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组PAH和 第3组PH由缺氧、HIF-2 α和非编码RNA驱动。我们发现，lncRNA KMT 2 E-AS 1是 在1/3 PH组中上调，并由HIF-2 α诱导。该lncRNA基因邻近KMT 2 E，一个基因 控制组蛋白3赖氨酸4三甲基化（H3 K4 me 3）和染色质重塑。在肺内皮细胞 (ECs)，KMT 2 E-AS 1稳定KMT 2 E以增加H3 K4 me 3，从而驱动HIF-2 α特异性代谢和 致病性改变KMT 2 E内的单核苷酸变体（SNV）rs73184087的G等位基因与KMT 2 E内的SNV基因的突变相关。 具有发生1组PAH的风险（在发现/验证队列和2，181例PAH vs. 10，060个对照）。rs73184087还显示出与HIF-2 α更亲合的等位基因（G）特异性关联，导致 这种lncRNA-KMT 2 E对的诱导。在保守lncRNA序列中缺陷的小鼠被保护免于 组1/3 PH;这是通过抑制PAH大鼠中的组蛋白甲基化进行的表型模拟。我们假设， KMT 2 E-AS 1/KMT 2 E轴是EC中致病性重编程的中心关键，促进PH。 1)定义KMT 2 E SNV rs73184087在控制HIF-2 α依赖性EC中的等位基因特异性作用 lncRNA-KMT 2 E表达和PH病理表型。使用源自基因组编辑的诱导型 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-2 β结合增加lncRNA-KMT 2 E并驱动更严重的EC表型。我们还将 在PAH患者的血液样本中进行表达数量性状基因座（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-KMT 2 E轴和H3 K4 me 3在体内促进PH中的作用。我们将量化1/3 PH组 在EC特异性敲除该lncRNA相对于lncRNA+ KMT 2 E后以及在AAV驱动的EC- lncRNA相对于lncRNA+ KMTE 2的特异性表达。我们还将确定MM-589，一种特定的H3 K4 me 3 抑制剂，逆转大鼠PAH。因此，我们的目标是确定lncRNA+ KMT 2 E是否是必要的， 足以驱动组1/3 PH，如果PAH依赖于H3 K4 me 3活性，从而提供新的表观遗传学 PH治疗。目的3）明确rs73184087基因G等位基因在肺血管病变中的作用 重塑和体内PH。培养人精确切割的肺切片，我们将确定rs73184087 G 等位基因通过调节lncRNA-KMT 2 E轴和H3 K4 me 3驱动血管重塑。我们还插入了 人rs73184087 G对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-2 β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， 和新陈代谢以及引入新的表观遗传疗法。通过确定rs73184087的致病作用， 在PH中，我们准备利用功能基因组学来获得专门针对人类的PH的机制见解。