Role of MicroRNAs 424 and 503 in Pulmonary Arterial Hypertension

描述（由申请人提供）：肺动脉高压（PAH）是一种以肺小动脉血管重塑为特征的疾病，包括由增殖内皮细胞组成的丛状和同心性病变的形成。我们最近发现，由配体apelin和受体APJ组成的G蛋白偶联受体信号传导轴的破坏导致小鼠缺氧诱导的肺动脉高压（PH）显著恶化，并发现该途径在临床PAH中也受到干扰。为了进一步表征下游内皮靶点，其可被爱帕琳-APJ信号传导利用以保护免于PAH中的重构过程，我们对经受爱帕琳、APJ或两者的敲低的肺动脉内皮细胞（PAEC）进行了microRNA（miRNA）阵列分析。我们鉴定了两种在PAEC中高度表达的miRNA（miR-424和miR-503），但在apelin/APJ敲低的情况下显著下调。值得注意的是，这些miRNAs在实验性肺动脉高压大鼠模型以及从临床诊断为肺动脉高压的患者中分离的PAEC中也显著减少。我们的体外功能研究表明，miR-424和miR-503都通过至少部分涉及靶向FGF信号通路的机制发挥抗增殖、抗血管生成作用。我们发现，通过鼻内慢病毒递送在野百合碱诱导的PH模型中恢复miR-424/503表达可以显著改善疾病的严重程度。基于这些初步数据，我们假设miR-424/503在维持肺血管稳态中起关键作用，并且其表达的破坏对作为PAH标志的异常内皮增殖起关键作用。我们提出以下目的以进一步评估miR-424/503在PAH中的作用：1）确定PAEC中miR-424/503的转录调节机制，其涉及通过apelin-APJ信号传导靶向肌细胞特异性增强因子2（MEF 2），2）评估PH实验模型中miR-424/503的内皮特异性表达的功效，3）进一步验证miR-424/503在临床PAH中的作用。

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a disease characterized by the vascular remodeling of the pulmonary arterioles, including formation of plexiform and concentric lesions comprised of proliferative endothelial cells. We recently found that disruption of the G protein-coupled receptor signaling axis comprised of the ligand apelin and the receptor APJ leads to significant worsening of hypoxia induced pulmonary hypertension (PH) in mice, and found that the pathway is also perturbed in clinical PAH. To further characterize the downstream endothelial targets which may be utilized by apelin-APJ signaling to protect against the remodeling processes in PAH, we carried out a microRNA (miRNA) array analysis of pulmonary artery endothelial cells (PAECs) subjected to knockdown of apelin, APJ, or both. We identified two miRNAs (miR-424 and miR-503) that are highly expressed in PAECs, but are significantly downregulated in the context of apelin/APJ knockdown. Remarkably, these miRNAs are also markedly decreased in the experimental monocrotaline rat model of PH, as well as in PAECs isolated from patients with clinically diagnosed PAH. Our in vitro functional studies demonstrated that both miR-424 and miR-503 exert anti-proliferative, anti-angiogenic effects, via a mechanism that at least in part involves targeting of the FGF signaling pathway. We found that restoration of miR-424/503 expression in the monocrotaline induced model of PH by intranasal lentiviral delivery can significantly ameliorate the severity of the disease. Based o these preliminary data, we hypothesize that miR-424/503 play a critical role in maintaining pulmonary vascular homeostasis, and that disruption of their expression serves a critical contribution to the aberrant endothelial proliferation which is a hallmark of PAH. We propose the following aims to further evaluate the role of miR-424/503 in PAH: 1) Determine the transcriptional regulatory mechanisms of miR-424/503 in PAECs that involves targeting of the myocyte-specific enhancer factor 2 (MEF2) by apelin-APJ signaling, 2) Evaluate the efficacy of endothelial specific expression of miR-424/503 in experimental models of PH, and 3) Further validate the role for miR-424/503 in clinical PAH.