MicroRNA-98 reduces nerve growth factor expression in nicotine-induced airway remodeling.

Evolving evidence suggests that nicotine may contribute to impaired asthma control by stimulating expression of nerve growth factor (NGF), a neurotrophin associated with airway remodeling and airway hyperresponsiveness. We explored the hypothesis that nicotine increases NGF by reducing lung fibroblast (LF) microRNA-98 (miR-98) and PPARγ levels, thus promoting airway remodeling. Levels of NGF, miR-98, PPARγ, fibronectin 1 (FN1), endothelin-1 (EDN1, herein referred to as ET-1), and collagen (COL1A1 and COL3A1) were measured in human LFs isolated from smoking donors, in mouse primary LFs exposed to nicotine (50 µg/ml), and in whole lung homogenates from mice chronically exposed to nicotine (100 µg/ml) in the drinking water. In selected studies, these pathways were manipulated in LFs with miR-98 inhibitor (anti-miR-98), miR-98 overexpression (miR-98 mimic), or the PPARγ agonist rosiglitazone. Compared with unexposed controls, nicotine increased NGF, FN1, ET-1, COL1A1, and COL3A1 expression in human and mouse LFs and mouse lung homogenates. In contrast, nicotine reduced miR-98 levels in LFs in vitro and in lung homogenates in vivo Treatment with anti-miR-98 alone was sufficient to recapitulate increases in NGF, FN1, and ET-1, whereas treatment with a miR-98 mimic significantly suppressed luciferase expression in cells transfected with a luciferase reporter linked to the putative seed sequence in the NGF 3'UTR and also abrogated nicotine-induced increases in NGF, FN1, and ET-1 in LFs. Similarly, rosiglitazone increased miR-98 and reversed nicotine-induced increases in NGF, FN1, and ET-1. Taken together, these findings demonstrate that nicotine-induced increases in NGF and other markers of airway remodeling are negatively regulated by miR-98.

UPR modulation of host immunity by Pseudomonas aeruginosa in cystic fibrosis.

Cystic fibrosis (CF) is a progressive multiorgan autosomal recessive disease with devastating impact on the lungs caused by derangements of the CF transmembrane conductance regulator (CFTR) gene. Morbidity and mortality are caused by the triad of impaired mucociliary clearance, microbial infections and chronic inflammation. Pseudomonas aeruginosa is the main respiratory pathogen in individuals with CF infecting most patients in later stages. Despite its recognized clinical impact, molecular mechanisms that underlie P. aeruginosa pathogenesis and the host response to P. aeruginosa infection remain incompletely understood. The nuclear hormone receptor peroxisome proliferator-activated receptor (PPAR) γ (PPARγ), has shown to be reduced in CF airways. In the present study, we sought to investigate the upstream mechanisms repressing PPARγ expression and its impact on airway epithelial host defense. Endoplasmic reticulum-stress (ER-stress) triggered unfolded protein response (UPR) activated by misfolded CFTR and P. aeruginosa infection contributed to attenuated expression of PPARγ. Specifically, the protein kinase RNA (PKR)-like ER kinase (PERK) signaling pathway led to the enhanced expression of the CCAAT-enhancer-binding-protein homologous protein (CHOP). CHOP induction led to the repression of PPARγ expression. Mechanistically, we showed that CHOP induction mediated PPARγ attenuation, impacted the innate immune function of normal and ∆F508 primary airway epithelial cells by reducing expression of antimicrobial peptide (AMP) and paraoxanse-2 (PON-2), as well as enhancing IL-8 expression. Furthermore, mitochondrial reactive oxygen species production (mt-ROS) and ER-stress positive feedforward loop also dysregulated mitochondrial bioenergetics. Additionally, our findings implicate that PPARγ agonist pioglitazone (PIO) has beneficial effect on the host at the multicellular level ranging from host defense to mitochondrial re-energization.

Pseudomonas aeruginosa Induced Host Epithelial Cell Mitochondrial Dysfunction.

