Vascular Dysfunction and Inflammation

血管功能障碍和炎症

• 批准号: 10262624
• 负责人: ROBERT L DANNER
• 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/10262624
• 关键词: Acute; Affect; Agonist; Aldosterone; Antiinflammatory Effect; Apoptosis; Arginine; BMPR2 gene; Binding Sites; Biological Models; Blood; Blood Circulation; Blood Vessels; Cardiac; Catalytic Domain; Cell Adhesion Molecules; Cell model; Cells; Cessation of life; Chronic; Clinical Protocols; Complex; Cyclic GMP; DNA Binding; DNA Sequence; Defect; Disease; Disease Progression; Drug Targeting; EP300 gene; ERCC3 gene; Endothelial Cells; Endothelium; Event; Exhibits; Expression Profiling; Failure; Family member; Female; Fibroblasts; Flow Cytometry; G-Protein-Coupled Receptors; G6PC3 gene; Gene Expression; Gene Expression Regulation; Genetic; Genetic Predisposition to Disease; Genetic Transcription; Genomics; Glucocorticoid Receptor; Goals; HIV; Hemostatic function; Human; Hypoxia; IL8 gene; Immune response; Immunochemistry; Impairment; In Vitro; Individual; Inflammation; Inflammatory; Inflammatory Response; Injury; Interferons; Interruption; Investigation; Laboratory Study; Ligands; Link; Lipoproteins; Lung; MAP Kinase Gene; MAPK3 gene; MAPK8 gene; Magnetic Resonance Imaging; Malignant Neoplasms; Manuscripts; Mediating; Mediator of activation protein; Mesenchymal; Messenger RNA; Meta-Analysis; Metabolism; Mitochondria; Modeling; Molecular; Molecular Conformation; Monounsaturated Fatty Acids; Mus; Mutation; NF-kappa B; Natural History; Nitric Oxide; Nitric Oxide Signaling Pathway; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Receptors; Nucleic Acids; Oncogenic; Output; Oxidants; PI3K/AKT; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Peroxisome Proliferator-Activated Receptors; Phenotype; Pilot Projects; Placebos; Play; Preparation; Procollagen-Proline Dioxygenase; Production; Proteins; Proto-Oncogene Proteins c-akt; Pulmonary artery structure; Ras/Raf; Rattus; Reactive Oxygen Species; Receptor Activation; Receptor Signaling; Refractory; Relaxation; Research; Resistance; Resolution; Role; SU 5416; Safety; Scleroderma; Septic Shock; Sickle Cell Anemia; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Source; Specimen; Spironolactone; Stress; Tertiary Protein Structure; Testing; Therapeutic; Therapy trial; Thrombosis; Transcript; Transcription Factor AP-1; Translations; Vascular Diseases; Vascular remodeling; Ventricular; Work; apoAI regulatory protein-1; biological adaptation to stress; caveolin 1; chemokine; chicken ovalbumin upstream promoter-transcription factor; circulating biomarkers; congenital heart disorder; constriction; cytokine; endothelial dysfunction; eplerenone; glucose-6-phosphatase; hemodynamics; knock-down; loss of function; loss of function mutation; meetings; mortality; organ injury; p38 MAPK Signaling Pathway; p38 Mitogen Activated Protein Kinase; promoter; prototype; pulmonary arterial hypertension; pulmonary artery endothelial cell; repaired; rosiglitazone; symposium; therapeutic target; transcription factor TFIIH; vascular bed; vascular inflammation; vascular injury

