Beneficial effects of hepatic cyclooxygenase-2 expression against cholestatic injury after common bile duct ligation in mice.

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but little is known about the significance of COX-2 in cholestatic injury. This study was designed to elucidate the role of COX-2 expression in hepatocytes during the pathogenesis of obstructive cholestasis. METHODS: We used genetically modified mice constitutively expressing human COX-2 in hepatocytes. Transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either subjected to a mid-abdominal laparotomy or common bile duct ligation (BDL) for 2 or 5 days. Then, we explored the mechanisms underlying the role of COX-2 and its derived prostaglandins in liver function, and the synthesis and excretion of bile acids (BA) in response to cholestatic liver injury. RESULTS: After BDL, hCOX-2-Tg mice showed lower grades of hepatic necrosis and inflammation than Wt mice, in part by a reduced hepatic neutrophil recruitment associated with lower mRNA levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice displayed a differential metabolic pattern of BA synthesis that led to an improved clearance after BDL-induced accumulation. In addition, an enhanced response to the BDL-induced oxidative stress and hepatic apoptosis was observed. In vitro experiments using hepatic cells that stably express hCOX-2 confirmed the cytoprotective role of prostaglandin E2 against BA toxicity. CONCLUSIONS: Taken together, our data indicate that constitutive expression of COX-2 in hepatocytes ameliorates cholestatic liver injury in mice by reducing inflammation and cell damage and by modulating BA metabolism, pointing to a role for COX-2 as a defensive response against cholestasis-derived BA accumulation and injury.

Quantitative Proteomics Analysis Reveals That Cyclooxygenase-2 Modulates Mitochondrial Respiratory Chain Complex IV in Cardiomyocytes.

The biochemical mechanisms of cell injury and myocardial cell death after myocardial infarction remain unresolved. Cyclooxygenase 2 (COX-2), a key enzyme in prostanoid synthesis, is expressed in human ischemic myocardium and dilated cardiomyopathy, but it is absent in healthy hearts. To assess the role of COX-2 in cardiovascular physiopathology, we developed transgenic mice that constitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no apparent phenotype but were protected against ischemia-reperfusion injury in isolated hearts, with enhanced functional recovery and diminished cellular necrosis. To further explore the phenotype of this animal model, we carried out a differential proteome analysis of wild-type vs. transgenic cardiomyocytes. The results revealed a tissue-specific proteomic profile dominated by mitochondrial proteins. In particular, an increased expression of respiratory chain complex IV proteins was observed. This correlated with increased catalytic activity, enhanced respiratory capacity, and increased ATP levels in the heart of COX-2 transgenic mice. These data suggest a new link between COX-2 and mitochondria, which might contribute to the protective cardiac effects of COX-2 against ischemia-reperfusion injury.

Protective Role of Hepatocyte Cyclooxygenase-2 Expression Against Liver Ischemia-Reperfusion Injury in Mice.

Liver ischemia and reperfusion injury (IRI) remains a serious clinical problem affecting liver transplantation outcomes. IRI causes up to 10% of early organ failure and predisposes to chronic rejection. Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but the significance of COX-2 in IRI is a matter of controversy. This study was designed to elucidate the role of COX-2 induction in hepatocytes against liver IRI. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were subjected to IRI. hCOX-2-Tg mice exhibited lower grades of necrosis and inflammation than Wt mice, in part by reduced hepatic recruitment and infiltration of neutrophils, with a concomitant decrease in serum levels of proinflammatory cytokines. Moreover, hCOX-2-Tg mice showed a significant attenuation of the IRI-induced increase in oxidative stress and hepatic apoptosis, an increase in autophagic flux, and a decrease in endoplasmic reticulum stress compared to Wt mice. Interestingly, ischemic preconditioning of Wt mice resembles the beneficial effects observed in hCOX-2-Tg mice against IRI due to a preconditioning-derived increase in endogenous COX-2, which is mainly localized in hepatocytes. Furthermore, measurement of prostaglandin E2 (PGE2 ) levels in plasma from patients who underwent liver transplantation revealed a significantly positive correlation of PGE2 levels and graft function and an inverse correlation with the time of ischemia. Conclusion: These data support the view of a protective effect of hepatic COX-2 induction and the consequent rise of derived prostaglandins against IRI.

Advances in Understanding the Role of NRF2 in Liver Pathophysiology and Its Relationship with Hepatic-Specific Cyclooxygenase-2 Expression.

Oxidative stress and inflammation play an important role in the pathophysiological changes of liver diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes, thus playing a key role in protecting against oxidative damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis. Its expression has always been associated with the induction of inflammation, but we have shown that, in addition to possessing other benefits, the constitutive expression of COX-2 in hepatocytes is beneficial in reducing inflammation and oxidative stress in multiple liver diseases. In this review, we summarized the role of NRF2 as a main agent in the resolution of oxidative stress, the crucial role of NRF2 signaling pathways during the development of chronic liver diseases, and, finally we related its action to that of COX-2, where it appears to operate as its partner in providing a hepatoprotective effect.

