Jayashree Rao

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;)

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.

Lipopolysaccaride induces autophagic signaling in macrophages via a TLR4, heme oxygenase-1 dependent pathway

Toll-like receptor (TLR) signaling is an important part of the innate immune response. One of the downstream responses to TLR4 signaling upon lipopolysaccharide (LPS) stimulation is the induction of autophagy, which is a key response to multiple stressors. An additional adaptive signaling molecule that is involved in the response to stress is heme oxygenase-1 (HO-1). HO-1 signaling is essential to limit inflammation and restore homeostasis. We found that LPS induced autophagic signaling in macrophages via a TLR4, HO-1 dependent pathway in macrophages. These data add to the developing contribution of autophagic signaling as part of the inflammatory response.

Carbon Monoxide Activates NF-κB Via ROS Generation and Akt Pathways To Protect Against Cell Death Of Hepatocytes

Heme oxygenase overexpression or exogenous carbon monoxide (CO) protects against hepatocyte apoptosis and fulminant hepatitis. The prevention of hepatocyte apoptosis by CO has been shown to require activation of NF-kappaB. The purpose of these investigations was to determine the mechanism of CO-induced hepatocyte NF-kappaB activation and protection against apoptosis. Primary rat or mouse hepatocytes and Hep3B cells were utilized. CO exposure was performed at 250 parts per million. Main outcome measures included cell viability, reactive oxygen species (ROS) generation, and changes in the levels of the intracellular antioxidants glutathione and ascorbate. Western blotting was performed for phospho-Akt, total Akt, and IkappaBalpha. NF-kappaB activation was determined by electrophoretic mobility shift assay and luciferase reporter assays. We found that CO treatment of hepatocytes prevents spontaneous apoptosis and leads to an increase in ROS production in association with Akt phosphorylation and IkappaB degradation. CO did not increase ROS production in respiration-deficient (rho0) Hep3B cells. Both Akt phosphorylation and IkappaB degradation can be inhibited by the addition of antioxidants. Furthermore, CO-induced NF-kappaB activation is reversed by phosphatidylinositol 3-kinase (PI3-K) inhibitor (LY294002) or antioxidants. Additionally, prevention of spontaneous hepatocyte apoptosis by CO is reversed by PI3-K inhibition and antioxidants. In conclusion, these data implicate a survival pathway of CO-induced ROS, Akt phosphorylation, and NF-kappaB activation in cultured hepatocytes. This pathway may prove to be important in maintenance of hepatic function in both physiological and pathophysiological conditions.

Heme oxygenase 1 protects against hepatic hypoxia and injury from hemorrhage via regulation of cellular respiration

Heme oxygenase 1 (HO-1) is an important regulator of the cellular response to stress and inflammation. These investigations test the hypothesis that HO-1 overexpression protects against hemorrhage-induced hypoxia by regulating cellular respiration and oxygen availability. Male C57BL/6 mice or primary mouse hepatocytes were treated with adenoviral gene transfer of HO-1 (AdHO-1) or &bgr;-galactosidase (AdLacZ). Mice were subjected to hemorrhagic shock and resuscitation or cannulation without hemorrhage. AdHO-1 prevented hemorrhagic shock/resuscitation-induced liver injury. In addition, AdHO-1 prevented hemorrhage-induced liver hypoxia and depletion of adenosine triphosphate. In vitro, HO-1 overexpression resulted in decreased cellular respiration under hypoxic conditions as determined by oxygen consumption and cytochrome c oxidase activity. This resulted in increased intracellular oxygen levels in the setting of low oxygen tensions. In conclusion, HO-1 overexpression protects the liver against hemorrhage-induced injury. This may be secondary to the ability of HO-1 to protect against bioenergetic failure via regulation of cellular respiration.