Nitric oxide: Our work has focused on cGMP-independent, non-canonical NO signaling and inflammatory gene regulation. NO up-regulated TNFa production (J Immunol 1994; Blood 1997) through a cGMP-independent signaling pathway (J Biol Chem 1997) that utilized NO-responsive Sp1 promoter binding sites (J Biol Chem 1999; J Biol Chem 2003). Dysfunctional eNOS upregulated TNFa (J Biol Chem 2000) through ROS and ERK1/2 (Am J Physiol 2001). NO activation of p38 MAPK stabilized IL-8 mRNA (J Infect Dis 1998; J Leuk Biol 2004). NO has diverse effects on transcript stability and translation (Nucleic Acids Research 2006; J Leuk Biol 2008). Sickle cell disease caused oxidant and inflammatory stress in the vasculature (Blood, 2004). This circulatory stress altered gene expression and arginine metabolism (Circulation, 2007). Anti-proliferative effects of NO were linked to p38 MAPK activation and p21 mRNA stabilization (BMC Genomics 2005; J Biol Chem 2006). Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. PPARg was activated by NO through a p38 MAPK signaling pathway (FASEB J 2007). In contrast to the pro-inflammatory effects of high output NO, CO blocked proximal events in NF-kB signal transduction and broadly suppressed inflammation (PLoS One 2009). Nuclear receptors (NRs): The glucocorticoid receptor (GR) suppresses inflammatory responses by tethering to DNA-bound NF-kB and AP-1 complexes that broadly control the expression of cytokines, chemokines and adhesion molecules. Effects on inflammation of other NRs including PPARg, MR, AR, and COUP-TF are being investigated in human endothelial cells (ECs). Rosiglitazone (RGZ) is a PPARg ligand/agonist used to treat type 2 diabetes. G-protein coupled receptor 40 (GPR40)/p38 MAPK/PGC1a/EP300 activation by RGZ was shown in human ECs to augment RGZ/PPARg genomic signaling (J Biol Chem 2015). Cognate GPR and nuclear receptor signaling networks may explain differences in the safety and efficacy of nuclear receptor targeted drugs (Pharm Research 2016). MR agonists repressed NF-kB mediated gene transcription, but trans-activated inflammatory AP-1 signaling in a DNA sequence, MR conformation, and AP-1 family member dependent fashion (J Biol Chem 2016). Aldosterone/MR activation of AP-1 may contribute to harmful inflammatory effects in CHF and PAH. Long-chain monounsaturated fatty acids (LCMUFA; i.e., C20:1 and C22:1) benefits were associated with PPAR activation, possibly via the activation of GPR40, and favorable alterations in lipoproteins (Atheroscelerosis 2017). SPL, but not eplerenone was found to suppress both NF-kB and AP-1 inflammatory signaling independent of MR through the proteasomal degradation of XPB, a core subunit of the eukaryotic basal transcription TFIIH complex (Cardiovasc Res 2018). Loss of COUPTF2 (NR2F2) de-repressed JAK/STAT/interferon inflammatory responses in endothelial cells (ATS 2011; Aspen Lung Conference 2019; manuscript in preparation 2020-21). Selective AR modulators (SARMs) have been investigated to identify AR ligands with reduced pro-inflammatory potential and possibly net anti-inflammatory effects in the human vasculature. Pulmonary arterial hypertension (PAH): Two PAH clinical protocols, including a pilot study of spironolactone therapy (Trials 2013) and a natural history study investigating circulating markers of vascular inflammation and high-resolution cardiac magnetic resonance imaging (MRI), provide a source of patient specimens to support ongoing laboratory studies. Circulating ECs were identified by flow cytometry and their endothelial phenotype was validated using ultramicro analytical immunochemistry (Thrombosis and Haemostasis 2014). ECs with heterogeneous PAH-associated molecular defects including BMPR2, CAV1 and SMAD9, PHD2 (prolyl hydroxylase domain protein 2; EGLN1), COUPTF2 (NR2F2), and G6PC3 (glucose-6-phosphatase catalytic subunit 3) are being studied in vitro to create a comprehensive picture of pathogenic mechanisms and therapeutic targets. Loss-of-function mutations in bone morphogenetic protein type II receptor (BMPR2) are the most common genetic cause of PAH. BMPR2 knockdown (KD) in human pulmonary artery ECs (PAECs) activated Ras/Raf/ERK signaling, an oncogenic pathway, leading to proliferation, invasiveness and cytoskeletal abnormalities (Am J Physiol Lung Cell Mol Physiol 2016). A meta-analysis of peripheral blood mononuclear cell (PBMC) expression profiling studies in PAH patients from multiple centers and across various expression profiling platforms identified an interferon-driven systemic immunologic response as a fundamental component of PAH pathobiology that was previously unrecognized in the individual blood expression profiling studies (Am J Physiol Lung Cell Mol Physiol 2020). Caveolin-1 (CAV1) loss-of-function (LOF), similar to BMPR2, produced a proliferative, hyper-migratory and inflammatory PAEC phenotype (Grover Conference 2015; ATS 2017) with activation of JAK/STAT/interferon signaling and AKT. This inflammatory signature was also found in fibroblasts from PAH patients with CAV1 mutations and in CAV1-/- mice (Aspen Lung Conference 2019; ATS 2017; manuscript submitted 2020). A sugen (SU5416) hypoxia rat model of pulmonary arterial hypertension has been established and an initial study of spironolactone and eplerenone compared to placebo has been completed (AHA Meeting 2019; manuscript in preparation). SMAD9 LOF in human PAECs also produced an abnormal cellular phenotype characterized by proliferation, hypermigration, cytoskeletal and mitochondrial alterations and endothelial to mesenchymal transition, as well as non-canonical activation of AKT, ERK and p38 (ATS 2018; manuscript in preparation). Loss-of-function mutations in COUPTF2 (NR2F2) have been associated with congenital heart disease (CHD), which can result in PAH. COUPTF2 silencing in ECs produced an interferon inflammatory response and exhibited a hyper-proliferative, apoptosis-resistant, and invasive phenotype with AKT activation. Dickkopf-1 (DKK1), an upstream regulator of AKT and DKK1 knockdown abrogated the abnormal signaling associated with COUPTF2 loss (Aspen Lung Conference 2019: manuscript in preparation). An in vitro pseudohypoxia model of PAH was established by silencing PHD2 (prolyl hydroxylase domain protein 2; EGLN1) in LMVECs. PHD2-silencing stabilized HIF2alpha, decreased ASK-interacting protein 1 (AIP; DAB2IP), activating AKT and ERK (Aspen Lung Conference 2019; manuscript in preparation). Marked resistance to apoptosis has been a consistent feature of our endothelial cell models of PAH. Using the BMPR2 loss-of-function model as a prototype, apoptosis resistance was linked to vasohibin 1 (VASH1) and DLL4 loss, PI3K/AKT and ERK activation, and JNK suppression, (manuscript in preparation). Inhibiting PI3K/AKT restored apoptosis sensitivity in the three model systems tested to date, BMPR2, CAV1 and PHD2.

