The Role of iNOS in Cholesterol-induced Liver Fibrosis

Sarit Anavi; Michal Eisenberg-Bord; Michal Hahn-Obercyger; Olga Genin; Mark Pines; Oren Tirosh

Disclosures

Lab Invest. 2015;95(8):914-924. 

In This Article

Abstract and Introduction

Abstract

Accumulation of cholesterol in the liver is associated with the development of non-alcoholic steatohepatitis-related fibrosis. However, underlying mechanisms are not well understood. The present study investigated the role of inducible nitric oxide synthase (iNOS) in cholesterol-induced liver fibrosis by feeding wild-type (WT) and iNOS-deficient mice with control or high-cholesterol diet (HCD) for 6 weeks. WT mice fed with HCD developed greater liver fibrosis, compared with iNOS-deficient mice, as evident by Sirius red staining and higher expression levels of profibrotic genes. Enhanced liver fibrosis in the presence of iNOS was associated with hypoxia-inducible factor-1α stabilization, matrix metalloproteinase-9 expression, and enhanced hepatic DNA damage. The profibrotic role of iNOS was also demonstrated in vivo using a selective inhibitor of iNOS as well as in vitro in a rat liver stellate cell line (HSC-T6). In conclusion, these findings suggest that iNOS is an important mediator in HCD-induced liver fibrosis.

Introduction

Non-alcoholic fatty liver disease (NAFLD) is currently one of the most common liver diseases worldwide, affecting both adults and children. The disease encompasses a wide spectrum of liver damage ranging from simple hepatic steatosis and non-alcoholic steatohepatitis (NASH) to liver cirrhosis and increased risk for developing hepatocellular carcinoma. Pathological characterization of NASH includes the presence of steatosis and inflammation and recently it has also been recognized as a major cause of liver fibrosis. Liver fibrosis represents the liver's wound healing response to virtually all forms of chronic liver injury. This dynamic process involves the accumulation of the extracellular matrix (ECM) following injury. Under acute or limited insults, parenchymal cells regenerate and replace the necrotic or apoptotic cells. However, if the liver injury is sustained, regeneration fails and liver parenchymal cells are substituted by scar tissue.[1,2]

Fibrosis associated with NASH has been suggested to be the physiological consequence of chronic hepatic injury, necrosis, and inflammation as well as unbalanced intrahepatic lipid metabolism (steatosis), insulin resistance, oxidative stress, and lipid peroxidation.[3–5]

Different lipid molecules may mediate the pathogenesis and the progression of NASH. To date, the identity of a dominant lipid molecule/s, which causes liver lipotoxicity, is still unknown. Recently, attention has been given to the role of hepatic cholesterol content in NASH pathology.[6–10] Evidence collected from both human and animal models of NASH point to cholesterol as a potential mediator of lipotoxicity in NASH, including the development of hepatic fibrosis. In the first National Health and Nutrition Examination Survey in the United States, dietary cholesterol consumption was positively associated with the risk of cirrhosis or liver cancer. Consistent with these findings, cholesterol-lowering agents, such as statins and ezetimibe, were shown to improve liver fibrosis among patients with hypercholesterolemia and mice with NASH.[8–11] Enhanced liver fibrosis is also evident in experimental studies conducted in rodents and rabbits following the consumption of a high-cholesterol diet (HCD) containing cholic acid or a high-fat/HCD diet or methionine–choline-deficient diet supplemented with cholesterol.[8,9]

Initiation of chronic liver diseases commonly involves an inflammatory phase, which progresses to fibrosis after continuous oxidative stress. Under these conditions inducible nitric oxide synthase (iNOS) is upregulated, leading to the production of large amounts of nitric oxide (NO).[12] The role of iNOS in fibrosis formation is not clearly understood. Data from animal models of fibrosis or NASH-related liver fibrosis have shown conflicting results of both beneficial and deleterious effects of iNOS.[12–19] The exact reason/s for these paradoxical effects is difficult to explain. Therefore, the present study was undertaken to elucidate the role of iNOS in the pathophysiology of liver fibrosis due to consumption of a HCD.

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