The pathogenicity of P. aeruginosa is dependent on quorum sensing (QS), an inter-bacterial communication system that can also modulate host biology. The innate immune function of the lung mucosal barrier is dependent on proper mitochondrial function. The purpose of this study was to define the mechanism by which bacterial factors modulate host lung epithelial cell mitochondrial function and to investigate novel therapies that ameliorate this effect. 3-oxo-C12-HSL disrupts mitochondrial morphology, attenuates mitochondrial bioenergetics, and induces mitochondrial DNA oxidative injury. Mechanistically, we show that 3-oxo-C12-HSL attenuates expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis, antioxidant defense, and cellular respiration, and its downstream effectors in both BEAS-2B and primary lung epithelial cells. Overexpression of PGC-1α attenuates the inhibition in cellular respiration caused by 3-oxo-C12-HSL. Pharmacologic activation of PGC-1α restores barrier integrity in cells treated with 3-oxo-C12-HSL. These data demonstrate that the P. aeruginosa QS molecule, 3-oxo-C12-HSL, alters mitochondrial pathways critical for lung mucosal immunity. Genetic and pharmacologic strategies that activate the PGC-1α pathway enhance host epithelial cell mitochondrial function and improve the epithelial innate response to P. aeruginosa. Therapies that rescue PGC-1α function may provide a complementary approach in the treatment of P. aeruginosa infection.

Glial Cell Line-Derived Neurotrophic Factor Enhances Autophagic Flux in Mouse and Rat Hepatocytes and Protects Against Palmitate Lipotoxicity.

Glial cell line-derived neurotrophic factor (GDNF) is a protein that is required for the development and survival of enteric, sympathetic, and catecholaminergic neurons. We previously reported that GDNF is protective against high fat diet (HFD)-induced hepatic steatosis in mice through suppression of hepatic expression of peroxisome proliferator activated receptor-γ and genes encoding enzymes involved in de novo lipogenesis. We also reported that transgenic overexpression of GDNF in mice prevented the HFD-induced liver accumulation of the autophagy cargo-associated protein p62/sequestosome 1 characteristic of impaired autophagy. Here we investigated the effects of GDNF on hepatic autophagy in response to increased fat load, and on hepatocyte mitochondrial fatty acid ß-oxidation and cell survival. GDNF not only prevented the reductions in the liver levels of some key autophagy-related proteins, including Atg5, Atg7, Beclin-1 and LC3A/B-II, seen in HFD-fed control mice, but enhanced their levels after 12 weeks of HFD feeding. In vitro, GDNF accelerated autophagic cargo clearance in primary mouse hepatocytes and a rat hepatocyte cell line, and reduced the phosphorylation of the mechanistic target of rapamycin complex downstream-target p70S6 kinase similar to the autophagy activator rapamycin. GDNF also enhanced mitochondrial fatty acid ß-oxidation in primary mouse and rat hepatocytes, and protected against palmitate-induced lipotoxicity. Conclusion: We demonstrate a role for GDNF in enhancing hepatic autophagy and in potentiating mitochondrial function and fatty acid oxidation. Our studies show that GDNF and its receptor agonists could be useful for enhancing hepatocyte survival and protecting against fatty acid-induced hepatic lipotoxicity.

Peroxisome proliferator-activated receptor-γ enhances human pulmonary artery smooth muscle cell apoptosis through microRNA-21 and programmed cell death 4.

Pulmonary hypertension (PH) is a progressive disorder whose cellular pathogenesis involves enhanced smooth muscle cell (SMC) proliferation and resistance to apoptosis signals. Existing evidence demonstrates that the tumor suppressor programmed cell death 4 (PDCD4) affects patterns of cell growth and repair responses in the systemic vasculature following experimental injury. In the current study, the regulation PDCD4 and its functional effects on growth and apoptosis susceptibility in pulmonary artery smooth muscle cells were explored. We previously demonstrated that pharmacological activation of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) attenuated hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (HPASMCs) by inhibiting the expression and mitogenic functions of microRNA-21 (miR-21). In the current study, we hypothesize that PPARγ stimulates PDCD4 expression and HPASMC apoptosis by inhibiting miR-21. Our findings demonstrate that PDCD4 is reduced in the mouse lung upon exposure to chronic hypoxia (10% O2 for 3 wk) and in hypoxia-exposed HPASMCs (1% O2). HPASMC apoptosis was reduced by hypoxia, by miR-21 overexpression, or by siRNA-mediated PPARγ and PDCD4 depletion. Activation of PPARγ inhibited miR-21 expression and resultant proliferation, while restoring PDCD4 levels and apoptosis to baseline. Additionally, pharmacological activation of PPARγ with rosiglitazone enhanced PDCD4 protein expression and apoptosis in a dose-dependent manner as demonstrated by increased annexin V detection by flow cytometry. Collectively, these findings demonstrate that PPARγ confers growth-inhibitory signals in hypoxia-exposed HPASMCs through suppression of miR-21 and the accompanying derepression of PDCD4 that augments HPASMC susceptibility to undergo apoptosis.

Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a significant contributor to recalcitrant multidrug-resistant infections, especially in immunocompromised and hospitalized patients. The pathogenic profile of P. aeruginosa is related to its ability to secrete a variety of virulence factors and to promote biofilm formation. Quorum sensing (QS) is a mechanism wherein P. aeruginosa secretes small diffusible molecules, specifically acyl homo serine lactones, such as N-(3-oxo-dodecanoyl)-l-homoserine lactone (3O-C12-HSL), that promote biofilm formation and virulence via interbacterial communication. Strategies that strengthen the host's ability to inhibit bacterial virulence would enhance host defenses and improve the treatment of resistant infections. We have recently shown that peroxisome proliferator-activated receptor γ (PPARγ) agonists are potent immunostimulators that play a pivotal role in host response to virulent P. aeruginosa Here, we show that QS genes in P. aeruginosa (strain PAO1) and 3O-C12-HSL attenuate PPARγ expression in bronchial epithelial cells. PAO1 and 3O-C12-HSL induce barrier derangements in bronchial epithelial cells by lowering the expression of junctional proteins, such as zonula occludens-1, occludin, and claudin-4. Expression of these proteins was restored in cells that were treated with pioglitazone, a PPARγ agonist, before infection with PAO1 and 3O-C12-HSL. Barrier function and bacterial permeation studies that have been performed in primary human epithelial cells showed that PPARγ agonists are able to restore barrier integrity and function that are disrupted by PAO1 and 3O-C12-HSL. Mechanistically, we show that these effects are dependent on the induction of paraoxonase-2, a QS hydrolyzing enzyme, that mitigates the effects of QS molecules. Importantly, our data show that pioglitazone, a PPARγ agonist, significantly inhibits biofilm formation on epithelial cells by a mechanism that is mediated via paraoxonase-2. These findings elucidate a novel role for PPARγ in host defense against P. aeruginosa Strategies that activate PPARγ can provide a therapeutic complement for treatment of resistant P. aeruginosa infections.-Bedi, B., Maurice, N. M., Ciavatta, V. T., Lynn, K. S., Yuan, Z., Molina, S. A., Joo, M., Tyor, W. R., Goldberg, J. B., Koval, M., Hart, C. M., Sadikot, R. T. Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Peroxisome Proliferator-Activated Receptor γ Regulates the V-Ets Avian Erythroblastosis Virus E26 Oncogene Homolog 1/microRNA-27a Axis to Reduce Endothelin-1 and Endothelial Dysfunction in the Sickle Cell Mouse Lung.

Pulmonary hypertension (PH), a serious complication of sickle cell disease (SCD), causes significant morbidity and mortality. Although a recent study determined that hemin release during hemolysis triggers endothelial dysfunction in SCD, the pathogenesis of SCD-PH remains incompletely defined. This study examines peroxisome proliferator-activated receptor γ (PPARγ) regulation in SCD-PH and endothelial dysfunction. PH and right ventricular hypertrophy were studied in Townes humanized sickle cell (SS) and littermate control (AA) mice. In parallel studies, SS or AA mice were gavaged with the PPARγ agonist, rosiglitazone (RSG), 10 mg/kg/day, or vehicle for 10 days. In vitro, human pulmonary artery endothelial cells (HPAECs) were treated with vehicle or hemin for 72 hours, and selected HPAECs were treated with RSG. SS mice developed PH and right ventricular hypertrophy associated with reduced lung levels of PPARγ and increased levels of microRNA-27a (miR-27a), v-ets avian erythroblastosis virus E26 oncogene homolog 1 (ETS1), endothelin-1 (ET-1), and markers of endothelial dysfunction (platelet/endothelial cell adhesion molecule 1 and E selectin). HPAECs treated with hemin had increased ETS1, miR-27a, ET-1, and endothelial dysfunction and decreased PPARγ levels. These derangements were attenuated by ETS1 knockdown, inhibition of miR-27a, or PPARγ overexpression. In SS mouse lung or in hemin-treated HPAECs, activation of PPARγ with RSG attenuated reductions in PPARγ and increases in miR-27a, ET-1, and markers of endothelial dysfunction. In SCD-PH pathogenesis, ETS1 stimulates increases in miR-27a levels that reduce PPARγ and increase ET-1 and endothelial dysfunction. PPARγ activation attenuated SCD-associated signaling derangements, suggesting a novel therapeutic approach to attenuate SCD-PH pathogenesis.

