Endothelial Dysfunction and Vascular Inflammation

内皮功能障碍和血管炎症

• 批准号: 8565269
• 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/8565269
• 关键词: 2,4-thiazolidinedione; Acetates; Acetyltransferase; Acute; Adverse effects; Affect; Age; Agonist; American; Androgen Receptor; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Appearance; Arginine; Ascaridil; Atherosclerosis; B-Lymphocytes; BMPR2 gene; Binding Sites; Blood; Blood Circulation; Blood Platelets; Blood Vessels; COUP transcription factor I; Cardiovascular system; Cell Line; Cell-Cell Adhesion; Cells; Cessation of life; Chest; Chronic; Chronic Disease; Cytoskeleton; Databases; Development; Disease; Disease Progression; Down-Regulation; Drug usage; E-Selectin; Endothelial Cells; Endothelium; Estrogen Receptors; Ethnic Origin; Event; Failure; Family member; Focal Adhesion Kinase 1; Functional disorder; Future; G Protein-Coupled Receptor Genes; G-Protein-Coupled Receptors; Gender; Gene Expression; Gene Targeting; Genes; Genetic Predisposition to Disease; Genetic Transcription; Genomics; Glucocorticoids; Goals; HIV; Human; Human Cell Line; Hybrids; Hypotension; Illness Days; Infection; Inflammation; Inflammatory; Inflammatory Response; Inherited; Injury; Integrins; Interferons; Interleukin-1; Interleukin-6; Investigation; Lesion; Leukocytes; Ligands; Link; Lung; MAP Kinase Gene; MAPK14 gene; Mediating; Mediator of activation protein; Messenger RNA; Metabolism; Mineralocorticoid Receptor; Mineralocorticoids; Molecular Profiling; Mutation; Myristates; NADP; NF-kappa B; NOS2A gene; Nitric Oxide; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Receptors; Oxidants; P-Selectin; PPAR gamma; Pathogenesis; Pathway interactions; Patients; Peripheral Blood Mononuclear Cell; Peroxisome Proliferator-Activated Receptors; Phenotype; Phorbol; Phorbols; Phosphotransferases; Play; Preparation; Production; Protocols documentation; Pulmonary Vascular Resistance; Rare Diseases; Reactive Oxygen Species; Receptor Signaling; Refractory; Relative (related person); Relaxation; Risk; Role; Safety; Scleroderma; Sepsis; Septic Shock; Shock; Sickle Cell Anemia; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Societies; Spironolactone; Stress; Superoxide Dismutase; Superoxides; System; T-Lymphocyte; TNF gene; Testing; Therapeutic; Thiazolidinediones; Time; Transfection; U937 Cells; Umbilical vein; United States; Vasoconstrictor Agents; Ventricular; Work; Writing; abstracting; apoAI regulatory protein-1; base; cell motility; cell type; constriction; drug development; genome-wide; hemodynamics; human NOS2A protein; human NOS3 protein; human PLK1 protein; immune function; in vitro Model; inhibitor/antagonist; injury and repair; macrophage; monocyte; mortality; new therapeutic target; overexpression; p21 activated kinase; prevent; promoter; pulmonary arterial hypertension; pulmonary artery endothelial cell; receptor; receptor expression; repaired; response; rosiglitazone; sensor; symposium; vascular bed; vascular inflammation; volunteer

Transfection of monoblastoid U937 cells with inducible nitric oxide synthase (NOS2) resulted in a human cell line that spontaneously produced large amounts NO, a mediator of vascular dysfunction in septic shock. After differentiation with phorbol-12-acetate-13-myristate (PMA), iNOS expressing cells produced increased amounts of TNF by a mechanism that was dependent on NO. This demonstrated the ability of endogenously generated NO to augment the inflammatory response (Blood, 1997). Likewise, overexpression of endothelial NOS (eNOS) was shown to upregulate TNFalpha production, but neither N-methyl-L-arginine, a NOS inhibitor, nor mutation of its L-arginine binding site (rendering it incapable of producing nitric oxide) blocked this effect (J Biol Chem, 2000). However, co-transfection with superoxide dismutase or deletion of the NADPH binding site of eNOS completely prevented eNOS from upregulating TNF production. Collectively, our results suggested that eNOS regulates inflammatory responses through both NO (J Immunol, 1994; J Biol Chem, 1997) and reactive oxygen species-based signal transduction pathways (J Biol Chem, 2000). Superoxide produced by eNOS was subsequently shown to upregulate TNFalpha via p42/44 MAPK activation (J Biol Chem, 2001). Sp1 was identified as a bidirectional NO sensor, down regulating eNOS in endothelial cells and upregulating TNF, both via proximal Sp promoter-binding sites (J Biol Chem, 2003). Direction (up or down) of the response was controlled by promoter sequences adjacent to the Sp1 binding site. Sickle cell disease was characterized by oxidant and inflammatory stress