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于有效的心脏性能至关重要。钙化性主动脉瓣疾病（CAVD）的特征在于瓣膜开口的进行性狭窄（变窄）和瓣叶表面上厚钙化沉积物的形成。虽然高达10%的65岁以上的美国人受到AVD的影响，但几乎相同数量的美国人出生时患有先天性主动脉瓣畸形，导致过早变性。使用在动脉粥样硬化中有效的药物和生物标志物的临床试验在诊断、预测或阻止AVD进展方面基本上不成功。动脉粥样硬化和AVD最初都表现为炎症的内皮，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是未知的。已经假设了两种钙化机制（营养不良性和成骨性），但这些模式如何参与基质矿化（几乎总是发现CAVD时）的发生尚不清楚。最近的证据表明，瓣膜表型和钙沉积是由周期性机械应变调节的，但应变如何影响后期钙化环境中的细胞尚不清楚。绝 了解这种疾病过程的大多数研究工作利用非常长期的小鼠模型（>10个月），难以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，更快速的生理培养平台对于确定中期和晚期CAVD的机制至关重要。在本申请中，我们将实施一种新型的3D体外培养平台，该平台结合胶原蛋白基质和可调的合成羟基磷灰石晶体纳米颗粒，以模拟天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（维克）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 羟基磷灰石矿物结晶度和VEC和维克表型的负担，单独和共培养。第二个目标将评估这些关系是如何调制的不同模式的循环双轴应变在三维文化。在这两个目标中，我们将重点关注早期分化行为和晚期基质重塑。本申请的结果将验证一种新的3D体外策略，以快速识别瓣膜细胞特异性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.