Enhanced Clearance of Pseudomonas aeruginosa by Peroxisome Proliferator-Activated Receptor Gamma.

The pathogenic profile of Pseudomonas aeruginosa is related to its ability to secrete a variety of virulence factors. Quorum sensing (QS) is a mechanism wherein small diffusible molecules, specifically acyl-homoserine lactones, are produced by P. aeruginosa to promote virulence. We show here that macrophage clearance of P. aeruginosa (PAO1) is enhanced by activation of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ). Macrophages treated with a PPARγ agonist (pioglitazone) showed enhanced phagocytosis and bacterial killing of PAO1. It is known that PAO1 QS molecules are inactivated by PON-2. QS molecules are also known to inhibit activation of PPARγ by competitively binding PPARγ receptors. In accord with this observation, we found that infection of macrophages with PAO1 inhibited expression of PPARγ and PON-2. Mechanistically, we show that PPARγ induces macrophage paraoxonase 2 (PON-2), an enzyme that degrades QS molecules produced by P. aeruginosa Gene silencing studies confirmed that enhanced clearance of PAO1 in macrophages by PPARγ is PON-2 dependent. Further, we show that PPARγ agonists also enhance clearance of P. aeruginosa from lungs of mice infected with PAO1. Together, these data demonstrate that P. aeruginosa impairs the ability of host cells to mount an immune response by inhibiting PPARγ through secretion of QS molecules. These studies define a novel mechanism by which PPARγ contributes to the host immunoprotective effects during bacterial infection and suggest a role for PPARγ immunotherapy for P. aeruginosa infections.

Smooth Muscle-Targeted Overexpression of Peroxisome Proliferator Activated Receptor-γ Disrupts Vascular Wall Structure and Function.

Activation of the nuclear hormone receptor, PPARγ, with pharmacological agonists promotes a contractile vascular smooth muscle cell phenotype and reduces oxidative stress and cell proliferation, particularly under pathological conditions including vascular injury, restenosis, and atherosclerosis. However, pharmacological agonists activate both PPARγ-dependent and -independent mechanisms in multiple cell types confounding efforts to clarify the precise role of PPARγ in smooth muscle cell structure and function in vivo. We, therefore, designed and characterized a mouse model with smooth muscle cell-targeted PPARγ overexpression (smPPARγOE). Our results demonstrate that smPPARγOE attenuated contractile responses in aortic rings, increased aortic compliance, caused aortic dilatation, and reduced mean arterial pressure. Molecular characterization revealed that compared to littermate control mice, aortas from smPPARγOE mice expressed lower levels of contractile proteins and increased levels of adipocyte-specific transcripts. Morphological analysis demonstrated increased lipid deposition in the vascular media and in smooth muscle of extravascular tissues. In vitro adenoviral-mediated PPARγ overexpression in human aortic smooth muscle cells similarly increased adipocyte markers and lipid uptake. The findings demonstrate that smooth muscle PPARγ overexpression disrupts vascular wall structure and function, emphasizing that balanced PPARγ activity is essential for vascular smooth muscle homeostasis.

PPARγ Ligands Attenuate Hypoxia-Induced Proliferation in Human Pulmonary Artery Smooth Muscle Cells through Modulation of MicroRNA-21.

