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

项目摘要 肺动脉高压（PAH）是一种逐渐衰弱并最终致命的疾病， 由此只有症状被当前的治疗剂治疗。PAH的一个定义特征是 肺动脉（PA）的过度细胞增殖和重塑， 血管阻力和僵硬，最终导致右心室衰竭和死亡。PAH有一个 诊断后生存时间少于5年，目前的治疗策略是自限性的， 它们不能充分延长存活时间或逆转病理性血管重塑。在 在上一个资助期，我们确定了半乳糖凝集素-3（Gal-3）在介导异常 肺血管重塑，并证明Gal-3基因敲除大鼠免受PAH， 进一步显示了Gal-3的药理学抑制剂预防和逆转已建立的PAH的能力。 肺动脉平滑肌细胞（PASMC）中也发现了Gal-3表达，在那里它促进了肺动脉平滑肌细胞（PASMC）的生长。 增殖、迁移和抗凋亡。 在这种竞争性更新的初步数据中，我们发现HIF 2 α强烈上调Gal-3表达。 沿着这些线索，我们发现了一个新的HIF 2 α结合位点在LGALS 3启动子上，并使用一种新的方法， 衣壳修饰的AAV 2特异性靶向肺内皮细胞（EC），我们还表明， 在体内PAEC中HIF 2 α的表达增加Gal-3并促进小鼠肺动脉高压。在 共培养实验表明，内皮细胞中HIF 2 α的上调可以促进内皮细胞的增殖， Gal-3在基础PASMC中表达并增强增殖。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，DNMT 3A是 在PAH中下调。此外，沉默DNMT 3A增加Gal-3表达，并且dCas 9-DNMT 3A表达增加。 针对Gal-3启动子的融合降低了Gal-3表达。在体外，缺氧和 和DNA甲基化缺失协同驱动高水平的Gal-3表达，NEAT 1是最近发现的一种新的基因。 描述了低氧调节的lncRNA，其调节平滑肌细胞表型和增殖。 为此，我们发现NEAT 1在人肺动脉高压肺组织中的表达增加， 在PASMC中受Gal-3调节。我们的长期目标是1。确定小区 Gal-3在介导血管重塑和PAH中的特定作用，2.研究DNA的作用 甲基化和DNMT 3A调节Gal-3表达以及与HIF 2 α的协同作用，以及3.确定 Gal-3对PASMC中NEAT 1的调节是否有助于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.