The hypoxia inducible factor: Galectin 3 signaling axis in pulmonary hypertension

项目摘要 肺动脉高压（PAH）是一种复杂和致命的疾病，其特征是持续增加 肺动脉压肺动脉平滑肌细胞（PASMC）过度增殖导致 血管阻力增加最终导致右心室衰竭和死亡。我们的实验室发现 半乳糖凝集素-3（GAL-3或Lgals 3）在驱动PASMC增殖和肺压升高中的关键作用， 但GAL-3上调和功能的潜在机制仍不清楚。大量研究 表明缺氧诱导因子（HIF）有助于人类和动物模型中的PAH。我们还 我们发现在PAH大鼠模型的分离PA中HIF表达增加，我们的初步数据表明， HIF-2α上调GAL-3。GAL-3含有BH 1样结构域，该结构域与NWGR序列具有高度同源性 抗凋亡蛋白BCL 2我们的中心假设是内皮细胞HIF-2α上调GAL-3， 通过NWGR结构域抑制细胞凋亡，在驱动PASMC增殖中起重要作用。 我们将在两个具体目标中检验这一假设：目标1将检测HIF-2α如何上调PASMC GAL-3， 呸。我们将在PAH大鼠模型中给予选择性HIF-2α抑制剂，并预期GAL-3表达和 PAH血流动力学指标下降。使用一种新的GAL-3 floxed小鼠和一种新的肺内皮细胞， 表达HIF-2α组成型的特异性AAV，我们将检验内皮细胞HIF-2α 增加内皮GAL-3，其驱动PASMC增殖和肺血管重塑。我们将 进一步研究HIF-2α通过增加去泛素化蛋白表达上调的新机制。 酶Otud 7 b（Cezanne）。目标2将确定GAL-3中NWGR结构域作为 PASMC凋亡和增殖的调节因子。这将在GAL-3 KO大鼠培养的PASMC中进行检测， 使用腺病毒方法再表达WT或突变体（G182 A）GAL-3。在体内，我们将产生肺 内皮特异性AAV表达WT或G182 A GAL-3和G182 A GAL-3 KO大鼠， 给MCT。我们预期突变GAL-3将增加细胞凋亡和减少细胞增殖。 PASMC和体内这将导致肺血管重塑和PAH减少。拟议 项目旨在开发新的技能，我没有显着的经验，主要是动物处理， 血液动力学变量的测量和涉及动物模型的实验设计。该项目将 在赞助商大卫博士的指导下，在奥古斯塔大学血管生物学中心进行 富尔顿和共同赞助人斯科特·巴曼博士。拟议项目为期3年， 研究将在三年内进行，并计划在最后一年进行论文答辩。 总体而言，我们预计该应用将突出GAL-3在PAH中的重要性，并将进一步阐明 PASMC增殖增加的潜在机制可能有助于发展更多的 有效的治疗方法。

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) is a complex and fatal disorder characterized by an unrelenting increase in pulmonary arterial pressure. Excessive proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased vascular resistance and eventually right ventricular failure and death. Our laboratory has identified a key role for Galectin-3 (GAL-3 or Lgals3) in driving PASMC proliferation and elevated pulmonary pressures, but the mechanisms underlying GAL-3 upregulation and function remain unknown. Numerous studies have shown that hypoxia inducible factors (HIFs) contribute to PAH in humans and animal models. We have also found increased HIF expression in isolated PA from rat models of PAH and our preliminary data suggests that HIF-2α upregulates GAL-3. GAL-3 contains a BH1 like domain that has an NWGR sequence with high homology to the anti-apoptotic protein, BCL2. Our central hypothesis is that endothelial HIF-2α upregulates GAL-3 which plays an important role in driving PASMC proliferation through repression of apoptosis via the NWGR domain. We will test this hypothesis in 2 specific aims: Aim 1 will examine how HIF-2α upregulates PASMC GAL-3 in PAH. We will administer selective HIF-2α inhibitors in rat models of PAH and expect that GAL-3 expression and hemodynamic indices of PAH will decline. Using a novel GAL-3 floxed mice and a novel lung endothelium- specific AAV that expresses a constitutive form of HIF-2α, we will test the hypothesis that endothelial HIF-2α increases endothelial GAL-3 which drives PASMC proliferation and pulmonary vascular remodeling. We will further investigate a novel mechanism of HIF-2α upregulation via increased expression of the deubiquitinating enzyme Otud7b (Cezanne) in PAH. Aim 2 will determine the importance of the NWGR domain in GAL-3 as a regulator of PASMC apoptosis and proliferation. This will be tested in cultured PASMC from GAL-3 KO rats via re-expression of WT or mutant (G182A) GAL-3 using adenoviral approaches. In vivo, we will generate lung endothelium specific AAVs that express WT or G182A GAL-3 and transduce GAL-3 KO rats prior to administration of MCT. We anticipate that mutant GAL-3 will increase apoptosis and decrease proliferation in PASMC and in vivo this will result in a reduction in pulmonary vascular remodeling and PAH. The proposed project is designed to develop new skills that I do not have significant experience in, primarily animal handling, measurements of hemodynamic variables and experimental design involving animal models. This project will take place at Augusta University within the Vascular Biology Center under the mentorship of Sponsor Dr. David Fulton and Co-Sponsor Dr. Scott Barman. The proposed project is for 3 years of funding and the proposed research would be divided amongst the three years with plans for thesis defense during the final year of funding. Overall we expect this application will highlight the importance of GAL-3 in PAH and will further elucidate the mechanisms underlying increased PASMC proliferation which may be useful in the development of more effective therapeutic approaches.