Cellular Models of PAH-Associated Molecular Defects as a Tool for Identifying New Therapeutic Targets

PAH 相关分子缺陷的细胞模型作为识别新治疗靶点的工具

• 批准号: 10683667
• 负责人: Jason Matthew Elinoff
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10683667
• 关键词: Adenylate Cyclase; Affect; Animals; Apoptosis; Atrial Heart Septal Defects; BMPR2 gene; Biological Assay; Blood Vessels; Breeding; CAV1 gene; CXCL10 gene; Cardiac; Catalytic Domain; Cell Proliferation; Cell model; Cells; Child; Chronic; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Congenital Heart Defects; Cyclic AMP-Dependent Protein Kinases; Defect; Development; Distal; Drug Kinetics; Endoplasmic Reticulum; Endothelial Cells; Endothelium; Enzymes; Exhibits; Fibroblasts; Future; G6PC3 gene; Gene Silencing; Generations; Genetic; Genotype; Glucose; Glucose-6-Phosphate; Goals; Homeostasis; Human; Hydrolysis; Hypoxia; Immunofluorescence Immunologic; In Vitro; Incidence; Infiltration; Inflammatory; Inflammatory Response; Interferon Activation; Interferons; Investigation; Knock-out; Knockout Mice; Lead; Lung; Metabolic; Modeling; Molecular; Mus; Mutation; NADPH Oxidase; NOS3 gene; National Heart, Lung, and Blood Institute; Neutropenia; Nitric Oxide; Oral; PI3K/AKT; Pathogenicity; Pathologic; Pathway interactions; Patients; Permeability; Peroxonitrite; Phase; Phenotype; Phosphorylation; Platelet-Derived Growth Factor; Post-Translational Protein Processing; Production; Protein Isoforms; Proto-Oncogene Proteins c-akt; Pulmonary Hypertension; Pulmonary artery structure; Rattus; Reactive Oxygen Species; Reagent; Resistance; Rodent; Role; Route; STAT1 gene; SU 5416; Serum; Signal Transduction; Site; Small Interfering RNA; Stains; Superoxide Dismutase; Superoxides; Syndrome; Tail; Testing; Time; Transgenic Organisms; Translating; Triad Acrylic Resin; United States National Institutes of Health; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vascular remodeling; Vasodilator Agents; Western Blotting; Work; arteriole; blood glucose regulation; catalase; clinical center; cross reactivity; endothelial dysfunction; experimental study; glucose metabolism; glucose-6-phosphatase; in vitro Model; in vivo; in vivo evaluation; induced pluripotent stem cell; inhibitor; insight; knock-down; loss of function mutation; lung hypoxia; lung microvascular endothelial cells; mRNA Expression; mimetics; mitochondrial dysfunction; mortality; neutrophil; new therapeutic target; novel therapeutics; phosphatidylinositol 3-kinase gamma; pre-clinical; preclinical study; primary pulmonary hypertension; protein expression; pulmonary arterial hypertension; pulmonary vascular cells; pulmonary vascular remodeling; respiratory; small molecule inhibitor; sphingosine 1-phosphate; targeted treatment; therapeutic target; tool; transcriptomics; zygote

