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.

子项目 1：葡萄糖代谢中的罕见遗传缺陷作为研究 PAH 血管重塑机制的模型 葡萄糖-6-磷酸酶催化亚基 3 (G6PC3) 是一种普遍表达的酶，通过催化内质网中葡萄糖-6-磷酸水解成葡萄糖来维持细胞内葡萄糖稳态。 G6PC3 的功能丧失突变导致常染色体隐性遗传、严重先天性中性粒细胞减少症的多系统综合征，具有广泛的表型谱，包括高发生率的先天性心脏缺陷。一部分受影响的患者表现出 Dursun 综合征，即先天性中性粒细胞减少症、房间隔缺损和 PAH 的三联征。虽然 G6PC3 缺乏对中性粒细胞功能的影响已得到深入研究，但对其对脉管系统的影响知之甚少。我们假设，对细胞能量稳态破坏的一种罕见但特征明确的遗传原因的研究将为了解代谢重编程如何影响 PAH 病理学提供有价值的见解。 目标 1：确定 G6PC3 沉默对人肺动脉和人肺微血管内皮细胞 (EC) 的表型影响。 在 2021 财年，原代人肺血管 EC 中 G6PC3 的缺失产生了增殖、抗凋亡、过度迁移的表型。与线粒体功能障碍一致，原代人类 PAEC 在 G6PC3 敲低后备用呼吸能力降低。 目标 2：使用基因敲除小鼠在常氧和慢性缺氧条件下研究 G6pc3 缺乏对体内肺血管功能的影响。 在 2021 财年，G6pc3 敲除 (KO) 小鼠的纵向心脏评估显示，在含氧量正常的条件下，随着时间的推移，双心室逐渐扩张，但没有明显出现肺动脉高压 (PH)。 2022财年，对G6pc3全基因敲除小鼠进行了冷冻恢复、初始繁殖和集落扩增。未来的研究计划使用慢性缺氧以及 SU5416 和慢性缺氧的组合来诱导 PH。此外，我们与 NHLBI Transgenic Core 合作，开始了生成内皮特异性 G6pc3 敲除菌株所需的初始步骤，以便在诱导 PH 的条件下进行进一步研究。受精卵被注射 G6pc3 CRISPR 试剂以插入上游和下游 loxP 位点。从所得小鼠中收集尾巴并进行基因分型以检查是否正确插入。这些小鼠研究是根据动物研究提案 (ASP)# CCM 20-03 进行的。 目标 3：使用诱导多能干细胞 (iPSC) 衍生的内皮细胞开发并表征患者特异性的内皮功能障碍体外模型。 子项目 2：活性氧对 PAH 细胞模型中干扰素信号传导的激活作用 我们之前已经证明，人类 PAEC 中的 BMPR2 siRNA 基因沉默会产生表型、转录组和功能上显着的信号传导变化，这些变化密切概括了与晚期 PAH 相关的许多异常和致病机制（Awad 和 Elinoff 等人，AJP Lung 2016）。最近，人肺内皮细胞 CAV1 缺陷的综合体外表征揭示了由持续激活的 STAT 和 AKT 信号传导驱动的增殖性、干扰素 (IFN) 偏向的炎症表型。具有 CAV1 突变的 PAH 患者的血清 CXCL10 水平也升高，并且他们的成纤维细胞反映了 CAV1 缺陷的 PAEC 的表型和分子特征。此外，免疫荧光染色显示特发性 PAH 患者肺小动脉内皮 CAV1 缺失和 STAT1 激活，表明这种范例可能不仅限于罕见的 CAV1 突变。最后，抑制 JAK/STAT 和/或 PI3K/AKT 可以逆转这种异常细胞表型，并可能改善 PAH 中的血管重塑（Gairhe 等人，PNAS 2021）。 在 2021 财年，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，与转染非靶向 siRNA 的对照细胞相比，CAV1 沉默的 PAEC 中检测到更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渗透性超氧化物歧化酶模拟物被用来确定 CAV1 沉默的 PAEC 中 ROS 失活是否会改善 STAT1 激活（IFN 信号传导的替代物）。小分子抑制剂和 siRNA 基因沉默被用来确定 NOS3 和/或 NADPH 氧化酶对 CAV1 丢失后 ROS 产生的贡献。 在 2022 财年，额外的实验证实，根据 CellRox 测定的结果，CAV1 沉默后 PAEC 中的 ROS 增加。通过免疫印迹法评估 3-硝基酪氨酸化，过氧亚硝酸盐的产生在 CAV1 缺陷细胞中也有所升高。 MnTBAP 是一种细胞渗透性超氧化物歧化酶 (SOD) 模拟物，可有效清除 CAV1 缺陷 PAEC 中的 ROS，但不会减少 STAT1 磷酸化。相比之下，NOS3 共沉默不仅会阻断 STAT1 磷酸化（Gairhe 等人，PNAS 2021），还会减少细胞增殖，并且在初步实验中，会减少 CAV1 缺陷 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 缺陷的 PAEC 中也会增加。除了 AKT 之外，蛋白激酶 A (PKA) 也会在 S1177 处磷酸化 NOS3。使用小分子抑制剂，我们研究了 CAV1 沉默后腺苷酸环化酶 (AC) 和 PKA 对 PAEC 中 STAT1 和 NOS3 磷酸化的作用。有趣的是，虽然抑制跨膜 AC 不会改变 STAT1 和 NOS3 磷酸化水平，但两种不同的可溶性 AC 抑制剂（KH7 和 LRE1）可有效降低两个靶标的磷酸化并减少细胞增殖。同样，在用 PKA 抑制剂 H89 处理后，CAV1 缺陷的 PAEC 中 STAT1 和 NOS3 磷酸化也降低。 子项目 3：转化体外确定的有前景的治疗靶点 重要的是，PI3K/AKT 通路的激活是我们的 PAH 相关分子缺陷模型的一个突出的共同特征。 Leniolisib 是一种 PI3K-delta 抑制剂，在患有激活 PI3K-delta 综合征的儿童中具有良好的长期耐受性，并逆转了体外 PAH 细胞模型中观察到的过度增殖、凋亡抵抗的细胞表型。我们与 Novaris/Pharming 合作，获得了 RB-50-LV29（缩写为 RB），这是 leniolisib 的工具化合物，用于在我们的大鼠 SU5416 缺氧 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提案 (ASP) # CCM 19-03 和 CCM 19-07。 2021 财年，我们完成了啮齿类动物的药代动力学测试，最终选择口服灌胃作为首选途径。我们的大鼠 PAH 模型的体内测试正在进行中。 在 2022 财年，我们对 PAEC 中不同 PI3K 亚型的 mRNA 和蛋白质表达水平进行了检查。值得注意的是，PI3K beta 是表达最丰富的异构体，紧随其后的是 alpha，然后是 delta，PI3K gamma 表达最低。未来的工作将使用与 PAH 病理学相关的 PI3K/AKT 经典激活剂（例如 1 磷酸鞘氨醇、PDGF、VEGF）来研究 PAEC 中 PI3K/AKT 激活所必需的亚型。不同 PI3K 亚型的相对表达以及 PI3K 各种小分子抑制剂之间的交叉反应程度对于了解如何最好地靶向肺血管系统中的 PI3K/AKT 激活非常重要。