Pulmonary hypertension (PH) is a progressive and often fatal disorder whose pathogenesis involves pulmonary artery smooth muscle cell (PASMC) proliferation. Although modern PH therapies have significantly improved survival, continued progress rests on the discovery of novel therapies and molecular targets. MicroRNA (miR)-21 has emerged as an important non-coding RNA that contributes to PH pathogenesis by enhancing vascular cell proliferation, however little is known about available therapies that modulate its expression. We previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) agonists attenuated hypoxia-induced HPASMC proliferation, vascular remodeling and PH through pleiotropic actions on multiple targets, including transforming growth factor (TGF)-ß1 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is a validated target of miR-21. We therefore hypothesized that antiproliferative effects conferred by PPARγ activation are mediated through inhibition of hypoxia-induced miR-21 expression. Human PASMC monolayers were exposed to hypoxia then treated with the PPARγ agonist, rosiglitazone (RSG,10 µM), or in parallel, C57Bl/6J mice were exposed to hypoxia then treated with RSG. RSG attenuated hypoxic increases in miR-21 expression in vitro and in vivo and abrogated reductions in PTEN and PASMC proliferation. Antiproliferative effects of RSG were lost following siRNA-mediated PTEN depletion. Furthermore, miR-21 mimic decreased PTEN and stimulated PASMC proliferation, whereas miR-21 inhibition increased PTEN and attenuated hypoxia-induced HPASMC proliferation. Collectively, these results demonstrate that PPARγ ligands regulate proliferative responses to hypoxia by preventing hypoxic increases in miR-21 and reductions in PTEN. These findings further clarify molecular mechanisms that support targeting PPARγ to attenuate pathogenic derangements in PH.

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. METHODS: Primary lung fibroblasts isolated from wild type and α7 nicotinic acetylcholine receptor (α7 nAChR) deficient mice were treated with nicotine (50 µg/ml) in vitro for 72 hours. NGF levels were measured in culture media and in bronchoalveolar lavage (BAL) fluid from asthmatic, smoking and non-smoking subjects by ELISA. The role of the NFκB pathway in nicotine-induced NGF expression was investigated by measuring NFκB nuclear translocation, transcriptional activity, chromatin immunoprecipitation assays, and si-p65 NFκB knockdown. The ability of nicotine to stimulate a fibroblast-mediated, contractile ASM cell phenotype was confirmed by examining expression of contractile proteins in ASM cells cultured with fibroblast-conditioned media or BAL fluid. RESULTS: NGF levels were elevated in the bronchoalveolar lavage fluid of nicotine-exposed mice, current smokers, and asthmatic children. Nicotine increased NGF secretion in lung fibroblasts in vitro in a dose-dependent manner and stimulated NFκB nuclear translocation, p65 binding to the NGF promoter, and NFκB transcriptional activity. These responses were attenuated in α7 nAChR deficient fibroblasts and in wild type fibroblasts following NFκB inhibition. Nicotine-treated, fibroblast-conditioned media increased expression of contractile proteins in ASM cells. CONCLUSION: Nicotine stimulates NGF release by lung fibroblasts through α7 nAChR and NFκB dependent pathways. These novel findings suggest that the nicotine-α7 nAChR-NFκB- NGF axis may provide novel therapeutic targets to attenuate tobacco smoke-induced AHR.

Glutathione attenuates ethanol-induced alveolar macrophage oxidative stress and dysfunction by downregulating NADPH oxidases.

Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress by downregulating Noxes and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 wk) treated ± ethanol in drinking water (20% wt/vol, 12 wk) ± orally gavaged GSH in methylcellulose vehicle (300 mg x kg(-1) x day(-1), during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 days) ± GSH (500 µM, 3 days or last 1 day of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 colony-forming units, 28 h) ± orally gavaged GSH (300 mg/kg, 24 h). GSH levels (HPLC), Nox mRNA (quantitative RT-PCR) and protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia.

Polymerase delta interacting protein 2 sustains vascular structure and function.

