Development of Complex Culture Systems to Study Valvular Dysfunction

 描述：钙化性主动脉瓣病(CAVD)是最常见的主动脉瓣疾病，但除了全瓣膜置换术外，尚无已知的CAVD治疗方法。虽然我们对严重病变瓣膜的特征了解很多，但对CAVD的早期阶段知之甚少，这抑制了我们开发这种疾病的药物治疗的能力。与人体标本的可用性和动物模型的生理学相关性相关的重大限制促使我们提出开发替代方法来研究CAVD。在这项申请中，我们描述了我们的计划，以产生工程化的体外培养模型，模拟瓣膜特征，以确定早期CAVD的事件顺序。具体地说，我们将重点复制CAVD中的三个关键事件：细胞外基质(ECM)紊乱、炎症和脂质氧化。在糖胺多聚糖(GAG)沉积改变是CAVD最早事件之一的假说的指导下，我们提出了以下目标：目的1：评估GAG富集化启动CAVD元件的能力。在这一目标中，我们建议使用创新的ECM操作技术来产生3D瓣膜培养物，这种培养物显示出早期CAVD所特有的富含Gag的结构。然后，我们将调查这些病变的结构是否能够导致瓣膜间质细胞(VIC)功能障碍，反过来，健康的结构是否能够挽救患病的VIC。目的2：确定GAG增强是否是CAVD级联反应进展的先决条件。我们将为3D VIC培养提供CAVD的其他已知“构件”；具体地说，我们将确定巨噬细胞渗透和脂质氧化是否需要GAG浓缩，这是CAVD的另外两个关键特征，从而使我们能够表征这些事件的序列。我们还将确定有组织的ECM环境是否对暴露在其他疾病刺激下的VIC具有保护作用。我们研究细胞外基质调节细胞功能的新策略可能适用于广泛的疾病研究，并将特别使我们能够阐明早期CAVD的事件序列信息，从而实现开发这种疾病亟需的治疗方法的关键第一步。

 DESCRIPTION: Calcific aortic valve disease (CAVD) is the most prevalent aortic valve disorder, but there is no known treatment for CAVD other than total valve replacement. Although we know much about the characteristics of severely diseased valves, relatively little is known about the early stages of CAVD, which inhibits our ability to develop pharmacological treatments for this disease. Significant limitations associated with the availability of human specimens and the physiological relevance of animal models have motivated us to propose the development of alternative approaches to studying CAVD. In this application, we describe our plan to generate engineered in vitro culture models that mimic valve features in order to identify the sequence of events in early CAVD. Specifically, we will focus on replicating three critical events in CAVD: extracellular matrix (ECM) disarray, inflammation, and lipid oxidation. Guided by the hypothesis that altered glycosaminoglycan (GAG) deposition is one of the earliest events in CAVD and is sufficient and necessary for initiation of subsequent pathological events in the CAVD cascade, we propose the following Aims: Aim 1: Evaluate the ability of the GAG enrichment to initiate elements of CAVD. In this Aim, we propose to employ innovative ECM manipulation techniques to generate 3-D valve cultures that exhibit a GAG- enriched structure that is characteristic of early CAVD. We will then investigate whether these diseased constructs are able to induce valvular interstitial cell (VIC) dysfunction, and, conversely, whether healthy constructs can rescue diseased VICs. Aim 2: Determine whether GAG enrichment is a prerequisite for progression of the CAVD cascade. We will provide 3-D VIC cultures with other known "building blocks" of CAVD; specifically, we will determine whether GAG enrichment is required for macrophage infiltration and lipid oxidation, two other key features of CAVD, thereby allowing us to characterize the sequence of these events. We will also determine whether an organized ECM environment has a protective effect on VICs exposed to other disease stimuli. Our novel strategies for investigating ECM regulation of cell function may be applicable to a broad range of disease investigations, and will specifically enable us to elucidate information about the sequence of events in early-stage CAVD, thereby achieving a critical first step toward developing much-needed treatments for this disease.