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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

基本信息

批准号：
10646507
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
项目类别：
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/10646507
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

项目摘要

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_ 应用的中心焦点是建立一种新的小鼠肺动脉高压（PH）模型，靶向内皮，并揭示了一种新的内皮驱动的非偶联eNOS-ER应激信号网络，线粒体功能障碍轴在PH的发病机制中，靶向其将导致PH的新疗法。肺动脉高压是一种严重的人类疾病，其特征在于肺中小动脉的强烈重塑，导致血管收缩、血管阻力和肺动脉压升高，并最终导致右心失败在初步研究中，我们通过直接靶向eNOS来激发eNOS，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，肺动脉高压是特发性肺动脉高压（IPAH）最早发生的事件之一，可能是IPAH的起始步骤。重要的是，DAHP处理的小鼠出现了平均肺动脉压（mPAP）增加的稳健PH表型和右心室收缩压（RVSP），伴有广泛的血管重塑，其特征是典型的中膜厚度、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PH患者相比，DAHP模型具有更明显和实质性的调节基因与缺氧模型（217 vs. 92）。在初步研究中，我们还发现了新的分子介导非偶联eNOS下游PH发展的机制，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补救酶二氢叶酸还原酶的逆转， NADPH氧化酶（NOX）激活下游的DHFR缺陷在保护eNOS偶联方面是稳健有效的活性，将检测特异性靶向NOX亚型和DHFR的新型遗传菌株调节PH的功效表型（16种新的和独特的菌株，其中大部分是在内部制造的）。在目标1中，我们的目标是建立一种新的人类样通过充分表征DAHP处理小鼠的表型，并通过比较其基因调控，与患有PH的人类患者和Sugen 5416/低氧（SuHx）治疗的小鼠的情况相比。此外，为了进一步研究在以下方面的作用，通过RNA-seq分析鉴定的新候选基因的PH开发。在目标2中，我们将检查新的内皮细胞- 使用非偶联的eNOS-ER应激-线粒体功能障碍轴在PH发病机制中的驱动信号网络在目的3中，我们将研究靶向内皮DHFR的策略，如作为DHFR的内皮特异性转基因，或在内皮中特异性敲除NOX同种型以保护 DHFR功能，将是新的治疗潜力的PH。我们还将研究是否全球和条件 DHFR的敲除或NOX亚型的内皮特异性过表达导致PH的发展和过度表达。博士新型DHFR活化剂和NOX小分子抑制剂对PH发展的影响也将进行研究。这些研究在潜在确定治疗的新治疗选择方面具有高度意义和转化性和/或预防PH，即通过减弱NOX同种型活化以保持内皮DHFR功能， eNOS偶联活性以关闭ER应激和线粒体功能障碍，或通过直接激活DHFR。