Recep Nigdelioglu

Particulate Matter-Induced Lung Inflammation Increases Systemic Levels of PAI-1 and Activates Coagulation Through Distinct Mechanisms

Background Exposure of human populations to ambient particulate matter (PM) air pollution significantly contributes to the mortality attributable to ischemic cardiovascular events. We reported that mice treated with intratracheally instilled PM develop a prothrombotic state that requires the release of IL-6 by alveolar macrophages. We sought to determine whether exposure of mice to PM increases the levels of PAI-1, a major regulator of thrombolysis, via a similar or distinct mechanism. Methods and Principal Findings Adult, male C57BL/6 and IL-6 knock out (IL-6−/−) mice were exposed to either concentrated ambient PM less than 2.5 µm (CAPs) or filtered air 8 hours daily for 3 days or were exposed to either urban particulate matter or PBS via intratracheal instillation and examined 24 hours later. Exposure to CAPs or urban PM resulted in the IL-6 dependent activation of coagulation in the lung and systemically. PAI-1 mRNA and protein levels were higher in the lung and adipose tissue of mice treated with CAPs or PM compared with filtered air or PBS controls. The increase in PAI-1 was similar in wild-type and IL-6−/− mice but was absent in mice treated with etanercept, a TNF-α inhibitor. Treatment with etanercept did not prevent the PM-induced tendency toward thrombus formation. Conclusions Mice exposed to inhaled PM exhibited a TNF-α-dependent increase in PAI-1 and an IL-6-dependent activation of coagulation. These results suggest that multiple mechanisms link PM-induced lung inflammation with the development of a prothrombotic state.

Particulate matter air pollution causes oxidant-mediated increase in gut permeability in mice.

BackgroundExposure to particulate matter (PM) air pollution may be an important environmental factor leading to exacerbations of inflammatory illnesses in the GI tract. PM can gain access to the gastrointestinal (GI) tract via swallowing of air or secretions from the upper airways or mucociliary clearance of inhaled particles.MethodsWe measured PM-induced cell death and mitochondrial ROS generation in Caco-2 cells stably expressing oxidant sensitive GFP localized to mitochondria in the absence or presence of an antioxidant. C57BL/6 mice were exposed to a very high dose of urban PM from Washington, DC (200 μg/mouse) or saline via gastric gavage and small bowel and colonic tissue were harvested for histologic evaluation, and RNA isolation up to 48 hours. Permeability to 4kD dextran was measured at 48 hours.ResultsPM induced mitochondrial ROS generation and cell death in Caco-2 cells. PM also caused oxidant-dependent NF-κB activation, disruption of tight junctions and increased permeability of Caco-2 monolayers. Mice exposed to PM had increased intestinal permeability compared with PBS treated mice. In the small bowel, colocalization of the tight junction protein, ZO-1 was lower in the PM treated animals. In the small bowel and colon, PM exposed mice had higher levels of IL-6 mRNA and reduced levels of ZO-1 mRNA. Increased apoptosis was observed in the colon of PM exposed mice.ConclusionsExposure to high doses of urban PM causes oxidant dependent GI epithelial cell death, disruption of tight junction proteins, inflammation and increased permeability in the gut in vitro and in vivo. These PM-induced changes may contribute to exacerbations of inflammatory disorders of the gut.

β2-Adrenergic agonists augment air pollution–induced IL-6 release and thrombosis

Acute exposure to particulate matter (PM) air pollution causes thrombotic cardiovascular events, leading to increased mortality rates; however, the link between PM and cardiovascular dysfunction is not completely understood. We have previously shown that the release of IL-6 from alveolar macrophages is required for a prothrombotic state and acceleration of thrombosis following exposure to PM. Here, we determined that PM exposure results in the systemic release of catecholamines, which engage the β2-adrenergic receptor (β2AR) on murine alveolar macrophages and augment the release of IL-6. In mice, β2AR signaling promoted the development of a prothrombotic state that was sufficient to accelerate arterial thrombosis. In primary human alveolar macrophages, administration of a β2AR agonist augmented IL-6 release, while the addition of a beta blocker inhibited PM-induced IL-6 release. Genetic loss or pharmacologic inhibition of the β2AR on murine alveolar macrophages attenuated PM-induced IL-6 release and prothrombotic state. Furthermore, exogenous β2AR agonist therapy further augmented these responses in alveolar macrophages through generation of mitochondrial ROS and subsequent increase of adenylyl cyclase activity. Together, these results link the activation of the sympathetic nervous system by β2AR signaling with metabolism, lung inflammation, and an enhanced susceptibility to thrombotic cardiovascular events.

HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium

Hemodynamic forces regulate vascular functions. Disturbed flow (DF) occurs in arterial bifurcations and curvatures, activates endothelial cells (ECs), and results in vascular inflammation and ultimately atherosclerosis. However, how DF alters EC metabolism, and whether resulting metabolic changes induce EC activation, is unknown. Using transcriptomics and bioenergetic analysis, we discovered that DF induces glycolysis and reduces mitochondrial respiratory capacity in human aortic ECs. DF-induced metabolic reprogramming required hypoxia inducible factor-1α (HIF-1α), downstream of NAD(P)H oxidase-4 (NOX4)-derived reactive oxygen species (ROS). HIF-1α increased glycolytic enzymes and pyruvate dehydrogenase kinase-1 (PDK-1), which reduces mitochondrial respiratory capacity. Swine aortic arch endothelia exhibited elevated ROS, NOX4, HIF-1α, and glycolytic enzyme and PDK1 expression, suggesting that DF leads to metabolic reprogramming in vivo. Inhibition of glycolysis reduced inflammation suggesting a causal relationship between flow-induced metabolic changes and EC activation. These findings highlight a previously uncharacterized role for flow-induced metabolic reprogramming and inflammation in ECs. DOI: http://dx.doi.org/10.7554/eLife.25217.001

Transforming Growth Factor (TGF)-β Promotes de Novo Serine Synthesis for Collagen Production

TGF-β promotes excessive collagen deposition in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). The amino acid composition of collagen is unique due to its high (33%) glycine content. Here, we report that TGF-β induces expression of glycolytic genes and increases glycolytic flux. TGF-β also induces the expression of the enzymes of the de novo serine synthesis pathway (phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)) and de novo glycine synthesis (serine hydroxymethyltransferase 2 (SHMT2)). Studies in fibroblasts with genetic attenuation of PHGDH or SHMT2 and pharmacologic inhibition of PHGDH showed that these enzymes are required for collagen synthesis. Furthermore, metabolic labeling experiments demonstrated carbon from glucose incorporated into collagen. Lungs from humans with IPF demonstrated increased expression of PHGDH and SHMT2. These results indicate that the de novo serine synthesis pathway is necessary for TGF-β-induced collagen production and suggest that this pathway may be a therapeutic target for treatment of fibrotic diseases including IPF.

TGF-β Promotes de novo Serine Synthesis for Collagen Production

TGF-β promotes excessive collagen deposition in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). The amino acid composition of collagen is unique due to its high (33%) glycine content. Here, we report that TGF-β induces expression of glycolytic genes and increases glycolytic flux. TGF-β also induces the expression of the enzymes of the de novo serine synthesis pathway (phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)) and de novo glycine synthesis (serine hydroxymethyltransferase 2 (SHMT2)). Studies in fibroblasts with genetic attenuation of PHGDH or SHMT2 and pharmacologic inhibition of PHGDH showed that these enzymes are required for collagen synthesis. Furthermore, metabolic labeling experiments demonstrated carbon from glucose incorporated into collagen. Lungs from humans with IPF demonstrated increased expression of PHGDH and SHMT2. These results indicate that the de novo serine synthesis pathway is necessary for TGF-β-induced collagen production and suggest that this pathway may be a therapeutic target for treatment of fibrotic diseases including IPF.

Lung-Specific Loss of α3 Laminin Worsens Bleomycin-Induced Pulmonary Fibrosis

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.

