Brian S. Zuckerbraun

Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.

Nitrate and nitrite in biology, nutrition and therapeutics

Inorganic nitrate and nitrite from endogenous or dietary sources are metabolized in vivo to nitric oxide (NO) and other bioactive nitrogen oxides. The nitrate-nitrite-NO pathway is emerging as an important mediator of blood flow regulation, cell signaling, energetics and tissue responses to hypoxia. The latest advances in our understanding of the biochemistry, physiology and therapeutics of nitrate, nitrite and NO were discussed during a recent 2-day meeting at the Nobel Forum, Karolinska Institutet in Stockholm.

Carbon Monoxide Induces Cytoprotection in Rat Orthotopic Lung Transplantation via Anti-Inflammatory and Anti-Apoptotic Effects

Successful lung transplantation has been limited by the high incidence of acute graft rejection. There is mounting evidence that the stress response gene heme oxygenase-1 (HO-1) and/or its catalytic by-product carbon monoxide (CO) confers cytoprotection against tissue and cellular injury. This led us to hypothesize that CO may protect against lung transplant rejection via its anti-inflammatory and antiapoptotic effects. Orthotopic left lung transplantation was performed in Lewis rat recipients from Brown-Norway rat donors. HO-1 mRNA and protein expression were markedly induced in transplanted rat lungs compared to sham-operated control lungs. Transplanted lungs developed severe intraalveolar hemorrhage, marked infiltration of inflammatory cells, and intravascular coagulation. However, in the presence of CO exposure (500 ppm), the gross anatomy and histology of transplanted lungs showed marked preservation. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-exposed recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA and protein expression in lung and serum, respectively. Gene array analysis revealed that CO also down-regulated other proinflammatory genes, including MIP-1alpha and MIF, and growth factors such as platelet-derived growth factor, which were up-regulated by transplantation. These data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation.

Carbon Monoxide Protects against Liver Failure through Nitric Oxide–induced Heme Oxygenase 1

Carbon monoxide (CO) and nitric oxide (NO) each have mechanistically unique roles in various inflammatory disorders. Although it is known that CO can induce production of NO and that NO can induce expression of the cytoprotective enzyme heme oxygenase 1 (HO-1), there is no information whether the protective effect of CO ever requires NO production or whether either gas must induce expression of HO-1 to exert its functional effects. Using in vitro and in vivo models of tumor necrosis factor α–induced hepatocyte cell death in mice, we find that activation of nuclear factor κB and increased expression of inducible NO are required for the protective effects of CO, whereas the protective effects of NO require up-regulation of HO-1 expression. When protection from cell death is initiated by CO, NO production and HO-1 activity are each required for the protective effect showing for the first time an essential synergy between these two molecules in tandem providing potent cytoprotection.

Carbon Monoxide Inhalation Protects Rat Intestinal Grafts from Ischemia/Reperfusion Injury

Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to exert anti-inflammatory effects. This study examines the cytoprotective efficacy of inhaled CO during intestinal cold ischemia/reperfusion injury associated with small intestinal transplantation. Orthotopic syngenic intestinal transplantation was performed in Lewis rats after 6 hours of cold preservation in University of Wisconsin solution. Three groups were examined: normal untreated controls, control intestinal transplant recipients kept in room air, and recipients exposed to CO (250 ppm) for 1 hour before and 24 hours after surgery. In air grafts, mRNA levels for interleukin-6, cyclooxygenase-2, intracellular adhesion molecule (ICAM-1), and inducible nitric oxide synthase rapidly increased after intestinal transplant. Histopathological analysis revealed severe mucosal erosion, villous congestion, and inflammatory infiltrates. CO effectively blocked an early up-regulation of these mediators, showed less severe histopathological changes, and resulted in significantly improved animal survival of 92% from 58% in air-treated controls. CO also significantly reduced mRNA for proapoptotic Bax, while it up-regulated anti-apoptotic Bcl-2. These changes in CO-treated grafts correlated with well-preserved CD31(+) vascular endothelial cells, less frequent apoptosis/necrosis in intestinal epithelial and capillary endothelial cells, and improved graft tissue blood circulation. Protective effects of CO in this study were mediated via soluble guanylyl cyclase, because 1H-(1,2,4)oxadiazole (4,3-alpha) quinoxaline-1-one (soluble guanylyl cyclase inhibitor) completely reversed the beneficial effect conferred by CO. Perioperative CO inhalation at a low concentration resulted in protection against ischemia/reperfusion injury to intestinal grafts with prolonged cold preservation.

Diverting loop ileostomy and colonic lavage: an alternative to total abdominal colectomy for the treatment of severe, complicated Clostridium difficile associated disease.

