Role of SIN3a in the epigenetic regulation of the Bone Morphogenetic Protein Receptor Type 2 in pulmonary arterial hypertension

项目摘要 PAH是一种以远端肺血管动脉进行性重塑为特征的致死性疾病， 导致肺血管阻力和肺动脉压升高，这可能导致严重的 并发症，如右心衰竭，最终死亡。越来越多的证据表明， 功能障碍是PAH的第一个触发因素之一，导致血管细胞、血管 重塑和肺血管闭塞。PAH中最常见的病理机制之一 是血管细胞中骨形态发生蛋白2型受体（BMPR 2）信号传导的改变。的 由突变或表达缺失诱导的BMPR 2功能丧失与严重的血流动力学异常相关。 PAH患者的特征和不良结局。多项研究指出肺内皮细胞是 在PAH中受BMPR 2损失影响最严重。然而，潜在的分子机制， BMPR 2表达的调节及其相关的PAH样表型仍然是未知的。我们集团 最近表明，SIN 3a在BMPR 2启动子的DNA和组蛋白甲基化中起核心作用， 肺动脉平滑肌细胞与肺动脉高压的发病机制。然而，表观遗传和转录 SIN 3a调控PAEC中BMPR 2基因的机制仍有待阐明。 本研究旨在揭示SIN 3a在肺内皮功能障碍中的作用， 我们的目标是 在PAH中，确定 PAEC中BMPR 2表达调节的下游机制，并评估治疗效果 编码SIN 3a的修饰mRNA在PAH动物模型中的作用。我们的初步数据显示，SIN 3a是 在从PAH患者分离的hPAEC中显著下调。在体外，我们观察到SIN 3a沉默， 下调BMPR 2表达和信号传导，同时增强PAEC增殖和迁移。 从机制上讲，我们发现了一种新的分子途径，SIN 3a通过该途径调节hPAEC中的BMPR 2水平。 我们的数据表明，SIN 3a过表达通过抑制Zeste增强子同源物2上调FOXK 2 F0 XK 2启动子中的（EZH 2）介导的组蛋白甲基化。最终，SIN 3a过表达增加 FOXK 2与BMPR 2启动子结合并上调hPAEC中的BMPR 2水平。基于这些发现， 我们假设SIN 3a的缺失损害了PAEC中BMPR 2的表达，触发了内皮细胞的凋亡。 肺动脉高压中的功能障碍和血管重塑。在这个提议中，我们的假设将通过追求 以下三个具体目的：目的1）研究SIN 3a在内皮细胞功能障碍中的作用， BMPR 2在PAH-hPAEC中调控的分子机制。目的2）阐明影响 SIN 3a缺陷在PAH发作中在平滑肌细胞和内皮细胞中使用双重方法。目的 3)评价SIN 3a modRNA在PAH临床前模型中的治疗效果。定义监管 PAH中BMPR 2表达缺失的潜在机制将具有重要意义。最后，恢复 使用SIN 3a修饰的RNA在肺中表达SIN 3a可能是治疗PAH的新的有希望的策略。

Project Summary PAH is a fatal disease characterized by the progressive remodeling of the distal pulmonary vascular arteries, resulting in elevated pulmonary vascular resistance and pulmonary artery pressure, which may lead to serious complications, such as right heart failure, and ultimately death. Accumulating evidence indicates that endothelial dysfunction is one of the first triggers in PAH that leads to uncontrolled proliferation of vascular cells, vascular remodeling, and occlusion of the pulmonary blood vessels. One of the most common pathomechanisms in PAH is the alteration of the Bone Morphogenetic Protein Type 2 Receptor (BMPR2) signaling in vascular cells. The loss of BMPR2 function, induced by mutation or a loss of expression, is associated with a severe hemodynamic profile and poor outcomes in PAH patients. Multiple studies point to the pulmonary endothelium as the cell type that is most critically impacted by BMPR2 loss in PAH. However, the molecular mechanisms underlying the regulation of BMPR2 expression and its associated PAH-like phenotype remain largely unknown. Our group has recently shown that SIN3a plays a central role in the DNA and histone methylation of the BMPR2 promoter in pulmonary artery smooth muscle cells and the pathogenesis of PAH. However, the epigenetic and transcriptional mechanisms by which SIN3a regulates the BMPR2 gene in PAEC remain to be elucidated. proposal are to uncover the role of SIN3a in the pulmonary endothelial dysfunction Our objectives in this in PAH, identify the downstream mechanisms underlying the regulation of BMPR2 expression in PAEC, and evaluate the therapeutic effects of modified mRNA encoding SIN3a in animal models of PAH. Our preliminary data showed that SIN3a is significantly downregulated in hPAEC isolated from PAH patients. In vitro, we observed that SIN3a silencing downregulates BMPR2 expression and signaling while potentiating PAEC proliferation and migration. Mechanistically, we discovered a novel molecular pathway by which SIN3a modulates BMPR2 levels in hPAECs. Our data showed that SIN3a overexpression upregulates FOXK2 by repressing Enhancer of Zeste Homolog 2 (EZH2)-mediated histone methylation in the FOXK2 promoter. Ultimately, SIN3a overexpression increases FOXK2 binding to the BMPR2 promoter and upregulates BMPR2 levels in hPAECs. Based upon these findings, we hypothesize that the loss of SIN3a impairs BMPR2 expression in PAEC, triggers endothelial dysfunction and vascular remodeling in PAH. In this proposal, our hypothesis will be tested by pursuing the following three specific aims: Aim 1) To investigate the role of SIN3a in endothelial cell dysfunction and decipher the molecular mechanism underlying the regulation of BMPR2 in PAH-hPAEC. Aim 2) To elucidate the effects of SIN3a deficiency in the onset of PAH in smooth muscle cells and endothelial cells using a dual approach. Aim 3) To evaluate the therapeutic efficacy of SIN3a modRNA in preclinical models of PAH. Defining the regulatory mechanisms underlying the loss of BMPR2 expression in PAH will be of great relevance. Finally, restoring the expression of SIN3a in the lungs using SIN3a modified RNA might be a new promising strategy for treating PAH.