Table_2_The Role of Wnt/β-Catenin Pathway Mediators in Aortic Valve Stenosis.DOCX

Aortic valve stenosis (AVS) is a prevailing and life-threatening cardiovascular disease in adults over 75 years of age. However, the molecular mechanisms governing the pathogenesis of AVS are yet to be fully unraveled. With accumulating evidence that Wnt signaling plays a key role in the development of AVS, the involvement of Wnt molecules has become an integral study target in AVS pathogenesis. Thus, we hypothesized that the Wnt/β-catenin pathway mediators, SFRP2, DVL2, GSK3β and β-catenin are dysregulated in patients with AVS. Using immunohistochemistry, Real-Time qPCR and Western blotting, we investigated the presence of SFRP2, GSK-3β, DVL2, and β-catenin in normal and stenotic human aortic valves. Markedly higher mRNA and protein expression of GSK-3β, DVL2, β-catenin and SFRP2 were found in stenotic aortic valves. This was further corroborated by observation of their abundant immunostaining, which displayed strong immunoreactivity in diseased aortic valves. Proteomic analyses of selective GSK3b inhibition in calcifying human aortic valve interstitial cells (HAVICs) revealed enrichment of proteins involved organophosphate metabolism, while reducing the activation of pathogenic biomolecular processes. Lastly, use of the potent calcification inhibitor, Fetuin A, in calcifying HAVICs significantly reduced the expression of Wnt signaling genes Wnt3a, Wnt5a, Wnt5b, and Wnt11. The current findings of altered expression of canonical Wnt signaling in AVS suggest a possible role for regulatory Wnts in AVS. Hence, future studies focused on targeting these molecules are warranted to underline their role in the pathogenesis of the disease.

主动脉瓣狭窄（Aortic valve stenosis, AVS）是75岁以上成年人中高发且危及生命的心血管疾病。然而，调控AVS发病的分子机制尚未完全阐明。已有越来越多证据表明Wnt信号通路在AVS发生发展中发挥关键作用，因此Wnt家族分子已成为AVS发病机制研究的核心靶点。据此，我们提出假设：Wnt/β-连环蛋白（Wnt/β-catenin）通路介质分泌型卷曲相关蛋白2（SFRP2）、散乱蛋白2（DVL2）、糖原合成酶激酶3β（GSK3β）及β-连环蛋白（β-catenin）在AVS患者体内存在表达失调。我们采用免疫组化（immunohistochemistry）、实时定量聚合酶链反应（Real-Time qPCR）及蛋白质印迹法（Western blotting），检测了正常与狭窄人主动脉瓣中SFRP2、GSK-3β、DVL2及β-连环蛋白的表达情况。结果显示，狭窄主动脉瓣中GSK-3β、DVL2、β-连环蛋白及SFRP2的mRNA与蛋白质表达水平显著升高。这一结果进一步通过免疫染色观察得到验证：病变主动脉瓣中上述分子呈现出强烈的免疫反应性，染色信号丰富。对钙化人主动脉瓣间质细胞（HAVICs）进行选择性GSK3β抑制后的蛋白质组学分析显示，参与有机磷酸酯代谢的蛋白质发生富集，同时致病生物分子过程的激活受到抑制。最后，在钙化HAVICs中使用强效钙化抑制剂胎球蛋白A（Fetuin A），可显著降低Wnt信号通路基因Wnt3a、Wnt5a、Wnt5b及Wnt11的表达水平。本研究发现AVS患者体内经典Wnt信号通路的表达发生改变，提示调控性Wnt蛋白可能在AVS发病过程中发挥作用。因此，未来开展靶向上述分子的相关研究，对于明确其在该疾病发病机制中的作用具有重要价值。