Shear stress, endothelial miRNAs, and AV calcification

钙化性主动脉瓣疾病（CAVD）是老年人群中发病的重要原因， 其他心血管事件的强风险因素。目前，CAVD没有其他治疗选择。 部分原因是对潜在机制的不完全理解。 有趣的是，AV钙化以侧特异性方式发展，优先发生在纤维瘤侧 暴露于D流，而暴露于稳定流的心室侧则不受影响。另一种机械力， 通常在二尖瓣（BAV）和病变瓣膜中观察到的拉伸升高与高血压也相关 与CAVD。这表明，在CAVD的发病机制中，d-血流和牵张升高可能发挥作用， 其机制尚不清楚。我们的目标是确定CAVD中机械力的作用和机制 发病机制并利用知识开发新型抗CAVD疗法。在上一个供资周期， 我们已经在人类AV内皮细胞中鉴定出了几种流动和侧边依赖性microRNAs（miRNAs）， （HAVEC）和猪AV（PAV），并开始确定它们在CAVD中的作用。最近我们发现了一本小说 流动和伸展敏感的miR-483- 3 p（miR-483），其作为CAVD的关键调节剂具有令人兴奋的潜力 发病机制我们的初步数据显示，miR-483的表达减少1）通过d-flow（OS）相比， HAVEC中的稳定流动（LS），2）与人和猪AV中的心室相比，纤维层中的稳定流动（LS），以及3） 通过离体PAV中的病理性拉伸条件。进一步的数据表明miR-483抑制EC炎症 和内皮-间质转化（EndoMT），CAVD中的关键病理生物学事件， miR-483的基因靶点是Ube 2c（E2泛素结合酶），其反过来可以靶向缺氧-缺氧 诱导因子（HIF 1 α）通过调控其上游调控因子pVHL而发挥作用。HIF 1 α在CAVD中的作用尚不清楚，但其良好的- 已知靶基因包括VEGF（血管生成和炎症）、TGFβ（纤维化和钙化）、Runx 2 （钙化）和Twist 1（EndoMT），关键的CAVD致病过程。因此，我们的总体假设是 miR-483是一种抗CAVD miRNA，在OS/病理拉伸条件下减少，导致 Ube 2c增加，进而泛素化pVHL以降解并增加HIF 1 α水平。HIFα， 然后，刺激其靶基因，导致炎症，EndoMT，AV硬化和钙化。我们将测试 这分为三个目标。目的1将确定miR-483调节剪切依赖性反应的机制， HAVEC和PAV在体外和离体以Ube 2c和HIF 1 α依赖性方式表达。目标2将决定 miR-483通过Ube 2c和HIF 1 α依赖性在HAVIC和PAV离体牵张依赖性钙化中的作用 机制等目的3是一项体内研究，其中miR-483、Ube 2c和HIF 1 α将被遗传修饰， 我们刚刚通过治疗GATA 5-/- 用AAV-PCSK 9处理BAV小鼠以诱导高胆固醇血症。在这里，我们将测试他们的角色和他们的反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.