in the vasculature, even in the absence of an acute crisis (Blood, 2004). Circulatory stress in sickle cell disease was associated with gene expression and arginine metabolism abnormalities in platelets (Circulation, 2007). Anti-proliferative effects of NO that prevent the development of a chronic vascular injury phenotype was linked to p38 MAPK activation and p21 mRNA stabilization with subsequent down regulation of polo-like kinase 1 through a CDE/CHR proximal promoter site (BMC Genomics, 2005; J Biol Chem, 2006). Both NO and peroxisome proliferator-activated receptors (PPARs) protect the endothelium and regulate its function. Therefore, we tested for crosstalk between these signaling pathways using human umbilical vein and hybrid EA.hy926 endothelial cells (FASEB J, 2007). PPARgamma was activated by NO through a p38 MAPK dependent signal transduction pathway. This crosstalk mechanism may contribute to the anti-inflammatory and cytoprotective effects of NO in the vasculature and suggests new strategies for preventing and treating vascular dysfunction. The optimal activation of PPARgamma by thiazolidinediones (TZDs), drugs used in the treatment of type 2 diabetes mellitus, was found to similarly require p38 MAPK activation. TZDs, like homeostatic NO production, reduce markers of cardiovascular inflammation. We have subsequently identified a p38MAPK/acetyltransferase signal transduction mechanism that enhances the transcription of PPARgamma target genes (in preparation, 2012). PPARgamma agonist (rosiglitazone; RGZ) activation of p38 MAPK with downstream enhancement of PPARgamma signaling was linked to G-protein coupled receptor 40 (GPCR40). This finding defines PPARgamma signaling for the first time as a two receptor system. Cognate GPCR and nuclear receptor signaling networks may explain differences in safety and efficacy among nuclear receptor ligands and has implications for future drug development. Collectively, these findings suggest that PPARgamma may be a useful target for reducing endothelial dysfunction and vascular inflammation in septic shock and possibly chronic diseases such as PAH. Interrogation of our microarray database indicates that mineralocorticoid receptor (MR) is relatively overexpressed in endothelial cells and T-lymphocytes compared to monocytes and B-lymphocytes. Likewise, three other nuclear receptor family members, androgen receptor (AR), COUP-TFI and COUP-TFII were also found to be over-expressed in endothelial cells relative to leukocytes. In contrast, glucocorticoid (GR), a nuclear receptor target that can reverse shock but may lack overall benefit due to adverse effects on immune function, is expressed more uniformly across these cell types. Therefore, MR, AR, COUP-TFI and COUP-TFII may be useful targets for modulating endothelium inflammation. Using genome-wide expression profiling, COUP-TFII knockdown in a human endothelial cell line was shown to modulate 36% of all TNF-responsive genes (American Thoracic Society, abstract 2011). COUP-TFII suppression of TNFlapha-induced gene expression was associated with interferon signaling pathways (IRFs and STATs), while COUP-TFII amplification of TNFalpha responses was linked to NF-kappaB. Genome-wide gene expression differences in the peripheral blood mononuclear cells (PBMCs) of patients with PAH compared to healthy gender, age and ethnicity matched volunteers categorically identified alterations in inflammation, cell adhesion, cell motility, the cytoskeleton and apoptosis (American Thoracic Society, abstract 2011). Specific genes and canonical pathways overlapped with several previously proposed mechanisms and suggested novel therapeutic targets such as Ras, RhoA, integrin, focal adhesion kinase-1 (FAK), and p21-activated kinase (PAK). Recent work has investigated BMPR2 silencing in primary human pulmonary artery endothelial cells (PAECs) as an in vitro model of hereditary PAH. BMPR2 knockdown produces a phenotype with altered estrogen receptor expression, exaggerated inflammatory responses and dysregulated stress kinase signaling (Keystone Symposium on Pulmonary Arterial Hypertension 2012). In human endothelial cells both MR agonists and antagonists have been shown to repress NF-kappaB mediated gene transcription (Keystone Symposia on Nuclear Receptors, abstract, 2010; American Thoracic Society, abstract 2010), a finding with implications for the short-term use of mineralocorticoids in septic shock and long-term, early use of spironolactone in PAH. Based on this work, a pilot protocol to study early spironolactone therapy in PAH has been written and is undergoing scientific review.

