Emeka Ifedigbo

Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis.

Autophagy protein microtubule-associated protein 1 light chain-3B (LC3B) activates extrinsic apoptosis during cigarette smoke-induced emphysema

Chronic obstructive pulmonary disease (COPD) is a debilitating disease caused by chronic exposure to cigarette smoke (CS), which involves airway obstruction and alveolar loss (i.e., emphysema). The mechanisms of COPD pathogenesis remain unclear. Our previous studies demonstrated elevated autophagy in human COPD lung, and as a cellular and tissue response to CS exposure in an experimental model of emphysema in vivo. We identified the autophagic protein microtubule-associated protein 1 light chain-3B (LC3B) as a positive regulator of CS-induced lung epithelial cell death. We now extend these initial observations to explore the mechanism by which LC3B mediates CS-induced apoptosis and emphysema development in vivo. Here, we observed that LC3B−/− mice had significantly decreased levels of apoptosis in the lungs after CS exposure, and displayed resistance to CS-induced airspace enlargement, relative to WT littermate mice. We found that LC3B associated with the extrinsic apoptotic factor Fas in lipid rafts in an interaction mediated by caveolin-1 (Cav-1). The siRNA-dependent knockdown of Cav-1 sensitized epithelial cells to CS-induced apoptosis, as evidenced by enhanced death-inducing signaling complex formation and caspase activation. Furthermore, Cav-1−/− mice exhibited higher levels of autophagy and apoptosis in the lung in response to chronic CS exposure in vivo. In conclusion, we demonstrate a pivotal role for the autophagic protein LC3B in CS-induced apoptosis and emphysema, suggestive of novel therapeutic targets for COPD treatment. This study also introduces a mechanism by which LC3B, through interactions with Cav-1 and Fas, can regulate apoptosis.

Heme oxygenase-1-derived carbon monoxide protects hearts from transplant associated ischemia reperfusion injury

Heme oxygenase‐1 (HO‐1) degrades heme into iron, biliverdin, and carbon monoxide (CO). HO‐ 1 expression can be used therapeutically to ameliorate undesirable consequences of ischemia reperfusion injury (IRI), but the mechanism by which this occurs, remains to be established. Rat hearts, exposed to a prolonged period (24 h) of cold (4°C) ischemia, failed to function upon transplantation into syngeneic recipients. Induction of HO‐1 expression by administration of cobalt protoporphyrin IX (CoPPIX) to the graft donor restored graft function. Inhibition of HO‐1 enzymatic activity, by administration of zinc protoporphyrin (ZnPPIX) at the time of transplantation, reversed the protective effect of HO‐1. Exposure of the graft donor as well as the graft (during ischemia) to exogenous CO mimicked the protective effect of HO‐1. This was associated with a significant reduction in the number of cells undergoing apoptosis in the graft with no apparent decrease of intravascular fibrin polymerization, platelet aggregation, or P‐ selectin expression. In conclusion, HO‐1‐derived CO prevents IRI associated with cardiac transplantation based on its antiapoptotic action. The observation that exposure of the donor and the graft to CO is sufficient to afford this protective effect should have important clinical implications in terms of preventing IRI associated with heart transplantation in humans.

MKK3 mitogen-activated protein kinase pathway mediates carbon monoxide-induced protection against oxidant-induced lung injury.

The stress-inducible gene heme oxygenase (HO-1) has previously been shown to provide cytoprotection against oxidative stress. The mechanism(s) by which HO-1 provides this cytoprotection is poorly understood. We demonstrate here that carbon monoxide (CO), a byproduct released during the degradation of heme by HO, plays a major role in mediating the cytoprotection against oxidant-induced lung injury. We show in vitro that CO protects cultured epithelial cells from hyperoxic damage. By using dominant negative mutants and mice deficient in the genes for the various MAP kinases, we demonstrate that the cytoprotective effects of CO are mediated by selective activation of the MKK3/p38 beta protein MAP kinase pathway. In vivo, our experiments demonstrate that CO at a low concentration protects the lungs, extends the survival of the animals, and exerts potent anti-inflammatory effects with reduced inflammatory cell influx into the lungs and marked attenuation in the expression of pro-inflammatory cytokines.

