、A¿42、A¿寡聚物和/或A¿原纤维的抗A¿抗体进行免疫治疗；d) NADPH氧化酶的药理和遗传抑制作用；d) a2结合细胞表面受体、LRP1和HSPGs的药理学和遗传学抑制作用；e) CAA、A¿40、A¿42、APP、ApoE的定量。如果成功，这些研究将导致对A¿诱导的CV缺陷和CAA形成的机制的更好理解。这可能会促进针对A¿及其下游效应物的疗法的发展，最终可能改善AD、CAA或两者患者的预后。

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.