Prolonged Exposures to Intermittent Hypoxia Promote Visceral White Adipose Tissue Inflammation in a Murine Model of Severe Sleep Apnea: Effect of Normoxic Recovery

Study Objective Increased visceral white adipose tissue (vWAT) mass results in infiltration of inflammatory macrophages that drive inflammation and insulin resistance. Patients with obstructive sleep apnea (OSA) suffer from increased prevalence of obesity, insulin resistance, and metabolic syndrome. Murine models of intermittent hypoxia (IH) mimicking moderate-severe OSA manifest insulin resistance following short-term IH. We examined in mice the effect of long-term IH on the inflammatory cellular changes within vWAT and the potential effect of normoxic recovery (IH-R). Methods Male C57BL/6J mice were subjected to IH for 20 weeks, and a subset was allowed to recover in room air (RA) for 6 or 12 weeks (IH-R). Stromal vascular fraction was isolated from epididymal vWAT and mesenteric vWAT depots, and single-cell suspensions were prepared for flow cytometry analyses, reactive oxygen species (ROS), and metabolic assays. Results IH reduced body weight and vWAT mass and IH-R resulted in catch-up weight and vWAT mass. IH-exposed vWAT exhibited increased macrophage counts (ATMs) that were only partially improved in IH-R. IH also caused a proinflammatory shift in ATMs (increased Ly6c(hi)(+) and CD36(+) ATMs). These changes were accompanied by increased vWAT insulin resistance with only partial improvements in IH-R. In addition, ATMs exhibited increased ROS production, altered metabolism, and changes in electron transport chain, which were only partially improved in IH-R. Conclusion Prolonged exposures to IH during the sleep period induce pronounced vWAT inflammation and insulin resistance despite concomitant vWAT mass reductions. These changes are only partially reversible after 3 months of normoxic recovery. Thus, long-lasting OSA may preclude complete reversibility of metabolic changes.

Experimental Lung Injury Reduces Krüppel-like Factor 2 to Increase Endothelial Permeability via Regulation of RAPGEF3–Rac1 Signaling

Rationale: Acute respiratory distress syndrome (ARDS) is caused by widespread endothelial barrier disruption and uncontrolled cytokine storm. Genome‐wide association studies (GWAS) have linked multiple genes to ARDS. Although mechanosensitive transcription factor Krüppel‐like factor 2 (KLF2) is a major regulator of endothelial function, its role in regulating pulmonary vascular integrity in lung injury and ARDS‐associated GWAS genes remains poorly understood. Objectives: To examine KLF2 expression in multiple animal models of acute lung injury and further elucidate the KLF2‐mediated pathways involved in endothelial barrier disruption and cytokine storm in experimental lung injury. Methods: Animal and in vitro models of acute lung injury were used to characterize KLF2 expression and its downstream effects responding to influenza A virus (A/WSN/33 [H1N1]), tumor necrosis factor‐&agr;, LPS, mechanical stretch/ventilation, or microvascular flow. KLF2 manipulation, permeability measurements, small GTPase activity, luciferase assays, chromatin immunoprecipitation assays, and network analyses were used to determine the mechanistic roles of KLF2 in regulating endothelial monolayer integrity, ARDS‐associated GWAS genes, and lung pathophysiology. Measurements and Main Results: KLF2 is significantly reduced in several animal models of acute lung injury. Microvascular endothelial KLF2 is significantly induced by capillary flow but reduced by pathologic cyclic stretch and inflammatory stimuli. KLF2 is a novel activator of small GTPase Ras‐related C3 botulinum toxin substrate 1 by transcriptionally controlling Rap guanine nucleotide exchange factor 3/exchange factor directly activated by cyclic adenosine monophosphate, which maintains vascular integrity. KLF2 regulates multiple ARDS GWAS genes related to cytokine storm, oxidation, and coagulation in lung microvascular endothelium. KLF2 overexpression ameliorates LPS‐induced lung injury in mice. Conclusions: Disruption of endothelial KLF2 results in dysregulation of lung microvascular homeostasis and contributes to lung pathology in ARDS.

Impaired Clearance of Influenza A Virus in Obese, Leptin Receptor Deficient Mice Is Independent of Leptin Signaling in the Lung Epithelium and Macrophages

Rationale During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients. Methods We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepRfl/fl) or macrophages and alveolar type II cells (LysM-Cre/Leprfl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity. Results The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre+/+/LepRfl/fl and LysM-Cre+/+/LepRfl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre+/+/LepRfl/fl and LysM-Cre+/+/LepRfl /fl mice exhibited improved survival. Conclusions Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.