Carbon monoxide protection against endotoxic shock involves reciprocal effects on iNOS in the lung and liver

Carbon monoxide (CO) has recently emerged as having potent cytoprotective properties; the mechanisms underlying these effects, however, are just beginning to be elucidated. In a rat model of lipopolysaccharide (LPS)‐induced multiorgan failure, we demonstrate that exposure to a low concentration of CO for only 1 h imparts a potent defense against lethal endotoxemia and effectively abrogates the inflammatory response. Exposure to CO leads to long‐term survival of >80% of animals vs. 20% in controls. In the lung, CO suppressed LPS‐induced lung alveolitis and associated edema formation, while in the liver, it reduced expression of serum alanine aminotransferase, a marker of liver injury. This protection appears to be based in part on different mechanisms in the lung and liver in that CO had reciprocal effects on LPS‐induced expression of iNOS and NO production, important mediators in the response to LPS. CO prevented the up‐regulation of iNOS and NO in the lung while augmenting expression of iNOS and NO in the liver. Studies of primary lung macrophages and hepatocytes in vitro revealed a similar effect; CO inhibited LPS‐induced cytokine production in lung macrophages while reducing LPS‐induced iNOS expression and nitrite accumulation and protected hepatocytes from apoptosis while augmenting iNOS expression. Although it is unclear to which extent these changes in iNOS contribute to the cytoprotection conferred by CO, it is fascinating that in each organ CO influences iNOS in a manner known to be protective in that organ: NO is therapeutic in the liver while it is damaging in the lung.

Nitrite Potently Inhibits Hypoxic and Inflammatory Pulmonary Arterial Hypertension and Smooth Muscle Proliferation via Xanthine Oxidoreductase–Dependent Nitric Oxide Generation

Background— Pulmonary arterial hypertension is a progressive proliferative vasculopathy of the small pulmonary arteries that is characterized by a primary failure of the endothelial nitric oxide and prostacyclin vasodilator pathways, coupled with dysregulated cellular proliferation. We have recently discovered that the endogenous anion salt nitrite is converted to nitric oxide in the setting of physiological and pathological hypoxia. Considering the fact that nitric oxide exhibits vasoprotective properties, we examined the effects of nitrite on experimental pulmonary arterial hypertension. Methods and Results— We exposed mice and rats with hypoxia or monocrotaline-induced pulmonary arterial hypertension to low doses of nebulized nitrite (1.5 mg/min) 1 or 3 times a week. This dose minimally increased plasma and lung nitrite levels yet completely prevented or reversed pulmonary arterial hypertension and pathological right ventricular hypertrophy and failure. In vitro and in vivo studies revealed that nitrite in the lung was metabolized directly to nitric oxide in a process significantly enhanced under hypoxia and found to be dependent on the enzymatic action of xanthine oxidoreductase. Additionally, physiological levels of nitrite inhibited hypoxia-induced proliferation of cultured pulmonary artery smooth muscle cells via the nitric oxide–dependent induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1. The therapeutic effect of nitrite on hypoxia-induced pulmonary hypertension was significantly reduced in the p21-knockout mouse; however, nitrite still reduced pressures and right ventricular pathological remodeling, indicating the existence of p21-independent effects as well. Conclusion— These studies reveal a potent effect of inhaled nitrite that limits pathological pulmonary arterial hypertrophy and cellular proliferation in the setting of experimental pulmonary arterial hypertension.

Carbon monoxide reverses established pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by a progressive increase in pulmonary vascular resistance leading to right heart failure. Carbon monoxide (CO) has emerged as a potently protective, homeostatic molecule that prevents the development of vascular disorders when administered prophylactically. The data presented in this paper demonstrate that CO can also act as a therapeutic (i.e., where exposure to CO is initiated after pathology is established). In three rodent models of PAH, a 1 hour/day exposure to CO reverses established PAH and right ventricular hypertrophy, restoring right ventricular and pulmonary arterial pressures, as well as the pulmonary vascular architecture, to near normal. The ability of CO to reverse PAH requires functional endothelial nitric oxide synthase (eNOS/NOS3) and NO generation, as indicated by the inability of CO to reverse chronic hypoxia-induced PAH in eNOS-deficient (nos3−/−) mice versus wild-type mice. The restorative function of CO was associated with a simultaneous increase in apoptosis and decrease in cellular proliferation of vascular smooth muscle cells, which was regulated in part by the endothelial cells in the hypertrophied vessels. In conclusion, these data demonstrate that CO reverses established PAH dependent on NO generation supporting the use of CO clinically to treat pulmonary hypertension.

Autophagic Protein LC3B Confers Resistance against Hypoxia-induced Pulmonary Hypertension

