Critical role of autophagy regulator Beclin1 in endothelial cell inflammation and barrier disruption.

Recent studies have implicated autophagy in several inflammatory diseases involving aberrant endothelial cell (EC) responses, such as acute lung injury (ALI). However, the mechanistic basis for a role of autophagy in EC inflammation and permeability remain poorly understood. In this study, we impaired autophagy by silencing the essential Beclin1 autophagy gene in human pulmonary artery EC.

PPARγ Regulates Mitochondrial Structure and Function and Human Pulmonary Artery Smooth Muscle Cell Proliferation.

Pulmonary hypertension (PH) is a progressive disorder that causes significant morbidity and mortality despite existing therapies. PH pathogenesis is characterized by metabolic derangements that increase pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling. PH-associated decreases in peroxisome proliferator-activated receptor γ (PPARγ) stimulate PASMC proliferation, and PPARγ in coordination with PPARγ coactivator 1α (PGC1α) regulates mitochondrial gene expression and biogenesis.

Hypoxia inhibits expression and function of mitochondrial thioredoxin 2 to promote pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary vascular pressures that often result in right ventricular dysfunction, leading to right heart failure. Evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis by altering pulmonary vascular cell proliferation and intracellular signaling pathways. However, the role of mitochondrial antioxidants and oxidant-derived stress signaling in the development of hypoxia-induced PH is largely unknown.

From the Cover: Manganese Stimulates Mitochondrial H2O2 Production in SH-SY5Y Human Neuroblastoma Cells Over Physiologic as well as Toxicologic Range.

Manganese (Mn) is an abundant redox-active metal with well-characterized mitochondrial accumulation and neurotoxicity due to excessive exposures. Mn is also an essential co-factor for the mitochondrial antioxidant protein, superoxide dismutase-2 (SOD2), and the range for adequate intake established by the Institute of Medicine Food and Nutrition Board is 20% of the interim guidance value for toxicity by the Agency for Toxic Substances and Disease Registry, leaving little margin for safety. To study toxic mechanisms over this critical dose range, we treated human neuroblastoma SH-SY5Y cells with a series of MnCl concentrations (from 0 to 100 μM) and measured cellular content to compare to human brain Mn content.

Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells.

Background: Pathogenesis of pulmonary hypertension is complex and involves activation of the transcription factor, hypoxia-inducible factor-1 (HIF-1) that shifts cellular metabolism from aerobic respiration to glycolysis, in part, by increasing the expression of its downstream target pyruvate dehydrogenase kinase-1 (PDK-1), thereby promoting a proliferative, apoptosis-resistant phenotype in pulmonary vascular cells. Activation of the nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), attenuates pulmonary hypertension and pulmonary artery smooth muscle cell (PASMC) proliferation. In the current study, we determined whether PPARγ inhibits HIF-1α and PDK-1 expression in human PASMCs.

Phospholipase C-ε signaling mediates endothelial cell inflammation and barrier disruption in acute lung injury.

Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε(-/-) mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI.

Proline-rich tyrosine kinase 2 downregulates peroxisome proliferator-activated receptor gamma to promote hypoxia-induced pulmonary artery smooth muscle cell proliferation.

Hypoxia stimulates pulmonary hypertension (PH), in part by increasing the proliferation of human pulmonary artery smooth muscle cells (HPASMCs) via sustained activation of mitogen-activated protein kinase, extracellular signal-regulated kinases 1 and 2 (ERK 1/2), and nuclear factor-kappa B (NF-κB); elevated expression of NADPH oxidase 4 (Nox4); and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. However, the upstream mediators that control these responses remain largely unknown. We hypothesized that proline-rich tyrosine kinase 2 (Pyk2) plays a critical role in the mechanism of hypoxia-induced HPASMC proliferation.

Serpine2 deficiency results in lung lymphocyte accumulation and bronchus-associated lymphoid tissue formation.

Serine proteinase inhibitor, clade E, member 2 (SERPINE2), is a cell- and extracellular matrix-associated inhibitor of thrombin. Although SERPINE2 is a candidate susceptibility gene for chronic obstructive pulmonary disease, the physiologic role of this protease inhibitor in lung development and homeostasis is unknown. We observed spontaneous monocytic-cell infiltration in the lungs of Serpine2-deficient (SE2(-/-)) mice, beginning at or before the time of lung maturity, which resulted in lesions that resembled bronchus-associated lymphoid tissue (BALT).