Sub-Project 1: Rare Genetic Defect in Glucose Metabolism as a Model for Investigating Mechanisms Underlying Vascular Remodeling in PAH Glucose-6-phosphatase catalytic subunit 3 (G6PC3) is a ubiquitously expressed enzyme that maintains intracellular glucose homeostasis by catalyzing the hydrolysis of glucose-6-phosphate to glucose in the endoplasmic reticulum. Loss-of-function mutations in G6PC3 lead to an autosomal recessive, multi-system syndrome of severe congenital neutropenia with a broad phenotypic spectrum that includes a high incidence of congenital heart defects. A subset of affected patients exhibits Dursun syndrome, a triad of congenital neutropenia, atrial septal defect and PAH. While the effect of G6PC3 deficiency on neutrophil function has been thoroughly studied, little is known about its impact on the vasculature. We hypothesize that investigation of a rare but well-characterized genetic cause of disrupted cellular energy homeostasis will provide valuable insight into how metabolic reprogramming contributes to PAH pathobiology. Aim 1: Determine the phenotypic consequences of G6PC3-silencing in human pulmonary artery and human pulmonary microvascular endothelial cells (ECs). In FY21, G6PC3 loss in primary human pulmonary vascular ECs produced a proliferative, apoptosis-resistant, hypermigratory phenotype. Consistent with mitochondrial dysfunction, spare respiratory capacity was reduced in primary human PAECs following in G6PC3 knockdown . Aim 2: Investigate the impact of G6pc3 deficiency on pulmonary vascular function in vivo using knockout mice under both normoxic and chronic hypoxic conditions. In FY21, longitudinal cardiac assessments in G6pc3 knockout (KO) mice revealed gradual biventricular dilation over time without obvious development of pulmonary hypertension (PH) under normoxic conditions. In FY22, cryorecovery, initial breeding and colony expansion were performed for G6pc3 global knockout mice. Future studies are planned using both chronic hypoxia and the combination of SU5416 and chronic hypoxia to induce PH. Furthermore, in collaboration with the NHLBI Transgenic Core, we began the initial steps necessary for generating an endothelial-specific G6pc3 knockout strain for further study under conditions that induce PH. Zygotes were injected with G6pc3 CRISPR reagents in order to insert an upstream and downstream loxP site. Tails were collected from the resultant mice and genotyped to check for proper insertion. These murine studies are done under Animal Study Proposal (ASP)# CCM 20-03. Aim 3: Develop and characterize patient-specific in vitro models of endothelial dysfunction using induced pluripotent stem cell (iPSC)-derived endothelial cells. Sub-Project 2: The Contribution of Reactive Oxygen Species to Activation of Interferon Signaling in Cellular Models of PAH We have previously shown that BMPR2 siRNA gene silencing in human PAECs produced phenotypic, transcriptomic and functionally significant signaling changes that closely recapitulated many of the abnormalities and pathogenic mechanisms associated with advanced PAH (Awad and Elinoff et al. AJP Lung 2016). Recently, comprehensive in vitro characterization of CAV1 deficiency in human lung endothelium revealed a proliferative, interferon (IFN)-biased inflammatory phenotype driven by constitutively activated STAT and AKT signaling. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 mutations. Finally, inhibiting JAK/STAT and/or PI3K/AKT reversed this aberrant cell phenotype and may ameliorate vascular remodeling in PAH (Gairhe et al. PNAS 2021). In FY21, we continued investigations into the mechanisms underlying the activation of IFN signaling following CAV1 loss in human PAECs. In preliminary experiments, higher levels of cytosolic reactive oxygen species (ROS) were detected in CAV1-silenced PAECs compared to control cells transfected with a non-targeting siRNA. Catalase, superoxide dismutase and a cell permeable superoxide dismutase mimetic are being used to determine whether inactivating ROS in CAV1-silenced PAECs will ameliorate STAT1 activation, a surrogate for IFN signaling. Small molecule inhibitors and siRNA gene silencing are being utilized to determine the contribution of NOS3 and/or NADPH oxidase to ROS production following CAV1 loss. In FY22, additional experiments confirmed that ROS, as determined by the CellRox assay, are increased in PAECs following CAV1 silencing. Peroxynitrite production, assessed by immunoblotting for 3-nitrotyrosylated, was also elevated in CAV1-deficient cells. MnTBAP, a cell-permeable superoxide dismutase (SOD) mimetic, effectively scavenged ROS but did not reduce STAT1 phosphorylation in CAV1-deficient PAECs. In contrast NOS3 co-silencing not only blocks STAT1 phosphorylation (Gairhe et al. PNAS 2021), but also decreases cell proliferation, and in preliminary experiments, diminishes ROS generation in CAV1-deficient PAECs. NOS3 phosphorylation (Ser1177), a post-translational modification that can activate either nitric oxide and/or superoxide production depending on the cellular context, is also increased in CAV1-deficient PAECs. In addition to AKT, protein kinase A (PKA) phosphorylates NOS3 at S1177. Using small molecule inhibitors, we examined the role of adenylyl cyclase (AC) and PKA on phosphorylation of both STAT1 and NOS3 in PAECs following CAV1 silencing. Interestingly, while inhibiting transmembrane AC did not alter levels of STAT1 and NOS3 phosphorylation, two different soluble AC inhibitors (KH7 and LRE1) potently reduced phosphorylation of both targets and reduced cell proliferation. Similarly, STAT1 and NOS3 phosphorylation were also reduced in CAV1-deficient PAECs following treatment with H89, an inhibitor of PKA. Sub-Project 3: Translating Promising Therapeutic Targets Identified In Vitro Importantly, activation of the PI3K/AKT pathway is a prominent, shared feature across our models of PAH-associated molecular defects. Leniolisib is a PI3K-delta inhibitor that has been very well tolerated over long periods of time in children with activated PI3K-delta syndrome and reversed the hyperproliferative, apoptosis resistant cellular phenotype seen in our in vitro PAH cellular models. In collaboration with Novaris/Pharming, we have obtained RB-50-LV29 (abbreviated RB), a tool compound for leniolisib, for testing in our rat SU5416-hypoxia PAH model. The pre-clinical studies associated with this project are Animal Study Proposal (ASP) # CCM 19-03 and CCM 19-07. In FY21, we completed pharmacokinetic testing in rodents and oral gavage was ultimately selected as the preferred route. In vivo testing in our rat PAH model is underway. In FY22, mRNA and protein expression levels of different PI3K isoforms were examined in PAECs. Notably, PI3K beta was the most abundantly expressed isoform, followed closely