Prox1 and oscillatory shear stress in aortic valve development and homeostasis

项目摘要 先天性主动脉瓣畸形，包括二叶式主动脉瓣，发生在大约2%的活产婴儿中， 和获得性心脏瓣膜疾病，例如钙化性主动脉瓣疾病，发生在约2.5%的美国人中。 人口理论上，机械力影响先天性和成人瓣膜疾病。最近 使用单细胞RNA鉴定表型和转录多样性的主动脉瓣（AoV）细胞群 成熟的鼠主动脉瓣和二尖瓣的测序。这是第一次，三种不同的瓣膜内皮细胞 (VEC)在AoV上的特定位置确定了种群。每个地点都有独特的 这可能是重要的多样化VEC亚群的机械力的组合。重要的是，一 这些群体中的一个存在于AoV的纤维瘤侧，其中VEC经历OSS。该人群 优先表达转录因子Prox 1，一种发育过程的主要调节因子，包括 多器官系统中的细胞命运和基因表达。在淋巴系统中，Prox1是一个关键的调节因子， 淋巴阀（LymphV）的发展和稳态响应OSS。由于Prox1存在于 小鼠胚胎第13.5天（E）半月瓣和Prox 1的纤维侧仍然存在到成年期，它是 预测在AoV发育和稳态期间类似的机制是活跃的。核心假设 Prox 1介导主动脉瓣形态发生和稳态，以响应 剪应力目的1将确定纤维瘤侧特异性Prox 1是否促进了细胞的形态发生和稳态。 通过分析Prox 1在胚胎和成年小鼠主动脉瓣中的表达模式， 瓣膜发育和体内平衡。与此同时，Gata 2和Cx37、Prox 1的诱导物和 还将分析分别在LymphV内皮细胞中的下游靶标（1A）。此外，胚胎 和成年内皮特异性Prox 1功能丧失小鼠将被用于研究Prox 1缺失对 对Gata2和Cx37表达以及瓣膜形态的影响（1B）。在目标2中， 将分析Prox 1调节网络的压力及其与瓣膜稳态和疾病的关系。 首先，Prox 1、其淋巴诱导物（Gata 2）和下游靶点（Cx37）的表达模式将是 在猪AoV和健康和患病的人AoV中检查（2A）。接下来，生理学的影响 将使用离体培养的猪AoV瓣叶中Prox 1上的相关异常剪切应力模式进行研究， 单锥板生物反应器（2B）。我们的长期目标是确定AoV的机械敏感机制 发育、体内平衡和疾病，具有潜在的治疗应用。该培训计划结合了 研究瓣膜生物学和生物工程与翻译的影响。我的职业目标是成为一名领导者 在理解机械环境如何影响心血管发育、体内平衡和 疾病

Project Summary Congenital aortic valve malformations, including bicuspid aortic valve, occur in approximately 2% of live births, and acquired heart valve diseases, such as calcific aortic valve disease, occur in about 2.5% of the U.S. population. It is theorized that mechanical forces influence both congenital and adult valve disease. Recently phenotypic and transcriptionally diverse aortic valve (AoV) cell populations were identified using single-cell RNA sequencing of maturing murine aortic and mitral valves. For the first time, three distinct valve endothelial cell (VEC) populations were identified in specific locations on the AoV. Each location experiences a unique combination of mechanical forces which could be important in diversifying VEC subpopulations. Importantly, one of these populations is present on the fibrosa side of the AoV where the VECs experience OSS. This population preferentially expresses the transcription factor Prox1, a master regulator of developmental processes, including cell fate and gene expression, in multiple organ systems. In the lymphatic system, Prox1 acts as a key regulator of lymphatic valve (LymphV) development and homeostasis in response to OSS. As Prox1 is present on the fibrosa side of murine embryonic day (E)13.5 semilunar valves and Prox1 remains present into adulthood, it is predicted that a similar mechanism is active during AoV development and homeostasis. The central hypothesis of this proposal is that Prox1 mediates aortic valve morphogenesis and homeostasis in response to changes in shear stress. Aim 1 will determine if fibrosa side-specific Prox1 promotes morphogenesis and homeostasis in the aortic valve by analyzing the expression pattern of Prox1 in the AoV of embryonic and adult mice throughout valve development and homeostasis. In parallel, the expression pattern of Gata2 and Cx37, Prox1’s inducer and downstream target in LymphV endothelial cells respectively, will also be analyzed (1A). Additionally, embryonic and adult endothelial-specific Prox1 loss-of-function mice will be utilized to study the effect Prox1 deletion has on Gata2 and Cx37 expression, as well as on valve morphology (1B). In Aim 2, the effects of altered shear stress on a Prox1 regulatory network and its relationship to valve homeostasis and disease will be analyzed. First, the expression pattern of Prox1, its lymphatic inducer (Gata2) and downstream target (Cx37) will be examined in porcine AoVs and in healthy and diseased human AoVs (2A). Next, the impact of physiologically relevant aberrant shear stress patterns on Prox1 in porcine AoV leaflets cultured ex vivo will be studied using a single cone-and-plate bioreactor (2B). Our long-term goal is to identify mechanosensitive mechanisms of AoV development, homeostasis and disease with potential therapeutic applications. This training plan combines studies of valve biology and bioengineering with translational implications. My career goal is to become a leader in understanding how the mechanical environment influences cardiovascular development, homeostasis and disease.