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: 10247816
• 负责人: Hua Linda Cai
• 金额: $ 52.14万
• 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/10247816
• 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; Heart failure; 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; Pulmonary artery structure; 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; inhibitor/antagonist; innovation; mitochondrial dysfunction; molecular phenotype; mortality; mouse model; novel; novel therapeutics; overexpression; preservation; pressure; prevent; pulmonary arterial hypertension; 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.

摘要_ 应用的中心焦点是通过特异性的方法建立一种新的小鼠肺动脉高压模型。 靶向内皮，揭示了一个新的内皮驱动的解偶联eNOS-ER应激信号网络- 线粒体功能障碍轴在肺高压发病机制中的作用，靶向线粒体功能障碍将为肺高压的治疗开辟新的途径。 肺动脉高压是一种严重的人类疾病，其特征是肺内小动脉严重重塑， 导致血管收缩，血管阻力和肺动脉压升高，最终导致右心 失败了。在初步研究中，我们通过直接靶向eNOS来激发eNOS，建立了一种新的PH模型 解偶联(DAHP抑制GTPCHI)和eNOS解偶联依赖的内皮功能障碍。值得注意的是，内皮细胞 功能障碍是特发性肺动脉高压最早发生的事件之一，可能是其发病的始动步骤。 重要的是，DAHP治疗的小鼠出现了平均肺动脉压(MPAP)升高的强健的PH表型 和右室收缩压(RVSP)，伴有广泛的血管重构，其特征是典型的 中层增厚、新生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性和实质性 调控基因与低氧模型(217对92)。在初步研究中，我们还发现了新的分子 内质网应激和线粒体参与介导内皮型一氧化氮合酶下游PH发生的机制 功能障碍。此外，由于eNOS辅因子四氢生物蝶呤抢救酶二氢叶酸还原酶逆转 NADPH氧化酶(NOX)激活下游的DHFR缺乏症在保护eNOS偶联方面非常有效 活性，针对NOX异构体和DHFR的新遗传菌株将被检查调节PH值的效果 表型(16个新的和独特的菌株，其中大部分是在室内制造的)。在目标1中，我们的目标是建立一个新的类似人类的 通过比较DAHP处理小鼠的表型及其基因调控，建立小鼠肺高压模型 与人类PH患者和Sugen5416/低氧(SuHx)治疗的小鼠的情况相同。还可以进一步研究在 通过rna-seq分析确定的新候选基因的ph值发展。在目标2中，我们将检查新的内皮- 非偶联eNOS-ER应激-线粒体功能障碍轴驱动的信号网络在PH发病机制中的作用 DAHP、缺氧和SU/HX模型。在目标3中，我们将检查针对内皮DHFR的策略，如 作为内皮特异性的dhfr转基因，或敲除内皮特异性的NOX异构体以保存 DHFR功能，将对PH具有新的治疗潜力。我们还将检查全局和有条件的 DHFR基因敲除，或内皮特异性NOX亚型的过度表达，会导致PH的发生和夸大 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。 这些研究在潜在地确定治疗的新的治疗方案方面具有非常重要的意义和转译意义 和/或预防PH，即通过减弱NOX异构体激活以保护内皮DHFR功能和 ENOS偶联活性，以关闭内质网应激和线粒体功能障碍，或通过直接激活DHFR。