Peroxisome Proliferator-Activated Receptor γ and microRNA 98 in Hypoxia-Induced Endothelin-1 Signaling.

Endothelin-1 (ET-1) plays a critical role in endothelial dysfunction and contributes to the pathogenesis of pulmonary hypertension (PH). We hypothesized that peroxisome proliferator-activated receptor γ (PPARγ) stimulates microRNAs that inhibit ET-1 and pulmonary artery endothelial cell (PAEC) proliferation. The objective of this study was to clarify molecular mechanisms by which PPARγ regulates ET-1 expression in vitro and in vivo.

Targeting mitochondrial reactive oxygen species to modulate hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O(2)(•-)) and hydrogen peroxide (H(2)O(2)) in pulmonary vascular cells.

Airway epithelial cell PPARγ modulates cigarette smoke-induced chemokine expression and emphysema susceptibility in mice.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent, chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-γ activity can modify inflammatory responses in several models of lung injury, the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema, with excessive macrophage accumulation associated with increased expression of chemokines, Ccl5, Cxcl10, and Cxcl15.

Peroxisome proliferator-activated receptor gamma depletion stimulates Nox4 expression and human pulmonary artery smooth muscle cell proliferation.

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia-induced cell proliferation include sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase 4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels associated with PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro under normoxic conditions.

Nicotine stimulates nerve growth factor in lung fibroblasts through an NFκB-dependent mechanism.

Rationale: Airway hyperresponsiveness (AHR) is classically found in asthma, and persistent AHR is associated with poor asthma control. Although airway smooth muscle (ASM) cells play a critical pathophysiologic role in AHR, the paracrine contributions of surrounding cells such as fibroblasts to the contractile phenotype of ASM cells have not been examined fully. This study addresses the hypothesis that nicotine promotes a contractile ASM cell phenotype by stimulating fibroblasts to increase nerve growth factor (NGF) secretion into the environment.

Regulation of endothelial cell inflammation and lung polymorphonuclear lymphocyte infiltration by transglutaminase 2.

We addressed the role of transglutaminase 2 (TG2), a calcium-dependent enzyme that catalyzes cross-linking of proteins, in the mechanism of endothelial cell (EC) inflammation and lung polymorphonuclear lymphocyte (PMN) infiltration. Exposure of EC to thrombin, a procoagulant and proinflammatory mediator, resulted in activation of the transcription factor nuclear factor κB (NF-κB) and its target genes, vascular cell adhesion molecule 1, monocyte chemotactic protein 1, and interleukin 6. RNAi knockdown of TG2 inhibited these responses.

Hypoxia mediates mutual repression between microRNA-27a and PPARγ in the pulmonary vasculature.

Pulmonary hypertension (PH) is a serious disorder that causes significant morbidity and mortality. The pathogenesis of PH involves complex derangements in multiple pathways including reductions in peroxisome proliferator-activated receptor gamma (PPARγ). Hypoxia, a common PH stimulus, reduces PPARγ in experimental models.

Hypoxia downregulates PPARγ via an ERK1/2-NF-κB-Nox4-dependent mechanism in human pulmonary artery smooth muscle cells.

The ligand-activated transcription factor peroxisome proliferator-activated receptor γ (PPARγ) regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. This study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo.

Critical role of non-muscle myosin light chain kinase in thrombin-induced endothelial cell inflammation and lung PMN infiltration.

The pathogenesis of acute lung injury (ALI) involves bidirectional cooperation and close interaction between inflammatory and coagulation pathways. A key molecule linking coagulation and inflammation is the procoagulant thrombin, a serine protease whose concentration is elevated in plasma and lavage fluids of patients with ALI and acute respiratory distress syndrome (ARDS). However, little is known about the mechanism by which thrombin contributes to lung inflammatory response.

Regulation of Rela/p65 and endothelial cell inflammation by proline-rich tyrosine kinase 2.

We investigated the role of proline-rich tyrosine kinase 2 (Pyk2) in the mechanism of NF-κB activation and endothelial cell (EC) inflammation induced by thrombin, a procoagulant serine protease released in high amounts during sepsis and other inflammatory conditions. Stimulation of ECs with thrombin resulted in a time-dependent activation of Pyk2. RNA interference knockdown of Pyk2 attenuated thrombin-induced activity of NF-κB and expression of its target genes, vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1.

