Galectin-3: A mediator of vascular remodeling in pulmonary arterial hypertension

项目总结 肺动脉高压(PAH)是一种渐进性衰弱并最终致命的疾病， 只有症状才能用目前的疗法治疗。PAH的一个定义特征是 细胞过度增殖和肺动脉(PA)重塑，导致 血管阻力和僵硬，最终右室衰竭和死亡。PAH有一个 确诊后生存时间不到五年，目前的治疗策略是自我限制的 它们不能充分延长生存时间或逆转病理性血管重塑。在 在之前的资助期间，我们确定了Galectin-3(Gal-3)在调节异常中的重要作用 肺血管重构，并证明Gal-3基因敲除大鼠对PAH和PAH有保护作用 进一步表明Gal-3的药理抑制剂具有预防和逆转已建立的PAH的能力。 GAL-3在肺动脉平滑肌细胞(PASMC)中也有表达，并促进 增殖、迁移和抗凋亡。 在这一竞争性更新的初步数据中，我们发现HIF2α有力地上调了Gal-3的表达。 沿着这些思路，我们已经在LGALS3启动子上发现了一个新的hif2α结合位点，并使用了 衣壳蛋白修饰的AAV2特异性靶向肺内皮细胞(EC)，我们还显示增加了 体内表达HIF2α可增加Gal-3的表达，促进小鼠的肺动脉高压。在……里面 共培养实验表明，HIF_2α在血管内皮细胞中的表达上调可促进内皮细胞增殖 GAL-3在PASMC中的表达并促进其增殖。我们还鉴定了DNA 甲基化作为Gal-3表达的主要调节因子，作为DNA甲基转移酶，DNMT3A是 在PAH中表达下调。此外，沉默DNMT3A增加了Gal-3的表达，并增加了dCas9-DNMT3A 融合针对Gal-3启动子，降低Gal-3的表达。在体外，低氧与低氧 DNA甲基化缺失协同驱动Gal-3的高水平表达，而NEAT1是最近的 描述了低氧调节的lncRNA，它调节平滑肌细胞的表型和增殖。 为此，我们发现NEAT1在人PAH肺和PA中的表达增加 并受PASMC中Gal-3的调节。我们的长期目标是：1.确定细胞 Gal-3在介导血管重塑和PAH中的特殊作用，2.研究DNA的作用 甲基化和DNMT3A对Gal-3表达的调控及其与HIF2的协同作用，以及3.确定 Gal-3对PASMC中NEAT1的调节是否参与了PAH。

PROJECT SUMMARY Pulmonary Arterial Hypertension (PAH) is a progressively debilitating and eventually lethal disease, whereby only the symptoms are treated by current therapeutics. A defining characteristic of PAH is the excessive cellular proliferation and remodeling of pulmonary arteries (PA) that results in increased vascular resistance and stiffness, and eventually failure of the right ventricle and death. PAH has a survival time of less than five-years post diagnosis, and current treatment strategies are self-limiting in that they do not sufficiently prolong survival time or reverse the pathologic vascular remodeling. In the previous funding period we established an important role for Galectin-3 (Gal-3) in mediating aberrant pulmonary vascular remodeling, and demonstrated that Gal-3 knockout rats are protected from PAH and further showed the ability of pharmacological inhibitors of Gal-3 to prevent and reverse established PAH. Gal-3 expression was also found in pulmonary arterial smooth muscle cells (PASMC), where it promoted proliferation, migration and resistance to apoptosis. In preliminary data for this competitive renewal, we find that HIF2α robustly upregulates Gal-3 expression. Along these lines, we have uncovered a novel HIF2α binding site on the LGALS3 promoter, and using a capsid modified AAV2 that specifically targets lung endothelial cells (EC), we also show that increased expression of HIF2α in PAEC in vivo increases Gal-3 and promotes pulmonary hypertension in mice. In co-culture experiments we show that upregulation of HIF2α in the endothelium can promote increased Gal-3 expression in the underlying PASMC and enhance proliferation. We also identified DNA methylation as a major regulator of Gal-3 expression as the DNA methyltransferase, DNMT3A was downregulated in PAH. Further, silencing DNMT3A increased Gal-3 expression, and a dCas9-DNMT3A fusion directed to the Gal-3 promoter, decreased Gal-3 expression. In vitro, the combination of hypoxia and loss of DNA methylation synergize to drive high level Gal-3 expression, and NEAT1 is a recently described hypoxia regulated lncRNA that regulates smooth muscle cell phenotype and proliferation. Towards this end, we found that NEAT1 expression is increased in human PAH lung as well as in PA from MCT rats and is regulated by Gal-3 in PASMC. Our long term objectives are to 1. Determine the cell specific role of Gal-3 in mediating vascular remodeling and PAH, 2. Investigate the role of DNA methylation and DNMT3A in regulating Gal-3 expression and synergy with HIF2, and 3. Determine whether Gal-3 regulation of NEAT1 in PASMC contributes to PAH.