COX-2 Expression in Hepatocytes Improves Mitochondrial Function after Hepatic Ischemia-Reperfusion Injury.

Cyclooxygenase 2 (COX-2) is a key enzyme in prostanoid biosynthesis. The constitutive hepatocyte expression of COX-2 has a protective role in hepatic ischemia-reperfusion (I/R) injury (IRI), decreasing necrosis, reducing reactive oxygen species (ROS) levels, and increasing autophagy and antioxidant and anti-inflammatory response. The physiopathology of IRI directly impacts mitochondrial activity, causing ATP depletion and being the main source of ROS. Using genetically modified mice expressing human COX-2 (h-COX-2 Tg) specifically in hepatocytes, and performing I/R surgery on the liver, we demonstrate that COX-2 expression has a beneficial effect at the mitochondrial level. Mitochondria derived from h-COX-2 Tg mice livers have an increased respiratory rate associated with complex I electron-feeding pathways compared to Wild-type (Wt) littermates, without affecting complex I expression or assembly. Furthermore, Wt-derived mitochondria show a loss of mitochondrial membrane potential (ΔΨm) that correlates to increased proteolysis of fusion-related OPA1 through OMA1 protease activity. All these effects are not observed in h-COX-2 Tg mitochondria, which behave similarly to the Sham condition. These results suggest that COX-2 attenuates IRI at a mitochondrial level, preserving the proteolytic processing of OPA1, in addition to the maintenance of mitochondrial respiration.

Chronic treatment with acetaminophen protects against liver aging by targeting inflammation and oxidative stress.

The liver exhibits a variety of functions that are well-preserved during aging. However, the cellular hallmarks of aging increase the risk of hepatic alterations and development of chronic liver diseases. Acetaminophen (APAP) is a first choice for relieving mild-to-moderate pain. Most of the knowledge about APAP-mediated hepatotoxicity arises from acute overdose studies due to massive oxidative stress and inflammation, but little is known about its effect in age-related liver inflammation after chronic exposure. Our results show that chronic treatment of wild-type mice on the B6D2JRcc/Hsd genetic background with APAP at an infratherapeutic dose reduces liver alterations during aging without affecting body weight. This intervention attenuates age-induced mild oxidative stress by increasing HO-1, MnSOD and NQO1 protein levels and reducing ERK1/2 and p38 MAPK phosphorylation. More importantly, APAP treatment counteracts the increase in Cd8+ and the reduction in Cd4+ T lymphocytes observed in the liver with age. This response was also found in peripheral blood mononuclear cells. In conclusion, chronic infratherapeutic APAP treatment protects mice from age-related liver alterations by attenuating oxidative stress and inflammation.

Succinate dehydrogenase inhibition with malonate during reperfusion reduces infarct size by preventing mitochondrial permeability transition.

AIMS: Previous studies demonstrated that pre-treatment with malonate, a reversible inhibitor of succinate dehydrogenase, given before ischaemia, reduces infarct size. However, it is unknown whether administration of malonate may reduce reperfusion injury. METHODS AND RESULTS: Isolated mice hearts were treated, under normoxic conditions, with increasing concentrations of disodium malonate (0.03-30 mmol/L, n = 4). Malonate induced a concentration-dependent decrease in left ventricular developed pressure (LVdevP) (EC50 = 8.05 ± 2.11 mmol/L). In isolated hearts submitted to global ischaemia (35 min) followed by reperfusion (60 min), malonate 3 mmol/L given only during the first 15 min of reperfusion reduced lactate dehydrogenase release (125.41 ± 16.82 vs. 189.20 ± 13.74 U/g dry tissue/15 min in controls, P = 0.015) and infarct size (24.57 ± 2.32 vs. 39.84 ± 2.78%, P = 0.001, n = 7-8 per group) and improved recovery of LVdevP (20.06 ± 3.82 vs 7.76 ± 2.53% of baseline LVdevP, P = 0.017). (1)H NMR spectroscopy demonstrated marked changes in the metabolic profile of malonate-treated hearts, including increased accumulation of succinate. Furthermore, malonate reduced reactive oxygen species (ROS) production, as measured by MitoSOX staining in myocardial samples obtained after 5 min of reperfusion and in mitochondrial preparations from these samples, preserved mitochondrial respiration, and reduced mitochondrial permeabilization, assessed by calcein retention. Treatment with malonate did not result in activation of RISK or SAFE signalling pathways in tissue extracts obtained 5 min after reperfusion. CONCLUSION: Succinate dehydrogenase inhibition with malonate at the onset of reperfusion reduces infarct size in isolated mice hearts through reduction in ROS production and mitochondrial permeability transition pore opening.