Narci C. Teoh

Hepatic ischemia reperfusion injury: Pathogenic mechanisms and basis for hepatoprotection

Abstract  This review highlights recent advances in our understanding of mechanisms underlying reperfusion injury to the liver after warm hepatic ischemia. Sinusoidal endothelial cells and hepatocytes are targets of injury in the early ‘cytotoxic’ phase, although participation of apoptosis in the cell‐death process remains contentious. Kupffer cells may play an important role as the initial cytotoxic cell type and are likely a source of reactive oxygen species and proinflammatory mediators, particularly tumor necrosis factor (TNF)‐α. The latter are involved with subsequent neutrophil activation and recruitment. Microcirculatory disruption results from an imbalance between the actions of vasoconstrictors and vasodilators, such as nitric oxide, and also has a major impact on reperfusion injury. There is growing evidence that a brief prior ischemia–reperfusion period, termed ‘ischemic preconditioning’, is hepatoprotective. This can be mimicked by drugs that produce oxidative stress, and by interleukin‐6 and TNF‐α; both these cytokines are involved with priming hepatocytes to enter the cell cycle. Several mechanisms have been implicated including mobilization of adenosine and activation of adenosine type 2 receptors, nitric oxide, abrogation of TNF synthesis, preservation of energy metabolism, protection of the microcirculation and accelerated cell‐cycle entry. A better understanding of preconditioning mechanisms will lead to novel approaches to improve outcomes of liver surgery.

Hepatic Free Cholesterol Accumulates in Obese, Diabetic Mice and Causes Nonalcoholic Steatohepatitis

BACKGROUND & AIMS Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH. METHODS Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice. RESULTS Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis. CONCLUSIONS In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.

Dual role of tumor necrosis factor-α in hepatic ischemia-reperfusion injury: Studies in tumor necrosis factor-α gene knockout mice

Although hepatic ischemia‐reperfusion (IR) injury is partially mediated by tumor necrosis factor‐α (TNF), we recently found that low‐dose TNF before IR is hepatoprotective. We examined the seemingly conflicting roles of TNF in mediating liver injury in a partial hepatic IR model using TNF gene knockout (TNF ko) mice to allow TNF replacement at specified times. Compared with wild‐type mice, TNF ko mice exhibit minimal alanine aminotransferase release and few hepatonecrotic lesions during the early (time, 2 hours) and late (time, 24 hours) phases of IR. TNF ko mice differed from wild‐type mice in that TNF ko mice exhibited no activation or induction of nuclear factor‐κ B, p38, cyclin D1, or proliferating cell nuclear antigen after IR. A single low‐dose TNF injection 1 minute before the onset of hepatic ischemia restored hepatic IR injury in TNF ko mice. To clarify the importance of TNF for hepatoprotection, preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) was performed before the onset of IR for TNF ko mice whose capacity to undergo IR injury had been restored by TNF replacement. Ischemic preconditioning failed to protect these mice from TNF‐augmented IR injury; however, following the administration of intravenous TNF (1 μg per kg body weight, which mimics the early increase in hepatic and plasma TNF levels that is mobilized by ischemic preconditioning), significant hepatoprotection against both the early and late phases of TNF‐augmented IR injury was observed. In conclusion, TNF appears to mediate both the early and late phases of liver injury in hepatic IR, but it also is an essential mediator of hepatoprotective effects brought about by ischemic preconditioning. (HEPATOLOGY 2004;39:412–421.)

NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice

BACKGROUND & AIMS NOD-like receptor protein 3 (NLRP3) inflammasome activation occurs in Non-alcoholic fatty liver disease (NAFLD). We used the first small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters inflammatory recruitment and liver fibrosis in two murine models of steatohepatitis. METHODS We fed foz/foz and wild-type mice an atherogenic diet for 16weeks, gavaged MCC950 or vehicle until 24weeks, then determined NAFLD phenotype. In mice fed an methionine/choline deficient (MCD) diet, we gavaged MCC950 or vehicle for 6weeks and determined the effects on liver fibrosis. RESULTS In vehicle-treated foz/foz mice, hepatic expression of NLRP3, pro-IL-1β, active caspase-1 and IL-1β increased at 24weeks, in association with cholesterol crystal formation and NASH pathology; plasma IL-1β, IL-6, MCP-1, ALT/AST all increased. MCC950 treatment normalized hepatic caspase 1 and IL-1β expression, plasma IL-1β, MCP-1 and IL-6, lowered ALT/AST, and reduced the severity of liver inflammation including designation as NASH pathology, and liver fibrosis. In vitro, cholesterol crystals activated Kupffer cells and macrophages to release IL-1β; MCC950 abolished this, and the associated neutrophil migration. MCD diet-fed mice developed fibrotic steatohepatitis; MCC950 suppressed the increase in hepatic caspase 1 and IL-1β, lowered numbers of macrophages and neutrophils in the liver, and improved liver fibrosis. CONCLUSION MCC950, an NLRP3 selective inhibitor, improved NAFLD pathology and fibrosis in obese diabetic mice. This is potentially attributable to the blockade of cholesterol crystal-mediated NLRP3 activation in myeloid cells. MCC950 reduced liver fibrosis in MCD-fed mice. Targeting NLRP3 is a logical direction in pharmacotherapy of NASH. LAY SUMMARY Fatty liver disease caused by being overweight with diabetes and a high risk of heart attack, termed non-alcoholic steatohepatitis (NASH), is the most common serious liver disease with no current treatment. There could be several causes of inflammation in NASH, but activation of a protein scaffold within cells termed the inflammasome (NLRP3) has been suggested to play a role. Here we show that cholesterol crystals could be one pathway to activate the inflammasome in NASH. We used a drug called MCC950, which has already been shown to block NLRP3 activation, in an attempt to reduce liver injury in NASH. This drug partly reversed liver inflammation, particularly in obese diabetic mice that most closely resembles the human context of NASH. In addition, such dampening of liver inflammation in NASH achieved with MCC950 partly reversed liver scarring, the process that links NASH to the development of cirrhosis.

Roles of adipose restriction and metabolic factors in progression of steatosis to steatohepatitis in obese, diabetic mice

Background and Aims:  We previously reported that steatohepatitis develops in obese, hypercholesterolemic, diabetic foz/foz mice fed a high‐fat (HF) diet for 12 months. We now report earlier onset of steatohepatitis in relation to metabolic abnormalities, and clarify the roles of dietary fat and bodily lipid partitioning on steatosis severity, liver injury and inflammatory recruitment in this novel non‐alcoholic steatohepatitis (NASH) model.

Hepatotoxicity associated with non-steroidal anti-inflammatory drugs

NSAIDs are one of most frequently prescribed agents in clinical practice. Whereas hepatotoxicity is a rare complication of most NSAIDs (typically 1 to 10 per 100,000 persons exposed), the high level of usage means that these drugs cause liver disease. Because of their divergent chemical structures, the mechanisms and clinicopathological manifestations of hepatotoxicity vary widely. The reactive metabolite syndrome, in which serious rash, eosinophilia, and other forms of tissue injury are common, may be incited by several NSAIDs, including newer agents. Women, people aged more than 50 years, and for some drugs, the type of arthritis, may be risk factors for drug-induced liver injury. The spectrum of NSAID-drug related hepatotoxicity continues to expand, with reports of interactive toxicity in adults with hepatitis C and recognition of rare cases of liver disease associated with non-selective, selective, and preferential COX-2 inhibitors. Better outcomes require people taking NSAIDs to be aware of possible drug reactions involving the liver, and prescribers should be vigilant for early symptoms of hepatotoxicity so that incriminated agents are discontinued promptly.

Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH

Cholesterol crystals form within hepatocyte lipid droplets in human and experimental nonalcoholic steatohepatitis (NASH) and are the focus of crown-like structures (CLSs) of activated Kupffer cells (KCs). Obese, diabetic Alms1 mutant (foz/foz) mice were a fed high-fat (23%) diet containing 0.2% cholesterol for 16 weeks and then assigned to four intervention groups for 8 weeks: a) vehicle control, b) ezetimibe (5 mg/kg/day), c) atorvastatin (20 mg/kg/day), or d) ezetimibe and atorvastatin. Livers of vehicle-treated mice developed fibrosing NASH with abundant cholesterol crystallization within lipid droplets calculated to extend over 3.3% (SD, 2.2%) of liver surface area. Hepatocyte lipid droplets with prominent cholesterol crystallization were surrounded by TNFα-positive (activated) KCs forming CLSs (≥3 per high-power field). KCs that formed CLSs stained positive for NLRP3, implicating activation of the NLRP3 inflammasome in response to cholesterol crystals. In contrast, foz/foz mice treated with ezetimibe and atorvastatin showed near-complete resolution of cholesterol crystals [0.01% (SD, 0.02%) of surface area] and CLSs (0 per high-power field), with amelioration of fibrotic NASH. Ezetimibe or atorvastatin alone had intermediate effects on cholesterol crystallization, CLSs, and NASH. These findings are consistent with a causative link between exposure of hepatocytes and KCs to cholesterol crystals and with the development of NASH possibly mediated by NLRP3 activation.

