Effects of hydroxyapatite mineralization and valve cell phenotype

描述（由申请人提供）：主动脉瓣的正常功能对有效的心脏功能至关重要。主动脉瓣钙化病（CAVD）的特征是瓣口进行性狭窄（变窄），瓣叶表面形成厚的钙化沉积物。在65岁以上的美国人中，有10%的人患有AVD，而几乎相同数量的美国人出生时就患有导致主动脉过早退化的先天性主动脉瓣畸形。临床试验中使用的药物和生物标志物对动脉粥样硬化有效，但在诊断、预测或阻止AVD进展方面基本上是不成功的。动脉粥样硬化和AVD最初都表现为内皮炎症，但内皮细胞调节间质细胞表型和后期基质矿化的分子和细胞机制在很大程度上是未知的。钙化的两种机制已经被假设（营养不良和成骨），但这些模式在基质矿化（当CAVD几乎总是被发现时）发生后如何参与尚不清楚。最近的证据表明，瓣膜表型和钙沉积是由循环机械应变调节的，但应变如何影响后期钙化环境中的细胞尚不清楚。绝大

DESCRIPTION (provided by applicant): Proper function of the aortic valve is critically important for efficient cardiac performance. Calcific aortic valve disease (CAVD) is characterized by progressive stenosis (narrowing) of the valve opening and the formation of thick calcified deposits on the surfaces of the valve leaflets. While up to 10% of Americans over 65 are affected with AVD, a nearly equal number of Americans are born with congenital malformations of the aortic valve that result in premature degeneration. Clinical trials using drugs and biomarkers found effective in atherosclerosis have been largely unsuccessful in diagnosing, predicting, or halting AVD progression. Both atherosclerosis and AVD present initially with an inflamed endothelium, but the molecular and cellular mechanisms by which endothelial cells regulate interstitial cell phenotype and subsequent matrix mineralization at late stages are largely unknown. Two mechanisms of calcification have been hypothesized (dystrophic and osteogenic), but how these modes participate after the onset of matrix mineralization (when CAVD is almost always discovered) is unknown. Recent evidence suggests that valve phenotypes and calcium deposition are modulated by cyclic mechanical strain, but how strain affects cells in later stage calcification environments is unknown. The vast majority of research efforts to understand this disease process utilize very long-term mouse models (>10 months) that are difficult to control the local valve microenvironment, while current in vitro culture systems lack physiological cell interactions and 3D biological matrix components. A more rapid, physiological culture platform is therefore essential to identifying mechanisms of mid and late-term CAVD. In this application we will implement a novel 3D in vitro culture platform that incorporates collagen matrix and tunable synthetic hydroxyapatite crystal nanoparticles to mimic the natural calcified aortic valve environment. With this system we will test how aortic valve endothelial cells (VEC) and interstitial cells (VIC) respond to early and lat calcified tissue environments. Our application has two Aims. The first aim will explore the effects of hydroxyapatite mineral crystallinity and burden on VEC and VIC phenotype, both individually and in co-culture. The second aim will assess how these relationships are modulated by different patterns of cyclic biaxial strain in 3D culture. In both aims, we will focus on early differentiation behavior and late term matrix remodeling. The results of this application will validate a novel 3D in vitro strategy to rapidly identify novel molecular and cellular signatures o disease pathogenesis specific to valve cells, which will significantly inform future therapeutic strategies to prevent valve mineralization at each phase of the disease process.