Endothelium-driven signaling network in the development of pulmonary hypertension

肺动脉高压发生过程中内皮驱动的信号网络

• 批准号: 10434113
• 负责人: Hua Linda Cai
• 金额: $ 52.15万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10434113
• 关键词: Animal Disease Models; Animals; Arteries; Attenuated; Biological Availability; Blood Vessels; Candidate Disease Gene; Cellular biology; Collagen; Coupling; Data; Deposition; Development; Dihydrofolate Reductase; Disease; Disease Progression; Ear; Electron Spin Resonance Spectroscopy; Endothelial Cells; Endothelium; Enzymes; Event; GTP Cyclohydrolase I; Gene Expression Profile; Gene Expression Regulation; Gene Transfer Techniques; Genes; Human; Hypertension; Hypoxia; Interruption; Knock-out; Knockout Mice; Lesion; LoxP-flanked allele; Lung; Medial; Mediating; Modeling; Molecular; Mus; NADPH Oxidase; NG-Nitroarginine Methyl Ester; NOS3 gene; Nitric Oxide; Oxidation-Reduction; Pathogenesis; Pathologic; Pathologic Processes; Patients; Phenotype; Physiological; Prevention; Production; Protein Isoforms; Pulmonary Heart Disease; Pulmonary Hypertension; Pulmonology; Rodent; Role; Signal Transduction; Spectrophotometry; Superoxides; Systolic Pressure; Therapeutic; Thick; Time; Transgenic Mice; Vascular remodeling; Vascular resistance; Ventricular; attenuation; cardiopulmonary system; cofactor; conditional knockout; design; efficacy evaluation; endoplasmic reticulum stress; endothelial dysfunction; genetic strain; human disease; hypoxia-induced pulmonary hypertension; inhibitor; innovation; mitochondrial dysfunction; molecular phenotype; mortality; mouse model; novel; novel therapeutics; overexpression; preservation; prevent; pulmonary arterial hypertension; pulmonary arterial pressure; right ventricular failure; screening; small molecule; small molecule inhibitor; tetrahydrobiopterin; transcriptome sequencing; translational study; vasoconstriction

ABSTRACT ______ The central focus of application is to establish a novel pulmonary hypertension (PH) model in mice by specifically targeting the endothelium, and reveal a novel endothelium-driven signaling network of uncoupled eNOS-ER stress- mitochondrial dysfunction axis in the pathogenesis of PH, targeting of which would result in novel therapeutics for PH. Pulmonary hypertension is a severe human disease characterized by intensive remodeling of small arteries in the lung, resulting in vasoconstriction, elevated vascular resistance and pulmonary arterial pressure, and eventually right heart failure. In preliminary studies, we have generated a novel PH model by directly targeting eNOS to provoke eNOS uncoupling (DAHP to inhibit GTPCHI) and eNOS uncoupling-dependent endothelial dysfunction. Of note, endothelial dysfunction is one of the earliest events and may the initiating step of idiopathic pulmonary artery hypertension (IPAH). Importantly, DAHP-treated mice developed robust PH phenotypes of increased mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP), accompanied by extensive vascular remodel characterized by typical human like vascular lesions of medial thickness, neointimal formation, and plexiform features. RNA-sequencing (RNA- seq) data indicated that, comparing to human patients with PH, the DAHP model had more overlappingly and substantially regulated genes vs. the hypoxia model (217 vs. 92). In preliminary studies we have also revealed new molecular mechanisms mediating PH development downstream of uncoupled eNOS, involving ER stress and mitochondrial dysfunction. Additionally, since reversal of eNOS cofactor tetrahydrobiopterin salvage enzyme dihydrofolate reductase (DHFR) deficiency downstream of NADPH oxidase (NOX) activation is robustly effective in preserving eNOS coupling activity, novel genetic strains specifically targeting NOX isoforms and DHFR will be examined for efficacies modulating PH phenotypes (16 novel and unique strains, most of which made in house). In Aim 1 we aim to establish a novel human like murine model of PH by fully characterizing phenotypes of DAHP-treated mice, and by comparing its gene regulation profile to that of human patients with PH and of Sugen5416/Hypoxia (SuHx)-treated mice. Also to further examine roles in PH development of novel candidate genes identified by RNA-seq analyses. In Aim 2, we will examine novel endothelium- driven signaling network of uncoupled eNOS-ER stress-mitochondrial dysfunction axis in the pathogenesis of PH using DAHP, hypoxia and Su/Hx models of PH. In Aim 3, we will examine whether strategies targeting endothelial DHFR, such as endothelium-specific transgenesis of DHFR, or knockout of NOX isoforms specifically in the endothelium to preserve DHFR function, would be of novel therapeutic potential for PH. We will also examine whether global and conditional knockout of DHFR, or endothelium-specific overexpression of NOX isoforms, leads to PH development and exaggerated PH. Effects on PH development of novel DHFR activators, and small molecule inhibitors for NOX, will also be examined. These studies are highly significant and translational in potentially identifying novel therapeutic options for the treatment and/or prevention of PH, namely via attenuation of NOX isoform activation to preserve endothelial DHFR function and eNOS coupling activity to shut down ER stress and mitochondrial dysfunction, or via direct activation of DHFR.