OBJECTIVE: On the basis of previous evidence that polymerase delta interacting protein 2 (Poldip2) increases reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (Nox4) activity in vascular smooth muscle cells, we hypothesized that in vivo knockdown of Poldip2 would inhibit reactive oxygen species production and alter vascular function. APPROACH AND RESULTS: Because homozygous Poldip2 deletion is lethal, Poldip2(+/-) mice were used. Poldip2 mRNA and protein levels were reduced by ≈50% in Poldip2(+/-) aorta, with no change in p22phox, Nox1, Nox2, and Nox4 mRNAs. NADPH oxidase activity was also inhibited in Poldip2(+/-) tissue. Isolated aortas from Poldip2(+/-) mice demonstrated impaired phenylephrine and potassium chloride-induced contractions, increased stiffness, and reduced compliance associated with disruption of elastic lamellae and excessive extracellular matrix deposition. Collagen I secretion was elevated in cultured vascular smooth muscle cells from Poldip2(+/-) mice and restored by H2O2 supplementation, suggesting that this novel function of Poldip2 is mediated by reactive oxygen species. Furthermore, Poldip2(+/-) mice were protected against aortic dilatation in a model of experimental aneurysm, an effect consistent with increased collagen secretion. CONCLUSIONS: Poldip2 knockdown reduces H2O2 production in vivo, leading to increases in extracellular matrix, greater vascular stiffness, and impaired agonist-mediated contraction. Thus, unaltered expression of Poldip2 is necessary for vascular integrity and function.

Endothelial cell peroxisome proliferator-activated receptor γ reduces endotoxemic pulmonary inflammation and injury.

Bacterial endotoxin (LPS)-mediated sepsis involves severe, dysregulated inflammation that injures the lungs and other organs, often fatally. Vascular endothelial cells are both key mediators and targets of LPS-induced inflammatory responses. The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) exerts anti-inflammatory actions in various cells, but it is unknown whether it modulates inflammation through actions within endothelial cells. To determine whether PPARγ acts within endothelial cells to diminish endotoxemic lung inflammation and injury, we measured inflammatory responses and mediators in mice with endothelial-targeted deletion of PPARγ. Endothelial cell PPARγ (ePPARγ) knockout exacerbated LPS-induced pulmonary inflammation and injury as shown by several measures, including infiltration of inflammatory cells, edema, and production of reactive oxygen species and proinflammatory cytokines, along with upregulation of the LPS receptor TLR4 in lung tissue and increased activation of its downstream signaling pathways. In isolated LPS-stimulated endothelial cells in vitro, absence of PPARγ enhanced the production of numerous inflammatory markers. We hypothesized that the observed in vivo activity of the ligand-activated ePPARγ may arise, in part, from nitrated fatty acids (NFAs), a novel class of endogenous PPARγ ligands. Supporting this idea, we found that treating isolated endothelial cells with physiologically relevant concentrations of the endogenous NFA 10-nitro-oleate reduced LPS-induced expression of a wide range of inflammatory markers in the presence of PPARγ, but not in its absence, and also inhibited neutrophil mobility in a PPARγ-dependent manner. Our results demonstrate a key protective role of ePPARγ against endotoxemic injury and a potential ePPARγ-mediated anti-inflammatory role for NFAs.

Oxidative stress modulates PPAR gamma in vascular endothelial cells.

The peroxisome proliferator-activated receptor gamma (PPAR gamma) plays an important role in vascular regulation. However, the impact of oxidative stress on PPAR gamma expression and activity has not been clearly defined. Human umbilical vein endothelial cells (HUVECs) were exposed to graded concentrations of H(2)O(2) for 0.5-72h, or bovine aortic endothelial cells (BAECs) were exposed to alterations in extracellular thiol/disulfide redox potential (E(h)) of the cysteine/cystine couple. Within 2h, H(2)O(2) reduced HUVEC PPAR gamma mRNA and activity and reduced the expression of two PPAR gamma-regulated genes without altering PPAR gamma protein levels. After 4h H(2)O(2) exposure, mRNA levels remained reduced, whereas PPAR gamma activity returned to control levels. PPAR gamma mRNA levels remained depressed for up to 72 h after exposure to H(2)O(2), without any change in PPAR gamma activity. Catalase prevented H(2)O(2)-induced reductions in PPAR gamma mRNA and activity. H(2)O(2) (1) reduced luciferase expression in HUVECs transiently transfected with a human PPAR gamma promoter reporter, (2) failed to alter PPAR gamma mRNA half-life, and (3) transiently increased expression and activity of c-Fos and phospho-c-Jun. Treatment with the AP1 inhibitor curcumin prevented H(2)O(2)-mediated reductions in PPAR gamma expression. In addition, medium having an oxidized E(h) reduced BAEC PPAR gamma mRNA and activity. These findings demonstrate that oxidative stress, potentially through activation of inhibitory redox-regulated transcription factors, attenuates PPAR gamma expression and activity in vascular endothelial cells through suppression of PPAR gamma transcription.