RATIONALEnPulmonary hypertension (PH) is a progressive disease with unclear etiology. The significance of autophagy in PH remains unknown.nnnOBJECTIVESnTo determine the mechanisms by which autophagic proteins regulate tissue responses during PH.nnnMETHODSnLungs from patients with PH, lungs from mice exposed to chronic hypoxia, and human pulmonary vascular cells were examined for autophagy using electron microscopy and Western analysis. Mice deficient in microtubule-associated protein-1 light chain-3B (LC3B(-/-)), or early growth response-1 (Egr-1(-/-)), were evaluated for vascular morphology and hemodynamics.nnnMEASUREMENTS AND MAIN RESULTSnHuman PH lungs displayed elevated lipid-conjugated LC3B, and autophagosomes relative to normal lungs. These autophagic markers increased in hypoxic mice, and in human pulmonary vascular cells exposed to hypoxia. Egr-1, which regulates LC3B expression, was elevated in PH, and increased by hypoxia in vivo and in vitro. LC3B(-/-) or Egr-1(-/-), but not Beclin 1(+/-), mice displayed exaggerated PH during hypoxia. In vitro, LC3B knockdown increased reactive oxygen species production, hypoxia-inducible factor-1α stabilization, and hypoxic cell proliferation. LC3B and Egr-1 localized to caveolae, associated with caveolin-1, and trafficked to the cytosol during hypoxia.nnnCONCLUSIONSnThe results demonstrate elevated LC3B in the lungs of humans with PH, and of mice with hypoxic PH. The increased susceptibility of LC3B(-/-) and Egr-1(-/-) mice to hypoxia-induced PH and increased hypoxic proliferation of LC3B knockdown cells suggest adaptive functions of these proteins during hypoxic vascular remodeling. The results suggest that autophagic protein LC3B exerts a protective function during the pathogenesis of PH, through the regulation of hypoxic cell proliferation.

Carbon Monoxide Protects against Ventilator-induced Lung Injury via PPAR-γ and Inhibition of Egr-1

RATIONALEnVentilator-induced lung injury (VILI) leads to an unacceptably high mortality. In this regard, the antiinflammatory properties of inhaled carbon monoxide (CO) may provide a therapeutic option.nnnOBJECTIVESnThis study explores the mechanisms of CO-dependent protection in a mouse model of VILI.nnnMETHODSnMice were ventilated (12 ml/kg, 1-8 h) with air in the absence or presence of CO (250 ppm). Airway pressures, blood pressure, and blood gases were monitored. Lung tissue was analyzed for inflammation, injury, and gene expression. Bronchoalveolar lavage fluid was analyzed for protein, cell and neutrophil counts, and cytokines.nnnMEASUREMENTS AND MAIN RESULTSnMechanical ventilation caused significant lung injury reflected by increases in protein concentration, total cell and neutrophil counts in the bronchoalveolar lavage fluid, as well as the induction of heme oxygenase-1 and heat shock protein-70 in lung tissue. In contrast, CO application prevented lung injury during ventilation, inhibited stress-gene up-regulation, and decreased lung neutrophil infiltration. These effects were preceded by the inhibition of ventilation-induced cytokine and chemokine production. Furthermore, CO prevented the early ventilation-dependent up-regulation of early growth response-1 (Egr-1). Egr-1-deficient mice did not sustain lung injury after ventilation, relative to wild-type mice, suggesting that Egr-1 acts as a key proinflammatory regulator in VILI. Moreover, inhibition of peroxysome proliferator-activated receptor (PPAR)-gamma, an antiinflammatory nuclear regulator, by GW9662 abolished the protective effects of CO.nnnCONCLUSIONSnMechanical ventilation causes profound lung injury and inflammatory responses. CO treatment conferred protection in this model dependent on PPAR-gamma and inhibition of Egr-1.

Brief inhalation of low-dose carbon monoxide protects rodents and swine from postoperative ileus*

Objective:Carbon monoxide (CO), an endogenous byproduct of heme metabolism, is produced at high levels in injured tissue via induction of heme-oxygenase–1 activity, where it contributes to the modulation of proinflammatory processes. Alone, CO has potent anti-inflammatory effects in models of acute and chronic inflammation. In rodents, inhalation of low concentrations of CO (250 ppm) for 24 hrs protects against postoperative gastrointestinal ileus. The current study determined whether shorter exposures and lower concentrations were equally protective and whether CO treatment would be effective in a large animal species (swine) managed under conditions approximating the clinical setting. Design:Dosing studies were first performed in rats by exposing them to CO (30–250 ppm) or air by inhalation for 1 or 3 hrs before anesthesia. An effective dosing regimen was then selected for testing in swine. Postoperative ileus in both species was induced by laparotomy and mild compression (running) of the small intestine. Measurements and Main Results:In rats, inhalation of 75 ppm CO for 3 hrs before anesthesia and surgery ameliorated the surgically induced delay in gastrointestinal transit to levels achieved using 250 ppm for 24 hrs. Swine treated with 250 ppm CO for the same time period exhibited significantly improved postoperative intestinal circular muscle contractility in vitro and gastrointestinal transit in vivo. Carboxyhemoglobin concentrations measured after termination of CO exposure averaged 5.8% (baseline, 1.5%). No deleterious effects on heart rate, oxygen saturation, blood chemistries, and serum electrolytes were observed. Conclusions:These findings demonstrate that inhalation of a low concentration of CO before surgery attenuates postoperative ileus in rodents and, more importantly, in a large animal species without risk to well-being during surgery or perioperatively. Exposures need not be prolonged, with significant benefit occurring with a 3-hr pretreatment.