抽象的 ______ 应用的中心重点是通过特异性地建立一种新型的小鼠肺动脉高压（PH）模型 靶向内皮，揭示了一种新型内皮驱动的非偶联 eNOS-ER 应激信号网络 PH 发病机制中线粒体功能障碍轴，针对该轴将产生新的 PH 治疗方法。 肺动脉高压是一种严重的人类疾病，其特征是肺部小动脉的强烈重塑， 导致血管收缩、血管阻力和肺动脉压升高，最终导致右心升高 失败。在初步研究中，我们通过直接靶向 eNOS 来激发 eNOS，生成了一种新颖的 PH 模型 解偶联（DAHP 抑制 GTPCHI）和 eNOS 解偶联依赖性内皮功能障碍。值得注意的是，内皮 功能障碍是最早的事件之一，可能是特发性肺动脉高压（IPAH）的起始步骤。 重要的是，DAHP 治疗的小鼠出现了平均肺动脉压 (mPAP) 增加的强健 PH 表型 和右心室收缩压（RVSP），伴随着广泛的血管重塑，其特征是典型的 具有中层厚度、新内膜形成和丛状特征的类人血管病变。 RNA 测序（RNA- seq）数据表明，与人类PH患者相比，DAHP模型具有更多的重叠和实质性 调节基因与缺氧模型（217 vs. 92）。在初步研究中，我们还揭示了新的分子 介导未偶联 eNOS 下游 PH 发育的机制，涉及 ER 应激和线粒体 功能障碍。此外，由于 eNOS 辅因子四氢生物蝶呤挽救酶二氢叶酸还原酶的逆转 NADPH 氧化酶 (NOX) 激活下游 (DHFR) 缺陷对于维持 eNOS 偶联非常有效 活性，将检查专门针对 NOX 同工型和 DHFR 的新型基因菌株调节 PH 的功效 表型（16 个新颖且独特的菌株，其中大部分是自制的）。在目标 1 中，我们的目标是建立一种新颖的类人 通过全面表征 DAHP 治疗小鼠的表型并比较其基因调控，建立 PH 小鼠模型 与人类 PH 患者和 Sugen5416/缺氧 (SuHx) 治疗的小鼠的情况相似。还进一步考察了角色 通过 RNA-seq 分析鉴定出新候选基因的 PH 发展。在目标 2 中，我们将检查新型内皮细胞 - PH发病机制中非偶联eNOS-ER应激-线粒体功能障碍轴驱动的信号网络 PH 的 DAHP、缺氧和 Su/Hx 模型。在目标 3 中，我们将研究是否针对内皮 DHFR 的策略，例如 作为 DHFR 的内皮特异性转基因，或特异地在内皮中敲除 NOX 同种型以保留 DHFR 功能对于 PH 具有新的治疗潜力。我们还将检查全局和条件是否 DHFR 的敲除或 NOX 亚型的内皮特异性过度表达会导致 PH 发展和夸大 PH值。还将研究新型 DHFR 激活剂和 NOX 小分子抑制剂对 PH 发展的影响。 这些研究对于潜在确定新的治疗选择具有非常重要的意义和转化作用 和/或预防 PH，即通过减弱 NOX 同工型激活来保护内皮 DHFR 功能和 eNOS 偶联活性可关闭 ER 应激和线粒体功能障碍，或通过直接激活 DHFR。