用诱导型一氧化氮合酶 (NOS2) 转染单母细胞 U937 细胞，产生了一种能够自发产生大量 NO 的人类细胞系，NO 是感染性休克中血管功能障碍的介质。用佛波醇 12 乙酸酯 13 肉豆蔻酸酯 (PMA) 分化后，iNOS 表达细胞通过依赖于 NO 的机制产生更多的 TNF。这证明了内源性产生的 NO 能够增强炎症反应（Blood，1997）。 同样，内皮型一氧化氮合酶 (eNOS) 的过度表达会上调 TNFα 的产生，但一氧化氮合酶 (NOS) 抑制剂 N-甲基-L-精氨酸及其 L-精氨酸结合位点的突变（使其无法产生一氧化氮）都无法阻止这一作用。效应（J Biol Chem，2000）。然而，与超氧化物歧化酶共转染或删除 eNOS 的 NADPH 结合位点完全阻止了 eNOS 上调 TNF 的产生。总的来说，我们的结果表明，eNOS 通过 NO（J Nutrition，1994；J Biol Chem，1997）和基于活性氧的信号转导途径（J Biol Chem，2000）调节炎症反应。 随后显示 eNOS 产生的超氧化物可通过 p42/44 MAPK 激活上调 TNFα（J Biol Chem，2001）。 Sp1 被确定为双向 NO 传感器，通过近端 Sp 启动子结合位点下调内皮细胞中的 eNOS 并上调 TNF（J Biol Chem，2003）。响应的方向（向上或向下）由与 Sp1 结合位点相邻的启动子序列控制。 镰状细胞病的特征是脉管系统中的氧化和炎症应激，即使没有急性危象也是如此（Blood，2004）。镰状细胞病中的循环应激与血小板中的基因表达和精氨酸代谢异常有关（Circulation，2007）。 NO 预防慢性血管损伤表型发展的抗增殖作用与 p38 MAPK 激活和 p21 mRNA 稳定有关，随后通过 CDE/CHR 近端启动子位点下调 Polo 样激酶 1（BMC Genomics，2005；生物化学杂志，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。因此，我们使用人脐静脉和杂交 EA.hy926 内皮细胞测试了这些信号传导通路之间的串扰（FASEB J，2007）。 PPARγ 通过 p38 MAPK 依赖性信号转导途径被 NO 激活。这种串扰机制可能有助于血管系统中 NO 的抗炎和细胞保护作用，并提出了预防和治疗血管功能障碍的新策略。 研究发现，用于治疗 2 型糖尿病的药物噻唑烷二酮 (TZD) 对 PPARgamma 的最佳激活同样需要 p​​38 MAPK 激活。 TZD 与稳态 NO 产生一样，可以减少心血管炎症标志物。我们随后确定了一种 p38MAPK/乙酰转移酶信号转导机制，可增强 PPARgamma 靶基因的转录（准备中，2012 年）。 PPARgamma 激动剂（罗格列酮；RGZ）激活 p38 MAPK，并增强 PPARgamma 信号下游，与 G 蛋白偶联受体 40 (GPCR40) 相关。这一发现首次将 PPARgamma 信号传导定义为双受体系统。 同源 GPCR 和核受体信号网络可以解释核受体配体之间安全性和功效的差异，并对未来的药物开发具有影响。总的来说，这些发现表明 PPARgamma 可能是减少脓毒性休克和可能的慢性疾病（如 PAH）中的内皮功能障碍和血管炎症的有用靶标。 对我们的微阵列数据库的询问表明，与单核细胞和 B 淋巴细胞相比，盐皮质激素受体 (MR) 在内皮细胞和 T 淋巴细胞中相对过度表达。同样，其他三个核受体家族成员，雄激素受体（AR），COUP-TFI和COUP-TFII也被发现在内皮细胞中相对于白细胞过度表达。相比之下，糖皮质激素（GR）是一种核受体靶标，可以逆转休克，但由于对免疫功能的不利影响，可能缺乏整体益处，在这些细胞类型中的表达更加一致。因此，MR、AR、COUP-TFI 和 COUP-TFII 可能是调节内皮炎症的有用靶标。 使用全基因组表达谱，人内皮细胞系中的 COUP-TFII 敲低显示可调节所有 TNF 反应基因的 36%（美国胸科学会，摘要 2011）。 COUP-TFII 对 TNFlapha 诱导的基因表达的抑制与干扰素信号通路（IRF 和 STAT）相关，而 COUP-TFII 对 TNFα 反应的放大与 NF-κB 相关。 与健康性别、年龄和种族匹配的志愿者相比，PAH 患者外周血单核细胞 (PBMC) 的全基因组基因表达差异明确地确定了炎症、细胞粘附、细胞运动、细胞骨架和细胞凋亡的改变（美国胸科学会，摘要 2011）。特定基因和经典途径与先前提出的几种机制重叠，并提出了新的治疗靶点，例如 Ras、RhoA、整合素、粘着斑激酶-1 (FAK) 和 p21 激活激酶 (PAK)。 最近的工作研究了原代人肺动脉内皮细胞 (PAEC) 中 BMPR2 的沉默，作为遗传性 PAH 的体外模型。 BMPR2 敲低会产生雌激素受体表达改变、炎症反应加剧和应激激酶信号失调的表型（2012 年肺动脉高压 Keystone 研讨会）。 在人内皮细胞中，MR 激动剂和拮抗剂均已被证明可以抑制 NF-kappaB 介导的基因转录（Keystone Symposia on Nuclear Receptors，摘要，2010；美国胸科学会，摘要 2010），这一发现对短期使用 MR 具有重要意义。盐皮质激素治疗感染性休克，长期、早期使用螺内酯治疗肺动脉高压。 基于这项工作，研究 PAH 早期螺内酯治疗的试点方案已经制定，并正在进行科学审查。