Essential role of cofilin-1 in regulating thrombin-induced RelA/p65 nuclear translocation and intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells.

Activation of RhoA/Rho-associated kinase (ROCK) pathway and the associated changes in actin cytoskeleton induced by thrombin are crucial for activation of NF-kappaB and expression of its target gene ICAM-1 in endothelial cells. However, the events acting downstream of RhoA/ROCK to mediate these responses remain unclear. Here, we show a central role of cofilin-1, an actin-binding protein that promotes actin depolymerization, in linking RhoA/ROCK pathway to dynamic alterations in actin cytoskeleton that are necessary for activation of NF-kappaB and thereby expression of ICAM-1 in these cells.

Protein kinase C-delta and phosphatidylinositol 3-kinase/Akt activate mammalian target of rapamycin to modulate NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells.

We have shown that the mammalian target of rapamycin (mTOR) down-regulates thrombin-induced ICAM-1 expression in endothelial cells by suppressing the activation of NF-kappaB. However, the mechanisms by which mTOR is activated to modulate these responses remain to be addressed. Here, we show that thrombin engages protein kinase C (PKC)-delta and phosphattidylinositol 3-kinase (PI3K)/Akt pathways to activate mTOR and thereby dampens NF-kappaB activation and intercellular adhesion molecule 1 (ICAM-1) expression.

Activation of Syk by protein kinase C-delta regulates thrombin-induced intercellular adhesion molecule-1 expression in endothelial cells via tyrosine phosphorylation of RelA/p65.

Protein kinase C-delta (PKC-delta) plays a pivotal role in mediating thrombin-induced NF-kappaB activation and ICAM-1 expression in endothelial cells. However, the downstream mechanisms mediating its function are unclear. In this study, we show that PKC-delta-mediated activation of protein-tyrosine kinase Syk plays an important role in thrombin signaling of NF-kappaB activation and intercellular adhesion molecule-1 (ICAM-1) expression in endothelial cells.

Evidence for actin cytoskeleton-dependent and -independent pathways for RelA/p65 nuclear translocation in endothelial cells.

Activation of the transcription factor NF-kappaB involves its release from the inhibitory protein IkappaBalpha in the cytoplasm and subsequently, its translocation to the nucleus. Whereas the events responsible for its release have been elucidated, mechanisms regulating the nuclear transport of NF-kappaB remain elusive. We now provide evidence for actin cytoskeleton-dependent and -independent mechanisms of RelA/p65 nuclear transport using the proinflammatory mediators, thrombin and tumor necrosis factor alpha, respectively.

c-Src interacts with and phosphorylates RelA/p65 to promote thrombin-induced ICAM-1 expression in endothelial cells.

The procoagulant thrombin promotes polymorphonuclear leukocyte (PMN) adhesion to endothelial cells by a mechanism involving expression of intercellular adhesion molecule-1 (ICAM-1) via an NF-kappaB-dependent pathway. We now provide evidence that activation of c-Src is crucial in signaling thrombin-induced ICAM-1 expression via tyrosine phosphorylation of RelA/p65. Stimulation of human umbilical vein endothelial cells with thrombin resulted in a time-dependent activation of c-Src, with maximal activation occurring at 30 min after thrombin challenge.

Inhibition of mammalian target of rapamycin potentiates thrombin-induced intercellular adhesion molecule-1 expression by accelerating and stabilizing NF-kappa B activation in endothelial cells.

We addressed the regulatory function of mammalian target of rapamycin (mTOR) in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. Pretreatment of HUVECs with rapamycin, an inhibitor of mTOR, augmented thrombin-induced ICAM-1 expression. Inhibition of mTOR by this approach promoted whereas over-expression of mTOR inhibited thrombin-induced transcriptional activity of NF-kappaB, an essential regulator of ICAM-1 transcription.

cAMP targeting of p38 MAP kinase inhibits thrombin-induced NF-kappaB activation and ICAM-1 expression in endothelial cells.

We investigated the mechanisms by which elevated intracellular cAMP concentration inhibits the thrombin-induced ICAM-1 expression in endothelial cells. Exposure of human umbilical vein endothelial cells to forskolin or dibutyryl cAMP, which increase intracellular cAMP by separate mechanisms, inhibited the thrombin-induced ICAM-1 expression. This effect of cAMP was secondary to inhibition of NF-kappaB activity, the key regulator of thrombin-induced ICAM-1 expression in endothelial cells.