Genetic and hypoxic control of a lncRNA axis orchestrates endothelial reprogramming in pulmonary hypertension

lncRNA轴的遗传和缺氧控制协调肺动脉高压中的内皮重编程

• 批准号: 10622021
• 负责人: Stephen Y Chan
• 金额: $ 73.74万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622021
• 关键词: Alleles; American; Automobile Driving; Binding; Biology; Blood specimen; CRISPR/Cas technology; Caring; Cells; ChIP-seq; DNA; Data; Development; Disease; Endothelial Cells; Endothelium; Ensure; Epigenetic Process; Genes; Genetic; Genetic Carriers; Genomics; Genotype; Hematological Disease; Histone-Lysine N-Methyltransferase; Histones; Human; Hypoxia; Individual; Introns; Knock-out; Link; Lung; Lung diseases; Lysine; Medical; Meta-Analysis; Metabolic; Metabolic dysfunction; Metabolism; Methylation; Modeling; Molecular; Mus; Pathogenicity; Pathway interactions; Patients; Phenocopy; Phenotype; Positioning Attribute; Proteins; Publishing; Pulmonary Hypertension; Quantitative Trait Loci; RNA; Rattus; Regulation; Risk; Rodent; Role; Severities; Single Nucleotide Polymorphism; Slice; Small Interfering RNA; Specificity; Structure of parenchyma of lung; Transcript; Untranslated RNA; Validation; Vascular Diseases; Vascular remodeling; Work; adeno-associated viral vector; cadherin 5; chromatin remodeling; cohort; epigenetic therapy; functional genomics; genome editing; histone methylation; humanized mouse; in vivo; induced pluripotent stem cell; inhibitor; insight; knock-down; lung hypoxia; molecular targeted therapies; nanoparticle; promoter; pulmonary vascular remodeling; response; transcription factor

Background and Hypothesis: Pulmonary hypertension (PH) is a deadly disease, where Group 1 PAH and Group 3 PH are driven by hypoxia, HIF-2, and non-coding RNAs. We found that the lncRNA KMT2E-AS1 is up-regulated in Groups 1/3 PH and is induced by HIF-2. This lncRNA gene neighbors KMT2E, a gene controlling histone 3 lysine 4 trimethylation (H3K4me3) and chromatin remodeling. In pulmonary endothelial cells (ECs), KMT2E-AS1 stabilizes KMT2E to increase H3K4me3, thus driving HIF-2-specific metabolic and pathogenic alterations. The G-allele of single nucleotide variant (SNV) rs73184087 within KMT2E is associated with risk of developing Group 1 PAH (in discovery/validation cohorts and a meta-analysis of 2,181 PAH vs. 10,060 controls). rs73184087 also displays more avid allele (G)-specific association with HIF-2 leading to induction of this lncRNA-KMT2E pair. A mouse deficient in the conserved lncRNA sequence is protected against Groups 1/3 PH; this is phenocopied by inhibition of histone methylation in PAH rats. We postulate that the KMT2E-AS1/KMT2E axis is a central lynchpin in pathogenic reprogramming in ECs, promoting PH. Aim 1) Define the allele-specific role of the KMT2E SNV rs73184087 in controlling HIF-2-dependent EC lncRNA-KMT2E expression and PH pathophenotypes. Using ECs derived from genome-edited inducible pluripotent stem cells (iPSC) as well as primary lung ECs carrying rs73184087 A and G alleles, we will determine if (G) increases lncRNA-KMT2E by more HIF-2 binding and drives more severe EC phenotypes. We will also pursue expression quantitative trait loci (eQTL) analysis in blood samples from PAH patients (discovery/validation cohorts) and PAH lung tissues carrying A and G alleles of rs73184087. Aim 2) Define the role of this lncRNA-KMT2E axis and H3K4me3 in promoting PH in vivo. We will quantify Groups 1/3 PH severity in rodents after EC-specific knockdown of this lncRNA vs. lncRNA+KMT2E and after AAV-driven EC- specific expression of lncRNA vs. lncRNA+KMTE2. We will also determine if MM-589, a specific H3K4me3 inhibitor, reverses PAH in rats. Thus, we aim to determine if lncRNA+KMT2E together are necessary and sufficient to drive Group 1/3 PH and if PAH is dependent upon H3K4me3 activity, thus offering a new epigenetic PH therapy. Aim 3) Define the causative role of the G allele of rs73184087 on pulmonary vascular remodeling and PH in vivo. Culturing human precision cut lung slices, we will determine if the rs73184087 G allele drives vascular remodeling via regulation of the lncRNA-KMT2E axis and H3K4me3. We have also inserted the human rs73184087 G vs. A allele in mice and will use these “humanized” mice to study these alleles in vivo. With these 2 unique platforms, we will determine if the G allele drives HIF-2-specific EC phenotypes and PH. Significance: We plan to shift paradigms of lncRNA biology in PH, via defining the links of hypoxia to epigenetics and metabolism and by introducing new epigenetic therapies. By establishing the causative role of rs73184087 in PH, we are poised to leverage functional genomics to gain mechanistic insight in PH specifically for humans.