by alpha, then delta, and PI3K gamma expression was the lowest. Future work using canonical activators of PI3K/AKT relevent to PAH pathobiology (e.g. sphingosine-1 phosphate, PDGF, VEGF) will be used to investigate which isoform(s) are necessary for PI3K/AKT activation in PAECs. The relative expression of the different PI3K isoforms and the degree of cross-reactivity between the various small molecule inhibitors of PI3K will be important for understanding how to best target PI3K/AKT activation in the pulmonary vasculature.

亚项目1：葡萄糖代谢中的罕见遗传缺陷，作为研究PAH中血管重塑基础机制的模型 葡萄糖-6-磷酸酶催化亚基3（G6PC3）是一种普遍表达的酶，通过催化葡萄糖-6-磷酸盐的水解为内质质含量的葡萄糖通过催化葡萄糖的水解来维持细胞内葡萄糖的稳态。 G6PC3的功能丧失突变导致严重的先天性中性粒细胞减少症的常染色体隐性，多系统综合征，具有广泛的表型谱，其中包括先天性心脏缺陷的高发生率。一部分受影响的患者表现出Dursun综合征，先天性中性粒细胞减少症，心房间隔缺陷和PAH。尽管已经对G6PC3缺乏症对中性粒细胞功能的影响进行了彻底的研究，但对其对脉管系统的影响知之甚少。我们假设对罕见但特征良好的遗传学原因的调查破坏了细胞能量稳态，将为代谢重编程如何有助于PAH病理生物学有价值。 AIM 1：确定人类肺动脉和人肺微血管内皮细胞（EC）中G6PC3分解的表型后果。 在FY21中，原发性人类肺血管EC中的G6PC3损失产生了增生的，抗细胞凋亡的过度迁移表型。与线粒体功能障碍一致，在G6PC3敲低中，原发性人类PAEC的备用呼吸能力降低。 AIM 2：研究G6PC3缺乏症对在常氧和慢性低氧条件下使用基因敲除小鼠在体内对肺血管功能的影响。 在FY21中，G6PC3敲除（KO）小鼠中的纵向心脏评估显示，随着时间的流逝，逐渐培养了双心脑扩张，而没有明显的肺动脉高压（pH）发展。 在22财年，对G6PC3全球基因敲除小鼠进行了冷冻发现，最初的繁殖和菌落扩张。未来的研究是使用慢性缺氧和SU5416和慢性缺氧的组合来诱导pH的。此外，在与NHLBI转基因核心合作的情况下，我们开始了生成内皮特异性G6PC3敲除菌株所需的初始步骤，以在诱导pH的条件下进行进一步研究。为了插入上游和下游LOXP位点，向Zygotes注入了G6PC3 CRISPR试剂。从最终的小鼠中收集尾巴并进行基因分型以检查正确的插入。这些鼠研究是根据动物研究建议（ASP）＃CCM 20-03进行的。 AIM 3：使用诱导的多能干细胞（IPSC）衍生的内皮细胞发展并表征内皮功能障碍的患者特异性体外模型。 亚项目2：活性氧对PAH细胞模型中干扰素信号的激活的贡献 我们先前已经表明，人类PAEC中的BMPR2 siRNA基因沉默产生了表型，转录组和功能意义的信号传导变化，这些变化紧密地概括了与晚期PAH相关的许多异常和致病机制（Awad和Elinoff et al.Elinoff etal。AJPLung 2016）。最近，人类肺内皮中Cav1缺乏症的全面体外表征揭示了由组成型激活的统计和AKT信号传导驱动的增生性，干扰素（IFN）偏置的炎性表型。 PAH患有CAV1突变的患者还具有升高的血清CXCL10水平，其成纤维细胞反映了CAV1缺陷型PAEC的表型和分子特征。此外，免疫荧光染色显示特发性PAH患者的肺动脉动脉中的内皮CAV1损失和STAT1激活，这表明该范式可能不限于罕见的CAV1突变。最后，抑制jak/stat和/或pi3k/akt逆转了这种异常的细胞表型，并可以改善PAH中的血管重塑（Gairhe等人PNAS 2021）。 在FY21中，我们继续研究人类PAEC中Cav1丢失后IFN信号传导激活的机制。在初步实验中，与用非靶向siRNA转染的对照细胞相比，在CAV1脱毛的PAEC中检测到较高水平的胞质活性氧（ROS）。过氧化氢酶，超氧化物歧化酶和细胞可渗透的超氧化物歧化酶模拟酶用于确定在CAV1中脱落的PAEC中灭活ROS是否会改善STAT1激活，这是IFN信号传导的替代。小分子抑制剂和siRNA基因沉默正在用于确定NOS3和/或NADPH氧化酶对CAV1损失后ROS产生的贡献。 在FY22中，其他实验证实，在CAV1沉默后，PAECS中的Cellrox分析确定的ROS增加了。在CAV1缺陷型细胞中，通过免疫印迹评估的过氧亚硝酸盐产生也升高。 MNTBAP是一种可渗透的细胞超氧化物歧化酶（SOD）模拟物，有效地清除了ROS，但并未减少CAV1缺陷型PAEC中的STAT1磷酸化。相比之下，NOS3共沉降不仅阻止了STAT1磷酸化（Gairhe等人PNAS 2021），而且还会减少细胞增殖，并在初步实验中减少CAV1缺陷型PAEC中ROS的产生。 NOS3磷酸化（SER1177）是一种翻译后修饰，可以根据细胞环境激活一氧化氮和/或超氧化物的产生，在CAV1缺乏的PAEC中也增加了。除AKT外，蛋白激酶A（PKA）在S1177处磷酸化NOS3。使用小分子抑制剂，我们检查了Cav1沉默后，PAECS中PAEC中STAT1和NOS3的腺苷环酶（AC）（AC）和PKA的作用。有趣的是，虽然抑制跨膜AC并未改变STAT1和NOS3磷酸化水平，但两种不同的可溶性AC抑制剂（KH7和LRE1）有效地降低了两个靶标的磷酸化和细胞增殖的降低。同样，在用PKA的抑制剂H89处理后，在CAV1缺陷的PAEC中，STAT1和NOS3磷酸化也降低了。 亚项目3：翻译有前途的体外鉴定的有希望的治疗靶标 重要的是，PI3K/AKT途径的激活是我们与PAH相关的分子缺陷模型中的突出特征。 Leniolisib是一种PI3K-DELTA抑制剂，在活化的PI3K-DELTA综合征的儿童中长期耐受性良好，并逆转了我们的体外PAH PAH细胞模型中可见的过度增殖，抗凋亡的抗凋亡性细胞表型。与Novaris/Pharming合作，我们获得了RB-50-LV29（缩写RB），这是一种用于Leniolisib的工具化合物，用于在我们的大鼠SU5416-Hypoxia PAH模型中进行测试。与该项目相关的临床前研究是动物研究建议（ASP）＃CCM 19-03和CCM 19-07。 在FY21中，我们完成了啮齿动物的药代动力学测试，并最终选择了口服烤肉作为首选途径。我们的大鼠PAH模型中的体内测试正在进行中。 在FY22中，在PAEC中检查了不同PI3K同工型的mRNA和蛋白质表达水平。值得注意的是，PI3K Beta是最丰富的同工型，其次是Alpha，然后是Delta，PI3K伽马表达是最低的。未来使用与PAH病理生物学相关的PI3K/AKT的规范激活剂（例如，鞘氨氨酸-1磷酸盐，PDGF，VEGF）将使用PAEC中PI3K/AKT激活所必需的哪种同工型。不同PI3K同工型的相对表达以及PI3K的各种小分子抑制剂之间的交叉反应程度对于理解如何最好地靶向肺脉管系统中的PI3K/AKT激活。