一氧化氮：我们的工作重点是不依赖 cGMP 的、非规范的 NO 信号传导和炎症基因调控。 NO 通过利用 NO 响应性 Sp1 启动子结合位点 (J Biol Chem 1999; J Biol Chem 2003) 的不依赖于 cGMP 的信号通路 (J Biol Chem 1997) 上调 TNFa 的产生 (J Immunol 1994; Blood 1997)。功能失调的 eNOS 通过 ROS 和 ERK1/2 (Am J Physiol 2001) 上调 TNFa (J Biol Chem 2000)。 p38 MAPK 的 NO 激活稳定了 IL-8 mRNA（J Infect Dis 1998；J Leuk Biol 2004）。 NO 对转录稳定性和翻译有多种影响（Nucleic Acids Research 2006；J Leuk Biol 2008）。 镰状细胞病引起脉管系统中的氧化和炎症应激（Blood，2004）。这种循环应激改变了基因表达和精氨酸代谢（Circulation，2007）。 NO 的抗增殖作用与 p38 MAPK 激活和 p21 mRNA 稳定有关（BMC Genomics 2005；J Biol Chem 2006）。 NO 和过氧化物酶体增殖物激活受体 (PPAR) 均可保护内皮并调节其功能。 PPARg 通过 p38 MAPK 信号通路被 NO 激活 (FASEB J 2007)。与高输出 NO 的促炎作用相反，CO 阻断 NF-kB 信号转导中的近端事件并广泛抑制炎症 (PLoS One 2009)。 核受体 (NR)：糖皮质激素受体 (GR) 通过与 DNA 结合的 NF-kB 和 AP-1 复合物结合来抑制炎症反应，这些复合物广泛控制细胞因子、趋化因子和粘附分子的表达。正在人内皮细胞 (EC) 中研究其他 NR（包括 PPARg、MR、AR 和 COUP-TF）对炎症的影响。 Rosiglitazone (RGZ) 是一种 PPARg 配体/激动剂，用于治疗 2 型糖尿病。在人类 EC 中，RGZ 激活 G 蛋白偶联受体 40 (GPR40)/p38 MAPK/PGC1a/EP300，从而增强 RGZ/PPARg 基因组信号传导 (J Biol Chem 2015)。同源 GPR 和核受体信号网络可以解释核受体靶向药物的安全性和有效性的差异（Pharm Research 2016）。 MR 激动剂抑制 NF-kB 介导的基因转录，但以 DNA 序列、MR 构象和 AP-1 家族成员依赖性方式反式激活炎症 AP-1 信号传导 (J Biol Chem 2016)。 AP-1 的醛固酮/MR 激活可能会导致 CHF 和 PAH 的有害炎症反应。 长链单不饱和脂肪酸（LCMUFA；即 C20:1 和 C22:1）的益处与 PPAR 激活相关，可能通过 GPR40 的激活以及脂蛋白的有利改变（Atheroscelerosis 2017）。 研究发现，SPL（而非依普利农）可以通过 XPB（真核转录 TFIIH 复合物的核心亚基）的蛋白酶体降解来抑制独立于 MR 的 NF-kB 和 AP-1 炎症信号传导（Cardiovasc Res 2018）。 COUPTF2 (NR2F2) 的缺失会解除内皮细胞中 JAK/STAT/干扰素炎症反应的抑制（ATS 2011；阿斯彭肺会议 2019；手稿准备中，2020-21）。 选择性 AR 调节剂 (SARM) 已被研究来识别在人体脉管系统中具有降低的促炎潜力和可能的净抗炎作用的 AR 配体。 肺动脉高压 (PAH)：两项 PAH 临床方案，包括螺内酯治疗的试点研究（2013 年试验）和调查血管炎症循环标志物和高分辨率心脏磁共振成像 (MRI) 的自然史研究，为支持正在进行的实验室研究提供了患者标本来源。 通过流式细胞术鉴定循环 EC，并使用超微分析免疫化学验证其内皮表型（Thrombosis and Haemostasis 2014）。 具有异质 PAH 相关分子缺陷的 EC，包括 BMPR2、CAV1 和 SMAD9、PHD2（脯氨酰羟化酶结构域蛋白 2；EGLN1）、COUPTF2 (NR2F2) 和 G6PC3（葡萄糖 6 磷酸酶催化亚基 3）正在体外研究，以全面了解致病机制和 治疗目标。 II 型骨形态发生蛋白受体 (BMPR2) 的功能丧失突变是 PAH 最常见的遗传原因。人肺动脉 EC (PAEC) 中的 BMPR2 敲低 (KD) 激活 Ras/Raf/ERK 信号传导（一种致癌途径），导致增殖、侵袭和细胞骨架异常 (Am J Physiol Lung Cell Mol Physiol 2016)。 对来自多个中心和不同表达谱平台的 PAH 患者的外周血单核细胞 (PBMC) 表达谱研究进行荟萃分析，发现干扰素驱动的全身免疫反应是 PAH 病理学的基本组成部分，而这一点以前在个体血液表达谱研究中未被认识到 (Am J Physiol Lung Cell Mol Physiol 2020)。 Caveolin-1 (CAV1) 功能丧失 (LOF) 与 BMPR2 类似，会产生增殖、过度迁移和炎症性 PAEC 表型（Grover Conference 2015；ATS 2017），并激活 JAK/STAT/干扰素信号传导和 AKT。在患有 CAV1 突变的 PAH 患者和 CAV1-/- 小鼠的成纤维细胞中也发现了这种炎症特征（2019 年阿斯彭肺会议；2017 年 ATS；2020 年提交的手稿）。 已建立 Sugen (SU5416) 肺动脉高压缺氧大鼠模型，并完成螺内酯和依普利酮与安慰剂比较的初步研究（2019 年 AHA 会议；手稿正在准备中）。 人类 PAEC 中的 SMAD9 LOF 还产生异常细胞表型，其特征是增殖、过度迁移、细胞骨架和线粒体改变、内皮向间质转化，以及 AKT、ERK 和 p38 的非典型激活（ATS 2018；手稿正在准备中）。 COUPTF2 (NR2F2) 的功能丧失突变与先天性心脏病 (CHD) 相关，后者可能导致 PAH。 EC 中的 COUPTF2 沉默会产生干扰素炎症反应，并表现出过度增殖、抗凋亡和侵袭性表型，同时 AKT 激活。 Dickkopf-1 (DKK1) 是 AKT 的上游调节因子，DKK1 敲低消除了与 COUPTF2 丢失相关的异常信号传导（2019 年阿斯本肺会议：准备中的手稿）。 通过沉默 LMVEC 中的 PHD2（脯氨酰羟化酶结构域蛋白 2；EGLN1）建立 PAH 体外假性缺氧模型。 PHD2 沉默稳定了 HIF2α，减少了 ASK 相互作用蛋白 1（AIP；DAB2IP），激活了 AKT 和 ERK（阿斯彭肺会议 2019；手稿正在准备中）。 显着的细胞凋亡抗性是我们的 PAH 内皮细胞模型的一致特征。使用 BMPR2 功能丧失模型作为原型，细胞凋亡抵抗与血管抑制素 1 (VASH1) 和 DLL4 丧失、PI3K/AKT 和 ERK 激活以及 JNK 抑制相关（手稿正在准备中）。抑制 PI3K/AKT 可恢复迄今为止测试的三个模型系统（BMPR2、CAV1 和 PHD2）中的细胞凋亡敏感性。