Objective:To determine whether a minimally invasive, colon-preserving approach could serve as an alternative to total colectomy in the treatment of severe, complicated Clostridium difficile–associated disease (CDAD). Background:C. difficile is a significant cause of morbidity and mortality worldwide. Most cases will respond to antibiotic therapy, but 3% to 10% of patients progress to a severe, complicated, or “fulminant” state of life-threatening systemic toxicity. Although the advocated surgical treatment of total abdominal colectomy with end ileostomy improves survival in severe, complicated CDAD, outcomes remain poor with associated mortality rates ranging from 35% to 80%. Methods:All patients who were diagnosed with severe, complicated (“fulminant”) CDAD and were treated at the University of Pittsburgh Medical Center or VA Pittsburgh Healthcare System between June 2009 and January 2011 were treated with this novel approach. The surgical approach involved creation of a loop ileostomy, intraoperative colonic lavage with warmed polyethylene glycol 3350/electrolyte solution via the ileostomy and postoperative antegrade instillation of vancomycin flushes via the ileostomy. The primary end point for the study was resolution of CDAD. The matching number of patients treated with colectomy for CDAD preceding the initiation of this current treatment strategy was analyzed for historical comparison. Results:Forty-two patients were treated during this time period. There was no significant difference in age, sex, pharmacologic immunosuppression, and Acute Physiology and Chronic Health Evaluation-II scores between our current cohort and historical controls. The operation was accomplished laparoscopically in 35 patients (83%). This treatment strategy resulted in reduced mortality compared to our historical population (19% vs 50%; odds ratio, 0.24; P = 0.006). Preservation of the colon was achieved in 39 of 42 patients (93%). Conclusions:Loop ileostomy and colonic lavage are an alternative to colectomy in the treatment of severe, complicated CDAD resulting in reduced morbidity and preservation of the colon.

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.

Heme oxygenase‐1–mediated autophagy protects against hepatocyte cell death and hepatic injury from infection/sepsis in mice

Adaptive responses to sepsis are necessary to prevent organ failure and death. Cellular signaling responses that limit cell death and structural damage allow a cell to withstand insult from sepsis to prevent irreversible organ dysfunction. One such protective pathway to reduce hepatocellular injury is the up‐regulation of heme oxygenase‐1 (HO‐1) signaling. HO‐1 is up‐regulated in the liver in response to multiple stressors, including sepsis and lipopolysaccharide (LPS), and has been shown to limit cell death. Another recently recognized rudimentary cellular response to injury is autophagy. The aim of these investigations was to test the hypothesis that HO‐1 protects against hepatocyte cell death in experimental sepsis in vivo or LPS in vitro via induction of autophagy. These data demonstrate that both HO‐1 and autophagy are up‐regulated in the liver after cecal ligation and puncture (CLP) in C57BL/6 mice or in primary mouse hepatocytes after treatment with LPS (100 ng/mL). CLP or LPS results in minimal hepatocyte cell death. Pharmacological inhibition of HO‐1 activity using tin protoporphyrin or knockdown of HO‐1 prevents the induction of autophagic signaling in these models and results in increased hepatocellular injury, apoptosis, and death. Furthermore, inhibition of autophagy using 3‐methyladenine or small interfering RNA specific to VPS34, a class III phosphoinositide 3‐kinase that is an upstream regulator of autophagy, resulted in hepatocyte apoptosis in vivo or in vitro. LPS induced phosphorylation of p38 mitogen‐activated protein kinase (p38 MAPK), in part, via HO‐dependent signaling. Moreover, inhibition of p38 MAPK prevented CLP‐ or LPS‐induced autophagy. Conclusion: Sepsis or LPS‐induced autophagy protects against hepatocellular death, in part via an HO‐1 p38 MAPK‐dependent signaling. Further investigations are needed to elucidate how autophagic signaling prevents apoptosis and cell death. (HEPATOLOGY 2011;)

Role of Nitric Oxide in Liver Injury

The complex role of nitric oxide (NO) in the liver can be explained by its patterns of regulation and unique biochemical properties. With a broad range of direct and indirect molecular targets, NO acts as an inhibitor or agonist of cell signaling events. In the liver, constitutively generated NO maintains the hepatic microcirculation and endothelial integrity, while inducible NO synthase (iNOS)-governed NO production can be either beneficial or detrimental. For instance, NO potentiates the hepatic oxidative injury in warm ischemia/reperfusion, while iNOS expression protects against hepatic apoptotic cell death seen in models of sepsis and hepatitis. Anti-apoptotic actions are either cyclic nucleotide dependent or independent, including the expression of heat shock proteins, prevention of mitochondrial dysfunction, and inhibition of caspase activity by S-nitrosation. Whether NO protects or injures is probably determined by the type of insult, the abundance of reactive oxygen species (ROS), the source and amount of NO production and the cellular redox status of liver. Through the use of pharmacological NO donors or NOS gene transfer in conjunction with genetically altered knockout animals, the physiological and pathophysiological roles of NO in liver function can be explored in more detail. The purpose of this paper is to review the current understanding of the role of NO in liver injury.