Rosiglitazone attenuates chronic hypoxia-induced pulmonary hypertension in a mouse model.

Chronic hypoxia contributes to pulmonary hypertension through complex mechanisms that include enhanced NADPH oxidase expression and reactive oxygen species (ROS) generation in the lung. Stimulation of peroxisome proliferator-activated receptor gamma (PPARgamma) reduces the expression and activity of NADPH oxidase. Therefore, we hypothesized that activating PPARgamma with rosiglitazone would attenuate chronic hypoxia-induced pulmonary hypertension, in part, through suppressing NADPH oxidase-derived ROS that stimulate proliferative signaling pathways. Male C57Bl/6 mice were exposed to chronic hypoxia (CH, Fi(O2) 10%) or room air for 3 or 5 weeks. During the last 10 days of exposure, each animal was treated daily by gavage with either the PPARgamma ligand, rosiglitazone (10 mg/kg/d) or with an equal volume of vehicle. CH increased: (1) right ventricular systolic pressure (RVSP), (2) right ventricle weight, (3) thickness of the walls of small pulmonary vessels, (4) superoxide production and Nox4 expression in the lung, and (5) platelet-derived growth factor receptor beta (PDGFRbeta) expression and activity and reduced phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression. Treatment with rosiglitazone prevented the development of pulmonary hypertension at 3 weeks; reversed established pulmonary hypertension at 5 weeks; and attenuated CH-stimulated Nox4 expression and superoxide production, PDGFRbeta activation, and reductions in PTEN expression. Rosiglitazone also attenuated hypoxia-induced increases in Nox4 expression in pulmonary endothelial cells in vitro despite hypoxia-induced reductions in PPARgamma expression. Collectively, these findings indicate that PPARgamma ligands attenuated hypoxia-induced pulmonary vascular remodeling and hypertension by suppressing oxidative and proliferative signals providing novel insights for mechanisms underlying therapeutic effects of PPARgamma activation in pulmonary hypertension.

The role of NADPH oxidase in chronic intermittent hypoxia-induced pulmonary hypertension in mice.

Obstructive sleep apnea, characterized by intermittent periods of hypoxemia, is an independent risk factor for the development of pulmonary hypertension. However, the exact mechanisms of this disorder remain to be defined. Enhanced NADPH oxidase expression and superoxide (O2(-).) generation in the pulmonary vasculature play a critical role in hypoxia-induced pulmonary hypertension. Therefore, the current study explores the hypothesis that chronic intermittent hypoxia (CIH) causes pulmonary hypertension, in part, by increasing NADPH oxidase-derived reactive oxygen species (ROS) that contribute to pulmonary vascular remodeling and hypertension. To test this hypothesis, male C57Bl/6 mice and gp91phox knockout mice were exposed to CIH for 8 hours per day, 5 days per week for 8 weeks. CIH mice were placed in a chamber where the oxygen concentration was cycled between 21% and 10% O2 45 times per hour. Exposure to CIH for 8 weeks increased right ventricular systolic pressure (RVSP), right ventricle (RV):left ventricle (LV) + septum (S) weight ratio, an index of RV hypertrophy, and thickness of the right ventricular anterior wall as measured by echocardiography. CIH exposure also caused pulmonary vascular remodeling as demonstrated by increased muscularization of the distal pulmonary vasculature. CIH-induced pulmonary hypertension was associated with increased lung levels of the NADPH oxidase subunits, Nox4 and p22phox, as well as increased activity of platelet-derived growth factor receptor beta and its associated downstream effector, Akt kinase. These CIH-induced derangements were attenuated in similarly treated gp91phox knockout mice. These findings demonstrate that NADPH oxidase-derived ROS contribute to the development of pulmonary vascular remodeling and hypertension caused by CIH.

