Shear stress, endothelial miRNAs, and AV calcification

钙化性主动脉瓣病(CAVD)是老年人群发病率的一个重要原因，是一种 更多心血管事件的强烈危险因素。目前，CAVD尚无其他治疗方案。 而不是更换或修复瓣膜，部分原因是对潜在机制的不完全了解。 有趣的是，房室钙化是以侧方特有的方式发生的，优先发生在纤维侧。 暴露在d-血流中，而暴露在稳定血流中的心室侧则幸免于难。另一种机械力， 在二尖瓣(BAV)和病变瓣膜中常见的伸展升高与高血压也相关 对CAVD来说很好。这些提示d-血流和抬高的牵张在CAVD的发病机制中可能起作用，但是 其机制尚不清楚。我们的目标是明确机械力在CAVD中的作用和机制 发病机制，并利用这些知识开发新的抗CAVD治疗药物。在前一个供资周期中， 我们在人房室内皮细胞中发现了几种流动和侧向依赖的microRNAs(MiRNAs) (HAVECs)和猪AVs(PAV)，并开始确定它们在CAVD中的作用。最近，我们确定了一部小说 流动和拉伸敏感的miR-483-3P(miR-483)，具有作为CAVD关键调节因子的激发潜力 发病机制。我们的初步数据显示，与1)d-flow(OS)相比，miR-483的表达降低 HAVECs中的稳定流(LS)，2)人和猪AVs中纤维层与脑室层的比较，以及3) 在PAV体外的病理拉伸条件下。进一步的数据表明miR-483抑制EC炎症 和内皮-间充质转化(EndoMT)，CAVD的关键病理生物学事件，这是一个关键 MiR-483的基因靶点是Ube2c(E2泛素结合酶)，而Ube2c又可能靶向缺氧- 诱导因子(HIF1α)通过调控其上游调控因子pVHl。HIF1αS在心血管疾病中的作用尚不清楚，但其良好的 已知的靶基因包括血管生成和炎症，转化生长因子β(纤维化和钙化)，Runx2 (钙化)和Twist1(EndoMT)是CAVD的关键致病过程。因此，我们的首要假设是 MiR-483是一种抗CAVD的miRNA，在OS/病理拉伸条件下会减少，导致 Ube2c的增加，进而使pVHL无处不在，使其降解，并增加HIF1α水平。如果是α， 然后，刺激其靶基因，导致炎症、内皮细胞移植、房室硬化和钙化。我们将测试 这有3个目标。目标1将确定miR-483调节切变相关反应的机制 在体外和体外，HAVECs和PAV以Ube2C和HIF1α依赖的方式表达。目标2将决定 MiR-483通过Ube2c和Hif1α依赖在HAVICs和PAV体外牵张依赖性钙化中的作用 机械装置。AIM 3是一项体内研究，其中miR-483、Ube2c和Hif1α将被转基因， 我们刚刚通过治疗GATA5-/-建立的一种新的CAVD小鼠模型的分子或药理学研究 用AAV-PCSK9诱导BAV小鼠高胆固醇血症。在这里，我们将测试他们的角色和他们的抗CAVD 治疗潜力。

Calcific aortic valve disease (CAVD) is a significant cause of morbidity among the aging population and is a strong risk factor for additional cardiovascular events. Currently, there are no therapeutic options for CAVD other than valve replacement or repair due in part to the incomplete understanding of the underlying mechanisms. Interestingly, AV calcification develops in a side-specific manner, occurring preferentially on the fibrosa side exposed to d-flow while the ventricularis side exposed to stable flow is spared. Another mechanical force, elevated stretching commonly observed in bicuspid (BAV) and diseased valves and hypertension, also correlates well with CAVD. These suggest a potential role for d-flow and elevated stretch in the pathogenesis of CAVD, but the mechanism is unclear. Our goal is to define the role and mechanisms of the mechanical forces in CAVD pathogenesis and use the knowledge to develop novel anti-CAVD therapeutics. In the previous funding cycle, we have identified several flow- and side-dependent microRNAs (miRNAs) in human AV endothelial cells (HAVECs) and pig AVs (PAVs) and began determining their roles in CAVD. Recently, we have identified a novel flow- and stretch-sensitive miR-483-3p (miR-483), which has exciting potential as a critical regulator of CAVD pathogenesis. Our preliminary data show that miR-483 expression is decreased 1) by d-flow (OS) compared to stable flow (LS) in HAVECs, 2) in the fibrosa layer compared to the ventricularis in human and pig AVs, and 3) by pathological stretch conditions in PAVs ex vivo. Further data indicate that miR-483 inhibits EC inflammation and endothelial-to-mesenchymal transition (EndoMT), critical pathobiological events in CAVD, and that a key gene target of miR-483 is Ube2c (E2 ubiquitin-conjugating enzyme), which in turn may target the hypoxia- inducible factor (HIF1α) via controlling its upstream regulator pVHL. HIF1α's role in CAVD is unclear, but its well- known target genes include VEGF (angiogenesis and inflammation), TGFβ (fibrosis and calcification), Runx2 (calcification) and Twist1 (EndoMT), key CAVD pathogenic processes. Therefore, our overarching hypothesis is that miR-483 is an anti-CAVD miRNA, which is reduced under OS/pathological stretch conditions, leading to an increase in Ube2c, which in turn ubiquitinates pVHL for its degradation and increases the HIF1α level. HIFα, then, stimulates its target genes leading to inflammation, EndoMT, AV sclerosis and calcification. We will test this in 3 Aims. Aim 1 will determine the mechanisms by which miR-483 regulates shear-dependent responses of HAVECs and PAVs in a Ube2c- and the HIF1α-dependent manner in vitro and ex vivo. Aim 2 will determine the role of miR-483 in stretch-dependent calcification of HAVICs and PAVs ex vivo via Ube2c and HIF1α-dependent mechanisms. Aim 3 is an in vivo study where miR-483, Ube2c, and HIF1α will be modified genetically, molecularly or pharmacologically in a novel mouse model of CAVD that we just developed by treating GATA5-/- BAV mice with AAV-PCSK9 to induce hypercholesterolemia. Here, we will test their roles and their anti-CAVD therapeutic potential.