Short‐term therapy with peroxisome proliferation‐activator receptor‐α agonist Wy‐14,643 protects murine fatty liver against ischemia–reperfusion injury

Steatosis increases operative morbidity/mortality from ischemia–reperfusion injury (IRI); few pharmacological approaches have been protective. Using novel genetic/dietary models of nonalcoholic steatohepatitis (NASH) and simple steatosis (SS) in Alms1 mutant (foz/foz) mice, we characterized severity of IRI in NASH versus SS and lean liver and tested our hypothesis that the lipid‐lowering effects of the peroxisome proliferation‐activator receptor (PPAR)‐α agonist Wy‐14,643 would be hepatoprotective. Mice were subjected to 60‐minute partial hepatic IRI. Microvascular changes were assessed at 15‐minute reperfusion by in vivo microscopy, injury at 24 hours by serum alanine aminotransferase (ALT), and hepatic necrosis area. Injury and inflammation mediators were determined by way of immunoblotting for intercellular cellular adhesion molecule, vascular cellular adhesion molecule, p38, c‐jun N‐terminal kinase, IκB‐α, interleukin (IL)‐1a, IL‐12, tumor necrosis factor‐α (TNF‐α) and IL‐6, cell cycle by cyclin D1 and proliferating cell nuclear antigen immunohistochemistry. In foz/foz mice fed a high‐fat diet (HFD) to cause NASH or chow (SS), IRI was exacerbated compared with HFD‐fed or chow‐fed wild‐type littermates by ALT release; corresponding necrotic areas were 60 ± 22% NASH, 29 ± 9% SS versus 7 ± 1% lean. Microvasculature of NASH or SS livers was narrowed by enormous lipid‐filled hepatocytes, significantly reducing numbers of perfused sinusoids, all exacerbated by IRI. Wy‐14,643 reduced steatosis in NASH and SS livers, whereas PPAR‐α stimulation conferred substantial hepatoprotection against IRI by ALT release, with reductions in vascular cellular adhesion molecule‐1, IL‐1a, TNF‐α, IL‐12, activated nuclear factor‐κB (NF‐κB), p38, IL‐6 production and cell cycle entry. Conclusion: NASH and SS livers are both more susceptible to IRI. Mechanisms include possible distortion of the microvasculature by swollen fat‐laden hepatocytes, and enhanced production of several cytokines. The beneficial effects of Wy‐14,643 may be exerted by dampening adhesion molecule and cytokine responses, and activating NF‐κB, IL‐6 production, and p38 kinase to effect cell cycle entry. (HEPATOLOGY 2009.)

Atorvastatin protects obese mice against hepatic ischemia-reperfusion injury by Toll-like receptor-4 suppression and endothelial nitric oxide synthase activation.

Background and Aim:  Steatosis accentuates the severity of hepatic ischemia–reperfusion injury (IRI). 3‐Hydroxy‐3‐methylglutaryl‐coenzyme A reductase inhibitors (“statins”) protect the heart and brain against post‐ischemic injury, without necessarily lowering serum cholesterol. We tested whether 10‐day or 1‐day atorvastatin administration protects livers with fatty change or non‐alcoholic steatohepatitis (NASH) against IRI.

Mice deficient in the putative phospholipid flippase ATP11C exhibit altered erythrocyte shape, anemia, and reduced erythrocyte life span

Background: Asymmetrical distribution of specific phospholipids between the two leaflets of biological membranes is generated and maintained by transporters. Results: A mutation in murine Atp11c results in altered morphology and shortened life span of erythrocytes. Conclusion: Phospholipid transport by ATP11C maintains phospholipid asymmetry in erythrocytes. Significance: Defects in phospholipid transport across the cell membrane can lead to anemia. Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.