Development of Complex Culture Systems to Study Valvular Dysfunction

 产品说明：钙化性主动脉瓣疾病（CAVD）是最常见的主动脉瓣疾病，但除了全瓣膜置换术外，目前还没有已知的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方法的能力。与人类标本的可用性和动物模型的生理相关性相关的重大限制促使我们提出开发研究CAVD的替代方法。在本申请中，我们描述了我们的计划，以产生工程体外培养模型，模拟瓣膜功能，以确定在早期CAVD的事件序列。具体来说，我们将专注于复制CAVD中的三个关键事件：细胞外基质（ECM）混乱，炎症和脂质氧化。在假设糖胺聚糖（GAG）沉积改变是CAVD中最早的事件之一，并且是CAVD级联中引发后续病理事件的充分和必要条件的指导下，我们提出了以下目的：目的1：评价GAG富集启动CAVD元素的能力。在这个目标中，我们建议采用创新的ECM操作技术来产生3-D瓣膜培养物，其表现出早期CAVD特征的GAG富集结构。然后，我们将研究这些病变的结构是否能够诱导瓣膜间质细胞（维克）功能障碍，反之，健康的结构是否可以挽救病变的VIC。目的2：确定GAG富集是否是CAVD级联进展的先决条件。我们将提供含有其他已知CAVD“构建模块”的3-D维克培养物;具体而言，我们将确定巨噬细胞浸润和脂质氧化（CAVD的另外两个关键特征）是否需要GAG富集，从而使我们能够表征这些事件的序列。我们还将确定是否有组织的ECM环境暴露于其他疾病刺激的VIC有保护作用。我们研究ECM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有关早期CAVD事件序列的信息，从而为开发这种疾病急需的治疗方法迈出关键的第一步。

 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.