ACVRL1突变在肺动脉高压发生中的作用和机制

Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease with poor prognosis. Pulmonary vascular remodeling due to activation of the TGF-β signaling pathway is an important molecular mechanism of PAH. Activin A receptor type II-like 1 (ACVRL1) mutations are known to be associated with hereditary hemorrhagic telangiectasia (HHT) with PAH presentation. Preliminary study detected ACVRL1 mutations in the non-HHT PAH patients with poor prognosis, but the mechanism of how these mutations cause PAH is currently unknown. Subsequently, we demonstrated that these mutations caused abnormal SMAD1 / 5 phosphorylation, indicating the involvement of TGF-β pathway as the downstream consequence. Therefore we hypothesize that ACVRL1 mutations diminish the antagonistic role of TGF-β pathway through abnormal SMAD1 / 5 phosphorylation in the pulmonary vascular cells, and consequently, promote cellular proliferation that leads to PAH. In this study, we will use the CRISPR/Cas9 technology for site-directed mutagenesis in the ACVRL1 gene in both zebrafish and mouse models to perform the procedure of functional verification. We will examine the pulmonary vascular activity in the zebrafish and monitor pulmonary artery morphology and pressure changes in mice. We will also examine the function of endothelial cells, the proliferation, migration and differentiation of smooth muscle. We will be able to understand the mechanism of how ACVRL1 mutations cause PAH in non-HHT patients, which in turn will enable us to provide early diagnosis and personalized treatment.

肺动脉高压(PAH)是预后不良的严重肺血管疾病，TGF-β信号通路异常激活致肺血管重构是其发生的重要分子机制之一，其中包括激活素受体样激酶1（ACVRL1）突变。已知ACVRL1突变与出血性毛细血管扩张症（HHT）发病有关，我们在非HHT合并PAH家系中发现ACVRL1新突变，并与不良预后相关，而此突变导致PAH发生的机理不明，此外还发现该突变导致SAMD1/5异常活化，由此我们假设ACVRL1突变通过影响肺血管细胞SMAD1/5磷酸化，使TGF-β通路异常活化，促进细胞增殖而导致PAH。本研究将采用CRISPR/Cas9技术对斑马鱼及小鼠行ACVRL1定点突变，观察斑马鱼同源肺循环血管及小鼠肺动脉增殖及压力变化，检测内皮功能和平滑肌的增殖迁移分化，明确ACVRL1突变及新位点突变在非HHT患者PAH产生的作用及机制，结合患者临床转归，为早期诊断和个体化治疗提供遗传学及转化医学依据。

肺动脉高压已的发病机制尚不完全清楚，它涉及细胞、体液介质和分子遗传等多个途径。本课题拟阐明ACVRL1突变是否参与、怎样参与PAH的发生发展；我们的主要工作是：1.收集患者血液样本库及临床资料，利用目标基因捕获及二代高通量测序技术，进行候选基因筛查。找出与PAH相关的致病基因多态性，排查ACVRL1突变基因，解析突变新位点并与正常序列相对比，实现对PAH的基因诊断。2. 利用ACVRL1突变质粒转染细胞，建立ACVRL1基因突变功能验证体外细胞模型，在细胞水平上验证其对TGF-β/BMP信号通路的影响，确定ACVRL1突变的致病性；制备ACVRL1突变Knock in小鼠，建立PAH在体模型，观察肺血管内皮的病变情况，研究ACVRL1受体介导的TGF-β信号通路异常对PAH表型中内皮细胞、血管平滑肌细胞过度增殖的影响，并探讨ACVRL1突变对下游信号通路相关基因的作用。3. 分别评价ACVRL1基因突变阳性/阴性PAH患者的临床表型。通过数据分析研究，明确ACVRL1突变基因型与表现型（心肺功能指标、血液生化指标）的关系，以及对PAH预后的影响，有助于细化PAH患者危险分级及制定个体化治疗方案。我们确定了ACVRL1突变参与PAH的发病，是通过影响TGF-β/BMP信号通路而实现的。基因突变阳性患者临床指标及预后更差，为早期药物治疗和干预提供了实验依据。

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