Thiazolidinediones as anti-cancer agents.

The PPAR-gamma (PPAR-γ) activating thiazolidinedione (TZD) medications are a class of drugs used to improve lipid and glucose metabolism in type-2 diabetes. In addition to their known insulin sensitization action, these drugs have been shown to suppress tumor development in several in vitro and in vivo models. Among the proposed mechanisms for the anti-tumor effects of TZDs, apoptosis induction, cell cycle arrest, and differentiation have been extensively reported. Interestingly, some of the observed anti-tumor effects are independent of PPAR-γ activation. The following review will discuss studies employing TZDs as anti-cancer therapies for the most common types of cancers including, lung, breast, and colon and will explore the principal PPAR-γ-dependent and -independent mechanisms by which TZDs exert their anti-tumor effects.

Effects of dietary decosahexaenoic acid (DHA) on eNOS in human coronary artery endothelial cells.

Endothelial dysfunction occurs in heart disease and may reduce functional capacity via attenuations in peripheral blood flow. Dietary decosahexaenoic acid (DHA) may improve this dysfunction, but the mechanism is unknown. This study determined if DHA enhances expression and activity of eNOS in cultured human coronary artery endothelial cells (HCAEC). HCAEC from 4 donors were treated with 5 nM, 50 nM, or 1 microM DHA for 7 days to model chronic DHA exposure. A trend for increased expression of endothelial nitric oxide synthase (eNOS) and phospho-eNOS was observed with 5 and 50 nM DHA. DHA also enhanced expression of 2 proteins instrumental in activation of eNOS: phospho-Akt (5 and 50 nM) and HSP90 (50 nM and 1 microM). Vascular endothelial growth factor-induced activation of Akt increased NOx in treated (50 nM DHA) versus untreated HCAEC (9.2 +/- 1.0 vs 3.3 +/- 1.1 micromol/microg protein/microL). Findings suggest that DHA enhances eNOS and Akt activity, augments HSP90 expression, and increases NO bioavailability in response to Akt kinase activation.

Vascular oxidative stress and nitric oxide depletion in HIV-1 transgenic rats are reversed by glutathione restoration.

Human immunodeficiency virus (HIV)-infected patients have a higher incidence of oxidative stress, endothelial dysfunction, and cardiovascular disease than uninfected individuals. Recent reports have demonstrated that viral proteins upregulate reactive oxygen species, which may contribute to elevated cardiovascular risk in HIV-1 patients. In this study we employed an HIV-1 transgenic rat model to investigate the physiological effects of viral protein expression on the vasculature. Markers of oxidative stress in wild-type and HIV-1 transgenic rats were measured using electron spin resonance, fluorescence microscopy, and various molecular techniques. Relaxation studies were completed on isolated aortic rings, and mRNA and protein were collected to measure changes in expression of nitric oxide (NO) and superoxide sources. HIV-1 transgenic rats displayed significantly less NO-hemoglobin, serum nitrite, serum S-nitrosothiols, aortic tissue NO, and impaired endothelium-dependent vasorelaxation than wild-type rats. NO reduction was not attributed to differences in endothelial NO synthase (eNOS) protein expression, eNOS-Ser1177 phosphorylation, or tetrahydrobiopterin availability. Aortas from HIV-1 transgenic rats had higher levels of superoxide and 3-nitrotyrosine but did not differ in expression of superoxide-generating sources NADPH oxidase or xanthine oxidase. However, transgenic aortas displayed decreased superoxide dismutase and glutathione. Administering the glutathione precursor procysteine decreased superoxide, restored aortic NO levels and NO-hemoglobin, and improved endothelium-dependent relaxation in HIV-1 transgenic rats. These results show that HIV-1 protein expression decreases NO and causes endothelial dysfunction. Diminished antioxidant capacity increases vascular superoxide levels, which reduce NO bioavailability and promote peroxynitrite generation. Restoring glutathione levels reverses HIV-1 protein-mediated effects on superoxide, NO, and vasorelaxation.