Effects of hydroxyapatite mineralization and valve cell phenotype

描述(由申请人提供)：正确的主动脉瓣功能对于有效的心脏功能至关重要。钙化性主动脉瓣病(CAVD)的特征是瓣口进行性狭窄(变窄)，瓣叶表面形成厚厚的钙化沉积物。虽然65岁以上的美国人中有高达10%的人受到AVD的影响，但几乎同样数量的美国人出生时就患有导致过早退化的先天性主动脉瓣畸形。使用被发现对动脉粥样硬化有效的药物和生物标记物的临床试验在诊断、预测或阻止AVD进展方面大多不成功。动脉粥样硬化和AVD最初都表现为炎症的内皮细胞，但内皮细胞调节间质细胞表型和随后的晚期基质矿化的分子和细胞机制在很大程度上尚不清楚。钙化有两种机制(营养不良和成骨)，但这些机制在基质矿化开始后如何参与(几乎总是发现CAVD)尚不清楚。最近的证据表明，瓣膜表型和钙沉积受到周期性机械应变的调节，但应变如何影响后期钙化环境中的细胞尚不清楚。浩瀚无边 大多数了解这种疾病过程的研究工作都使用非常长期的小鼠模型(&gt；10个月)，这些模型很难控制局部瓣膜微环境，而目前的体外培养系统缺乏生理细胞相互作用和3D生物基质成分。因此，一个更快速、更具生理性的培养平台对于确定中晚期CAVD的发病机制至关重要。在这项应用中，我们将实现一种新型的3D体外培养平台，该平台结合了胶原基质和可调节的合成羟基磷灰石晶体纳米颗粒，以模拟自然钙化的主动脉瓣环境。利用该系统，我们将测试主动脉瓣内皮细胞(VEC)和间质细胞(VIC)对早期和晚期钙化组织环境的反应。我们的应用程序有两个目标。第一个目标是探索这些影响 羟基磷灰石矿物结晶度和负荷对血管内皮细胞和血管内皮细胞表型的影响，无论是单独培养还是联合培养。第二个目标将评估3D培养中循环双轴应变的不同模式如何调节这些关系。在这两个目标中，我们将重点放在早期分化行为和晚期基质重塑上。这项应用的结果将验证一种新的3D体外策略，以快速识别特定于瓣膜细胞的疾病发病机制的新分子和细胞特征，这将为未来的治疗策略提供重要信息，以防止疾病过程的每个阶段的瓣膜矿化。

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.