Novel roles of RNA modifications in the pathogenesis of pulmonary vascular remodeling and PAH

肺动脉高压（PAH）是一种以肺动脉高压为特征的疾病， 阻力和闭塞性血管重塑，导致右心衰竭和过早死亡。给定 闭塞性血管重塑的潜在分子机制仍然是个谜， 没有针对基本的疾病修饰机制，因此只导致了适度的 降低发病率和死亡率。而一些转录因子和转录辅因子， 活化剂已经在PAH的背景下进行了研究，转录后调节的mRNA，可以影响 关键蛋白质的表达仍在很大程度上未被探索。包括N6-甲基腺苷的RNA修饰 (m6A)最近被发现是基因表达的重要调节因子。M6 A改装 控制RNA的稳定性、运输和翻译，并与肥胖等人类疾病有关， 癌的尽管其在各种基本生物过程中的功能重要性，m6 A修饰的研究 PAH中的mRNA缺乏。我们的支持性数据表明，脂肪量和肥胖相关的表达， FTO蛋白（FTO）是一种特征性的RNA去甲基化酶，在肺血管内皮细胞中显著升高， 特发性PAH患者的内皮细胞（EC）。Tie 2Cre介导的内皮细胞Fto缺失（FtoTie 2Cre）抑制PH 慢性缺氧引起的我们的机制研究提供的证据表明，几个PAH致病基因是 人肺EC中的潜在FTO靶点。此外，药理学抑制FTO在野百合碱- 激发大鼠抑制肺血管重塑和PH。因此，我们假设，内皮细胞， FTO，作为一个主要的m6 A擦除器，作为一个关键的调节mRNA的稳定性和积累的关键PAH引起的， 基因调节肺血管收缩和血管重塑，因此是一种新的治疗方法， PAH的目标。我们提出以下三个具体目标。在目标1中，我们将定义 内皮FTO在PAH发病机制中的作用。在目标2中，我们将描述 FTO调节内皮功能障碍，导致肺血管收缩和血管重塑。 我们将确定EC中的关键FTO靶点，并解决挽救FtoTieCre表型的可能性。 通过新型纳米颗粒介导的体内EC特异性基因转移。在目标3中，我们将探索治疗 FTO抑制剂（包括FDA批准的再利用药物）治疗PAH的潜力， PAH的免费动物模型。基于我们新发现的明确临床相关性，我们预计 通过描述分子和细胞的结构， 内皮功能障碍导致肺血管收缩和血管重塑的机制， 确定可药物化的目标，并探索可以治疗癌症的药理学药物。 抑制/逆转血管重塑，并抑制血管收缩，用于预防和治疗PAH 在病人身上。因此，我们认为所提出的研究具有很大的转化潜力。

Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and obliterative vascular remodeling that causes right heart failure and premature death. Given the underlying molecular mechanisms of obliterative vascular remodeling remain enigmatic, current therapies have not targeted the fundamental disease modifying mechanisms and hence only resulted in a modest improvement in the morbidity and mortality. While several transcription factors and transcriptional co- activators have been studied in the context of PAH, post-transcriptional regulations of mRNAs that can affect expression of key proteins remain largely unexplored. RNA modifications including N6-methyladenosine (m6A) have recently been discovered as essential regulators of gene expression. The m6A modification controls RNA stability, transport, and translation and has been linked to human diseases such as obesity and cancers. Despite its functional importance in various fundamental bioprocesses, studies of m6A modification of mRNAs in PAH are lacking. Our Supporting Data show that expression of fat mass and obesity-associated protein (FTO), a well-characterized RNA demethylase, is markedly elevated in pulmonary vascular endothelial cells (ECs) of idiopathic PAH patients. Tie2Cre-mediated deletion of Fto in ECs (FtoTie2Cre) inhibited PH induced by chronic hypoxia. Our mechanistic studies provide evidence that several PAH-causing genes are potential FTO targets in human lung ECs. Furthermore, pharmacological inhibition of FTO in monocrotaline- challenged rats inhibited pulmonary vascular remodeling and PH. Thus, we hypothesize that endothelial FTO, as a major m6A eraser, acts as a key regulator of mRNA stability and accumulation of key PAH-causing genes in ECs to regulate pulmonary vasoconstriction and vascular remodeling and thus is a novel therapeutic target for PAH. We propose the following 3 Specific Aims. In Aim 1, we will define the fundamental role of endothelial FTO in the pathogenesis of PAH. In Aim 2, we will delineate the molecular mechanisms underlying FTO regulation of endothelial dysfunction leading to pulmonary vasoconstriction and vascular remodeling. We will identify the key FTO targets in ECs and address the possibility of rescuing the phenotype of FtoTieCre by novel nanoparticle-mediated in vivo EC-specific gene transfer. In Aim 3, we will explore the therapeutic potential of FTO inhibitors including a repurposed FDA-approved drug in the treatment of PAH employing 3 complimentary animal models of PAH. Based on the clear clinical relevance of our novel findings, we expect that the proposed studies have significant translational potential by delineating the molecular and cellular mechanisms of endothelial dysfunction leading to pulmonary vasoconstriction and vascular remodeling, identifying druggable targets, and exploring pharmacological agents that can pharmacologically inhibit/reverse vascular remodeling and also inhibit vasoconstriction for the prevention and treatment of PAH in patients. Thus, we believe the proposed studies have great translational potential.