背景和假设：肺动脉高压 (PH) 是一种致命的疾病，其中 1 类 PAH 和 第 3 组 PH 是由缺氧、HIF-2α 和非编码 RNA 驱动的。我们发现 lncRNA KMT2E-AS1 是由缺氧、HIF-2α 和非编码 RNA 驱动的。 在 1/3 PH 组中上调，并由 HIF-2α 诱导。该 lncRNA 基因与 KMT2E 基因相邻。 控制肺内皮细胞中的组蛋白 3 赖氨酸 4 三甲基化 (H3K4me3) 和染色质重塑。 (ECs)，KMT2E-AS1 稳定 KMT2E 以增加 H3K4me3，从而驱动 HIF-2α 特异性代谢和 KMT2E 内的单核苷酸变异 (SNV) rs73184087 的 G 等位基因相关。 有发生第 1 组 PAH 的风险（在发现/验证队列和对 2,181 例 PAH 与 2,181 例 PAH 的荟萃分析中） 10,060 个对照） rs73184087 还显示出与 HIF-2 更强烈的等位基因 (G) 特异性关联，从而导致 该 lncRNA-KMT2E 对的诱导可以保护缺乏保守 lncRNA 序列的小鼠。 组 1/3 PH；这是通过抑制 PAH 大鼠的组蛋白甲基化来表现的。 KMT2E-AS1/KMT2E 轴是 EC 致病性重编程的核心关键，可促进 PH。 1) 定义 KMT2E SNV rs73184087 在控制 HIF-2α 依赖性 EC 中的等位基因特异性作用 lncRNA-KMT2E 表达和 PH 病理表型使用源自基因组编辑诱导型的 EC。 多能干细胞 (iPSC) 以及携带 rs73184087 A 和 G 等位基因的原代肺 EC，我们将确定 如果 (G) 通过更多的 HIF-2α 结合来增加 lncRNA-KMT2E 并驱动更严重的 EC 表型，我们也会。 对 PAH 患者的血液样本进行表达数量性状位点 (eQTL) 分析 （发现/验证队列）和携带 rs73184087 的 A 和 G 等位基因的 PAH 肺组织 目标 2) 定义 该 lncRNA-KMT2E 轴和 H3K4me3 在促进体内 PH 中的作用我们将量化组 1/3 PH。 与 lncRNA+KMT2E 相比，以及 AAV 驱动的 EC 特异性敲低该 lncRNA 后，啮齿类动物的严重程度 lncRNA 与 lncRNA+KMTE2 的特异性表达 我们还将确定 MM-589 是否是特定的 H3K4me3。 抑制剂，逆转大鼠 PAH 因此，我们的目标是确定 lncRNA+KMT2E 一起是否是必要的并且 足以驱动 1/3 组 PH，并且如果 PAH 依赖于 H3K4me3 活性，从而提供新的表观遗传 PH 治疗。目标 3) 明确 rs73184087 的 G 等位基因对肺血管的致病作用。 培养人精密切割的肺切片，我们将确定rs73184087 G。 等位基因通过 lncRNA-KMT2E 轴和 H3K4me3 的调节驱动血管重塑。 在小鼠中比较人类 rs73184087 G 与 A 等位基因，并将使用这些“人源化”小鼠在体内研究这些等位基因。 通过这两个独特的平台，我们将确定 G 等位基因是否驱动 HIF-2α 特异性 EC 表型和 PH。 意义：我们计划通过定义缺氧与表观遗传学的联系来改变 PH 中 lncRNA 生物学的范式 和代谢并引入新的表观遗传疗法通过确定 rs73184087 的致病作用。 在PH方面，我们准备利用功能基因组学来深入了解人类PH的机制。