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

缺氧诱导因子：肺动脉高压中的半乳糖凝集素 3 信号轴

• 批准号: 9761053
• 负责人: Stephen Haigh
• 金额: $ 4.06万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9761053
• 关键词: Affect; Age; Age-Years; Animal Model; Animals; Apoptosis; Apoptotic; Automobile Driving; BCL2 gene; Binding; Biology; Blood Vessels; C-terminal; Carbohydrates; Cell Nucleus; Cell Proliferation; Cells; Cessation of life; Collaborations; Complex; DNA Sequence Alteration; Data; Dependovirus; Deubiquitinating Enzyme; Development; Diagnosis; Disease; Endothelial Cells; Endothelium; Experimental Designs; Experimental Models; Exposure to; Failure; Family; Funding; Future; Galactose; Galactose Binding Lectin; Galectin 3; Genes; Genetically Engineered Mouse; Germany; Goals; Human; Hypoxia; Hypoxia Inducible Factor; Induction of Apoptosis; Laboratories; Lectin; Link; LoxP-flanked allele; Lung; Measurement; Mediating; Mentorship; Mitochondria; Mitosis; Modeling; Monocrotaline; Mus; Mutation; N-terminal; Oxygen; Play; Procollagen-Proline Dioxygenase; Promoter Regions; Property; Protein Isoforms; Proteins; Proteolysis; Pulmonary Hypertension; Pulmonary artery structure; Rattus; Repression; Research; Resistance; Response Elements; Risk; Role; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Testing; Time; Treatment Efficacy; Ubiquitination; Universities; Up-Regulation; Vascular remodeling; Vascular resistance; Ventricular; Woman; base; design; endothelial dysfunction; experience; experimental study; hemodynamics; human model; in vivo; indexing; inhibitor/antagonist; insight; member; men; multicatalytic endopeptidase complex; mutant; novel; overexpression; pressure; protein expression; pulmonary arterial hypertension; reconstitution; skills; targeted treatment; therapeutic target; transcription factor

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.

项目概要 肺动脉高压（PAH）是一种复杂且致命的疾病，其特征是血压持续升高 在肺动脉压中。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。我们的实验室已经鉴定出 Galectin-3（GAL-3 或 Lgals3）在驱动 PASMC 增殖和肺压升高中发挥关键作用， 但 GAL-3 上调和功能的机制仍不清楚。许多研究已经 研究表明，缺氧诱导因子 (HIF) 会导致人类和动物模型中的 PAH。我们还有 发现从 PAH 大鼠模型中分离出的 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BH1样结构域，该结构域具有高度同源性的NWGR序列 抗凋亡蛋白 BCL2。我们的中心假设是内皮 HIF-2α 上调 GAL-3，从而 通过 NWGR 结构域抑制细胞凋亡，在驱动 PASMC 增殖中发挥重要作用。 我们将在 2 个具体目标中检验这一假设： 目标 1 将研究 HIF-2α 如何上调 PASMC GAL-3 多环芳烃。我们将在 PAH 大鼠模型中施用选择性 HIF-2α 抑制剂，并期望 GAL-3 表达和 PAH的血流动力学指标会下降。使用新型 GAL-3 floxed 小鼠和新型肺内皮- 表达 HIF-2α 组成型的特定 AAV，我们将检验内皮 HIF-2α 的假设 增加内皮 GAL-3，促进 PASMC 增殖和肺血管重塑。我们将 进一步研究通过增加去泛素化表达来上调 HIF-2α 的新机制 PAH 中的酶 Otud7b (Cezanne)。目标 2 将确定 GAL-3 中 NWGR 域作为 PASMC 细胞凋亡和增殖的调节因子。这将在 GAL-3 KO 大鼠培养的 PASMC 中进行测试 使用腺病毒方法重新表达 WT 或突变体 (G182A) GAL-3。在体内，我们将产生肺 表达 WT 或 G182A GAL-3 并在治疗前转导 GAL-3 KO 大鼠的内皮特异性 AAV MCT 的管理。我们预计突变体 GAL-3 将增加细胞凋亡并减少细胞增殖 PASMC 和体内这将导致肺血管重塑和 PAH 的减少。拟议的 项目旨在开发我没有丰富经验的新技能，主要是动物处理， 血流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 David 博士的指导下在奥古斯塔大学血管生物学中心进行 富尔顿和共同发起人斯科特·巴曼博士。拟议项目的资助期限为 3 年，拟 研究将分三年进行，并计划在资助的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，并进一步阐明 PASMC 增殖增加的机制可能有助于开发更多 有效的治疗方法。