ton. Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin. Am J Physiol Gastrointest Liver Physiol 287: G1035-G1043, 2004. First published July 15, 2004 doi:10.1152/ ajpgi.00199.2004.-Obesity and type 2 diabetes are associated with nonalcoholic steatohepatitis (NASH), but an obese/diabetic animal model that mimics human NASH remains undefined. We examined the induction of steatohepatitis and liver fibrosis in obese and type 2 diabetic db/db mice in a nutritional model of NASH and determined the relationship of the expressions of osteopontin (OPN) and leptin receptors to the pathogenesis of NASH. db/db mice and the corresponding lean and nondiabetic db/m mice were fed a diet deficient in methionine and choline (MCD diet) or control diet for 4 wk. Leptindeficient obese and diabetic ob/ob mice fed similar diets were used for comparison. MCD diet-fed db/db mice exhibited significantly greater histological inflammation and higher serum alanine aminotransferase levels than db/m and ob/ob mice. Trichrome staining showed marked pericellular fibrosis in MCD diet-fed db/db mice but no significant fibrosis in db/m or ob/ob mice. Collagen I mRNA expression was increased 10-fold in db/db mice, 4-fold in db/m mice, and was unchanged in ob/ob mice. mRNA expressions of OPN, TNF-␣, TGF-␤, and short-form leptin receptors (Ob-Ra) were significantly increased in db/db mice compared with db/m or ob/ob mice. Parallel increases in OPN and Ob-Ra protein levels were observed in db/db mice. Cultured hepatocytes expressed only Ob-Ra, and leptin stimulated OPN mRNA and protein expression in these cells. In conclusion, our results demonstrate the development of an obese/diabetic experimental model for NASH in db/db mice and suggest an important role for Ob-Ra and OPN in the pathogenesis of NASH. obesity; diabetes; insulin resistance; osteopontin; fibrosis NONALCOHOLIC STEATOHEPATITIS (NASH) is commonly associated with obesity, type 2 diabetes, and the metabolic syndrome (1,41,45,48). However, several studies examining the pathogenesis of NASH have employed lean and nondiabetic strains of mice fed a diet deficient in methionine and choline (MCD diet). We and others have demonstrated that this model produces steatohepatitis and liver fibrosis that is histologically similar to human NASH (11,13,23,25,26,46). In addition, mice lacking methionine adenosyltransferase have been shown to develop steatohepatitis similar to human NASH (31,34,47). The molecular signaling mechanisms that lead to the activation of inflammation and fibrosis in these models of NASH remain poorly defined. Several studies suggest that peroxidative injury may play a role in the development of steatohepatitis in human and in experimental NASH (11,26,47,48). However, we (46) recently assessed oxidative stress during the progression of steatosis to steatohepatitis in MCD diet-fed A/J mice and found that peroxidative injury occurs late in developing steatohepatitis. We have id...

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The pathogenesis of nonalcoholic steatohepatitis (NASH) is poorly defined. Feeding mice a diet deficient in methionine and choline (MCD diet) induces experimental NASH. Osteopontin (OPN) is a Th1 cytokine that plays an important role in several fibroinflammatory diseases. We examined the role of OPN in the development of experimental NASH. A/J mice were fed MCD or control diet for up to 12 wk, and serum alanine aminotransferase (ALT), liver histology, oxidative stress, and the expressions of OPN, TNF-alpha, and collagen I were assessed at various time points. MCD diet-fed mice developed hepatic steatosis starting after 1 wk and inflammation by 2 wk; serum ALT increased from day 3. Hepatic collagen I mRNA expression increased during 1-4 wk, and fibrosis appeared at 8 wk. OPN protein expression was markedly increased on day 1 of MCD diet and persisted up to 8 wk, whereas OPN mRNA expression was increased at week 4. TNF-alpha expression was increased from day 3 to 2 wk, and evidence of oxidative stress did not appear until 8 wk. Increased expression of OPN was predominantly localized in hepatocytes. Hepatocytes in culture also produced OPN, which was stimulated by transforming growth factor-beta and TNF-alpha. Moreover, MCD diet-induced increases in serum ALT levels, hepatic inflammation, and fibrosis were markedly reduced in OPN(-/-) mice when compared with OPN(+/+) mice. In conclusion, our results demonstrate an upregulation of OPN expression early in the development of steatohepatitis and suggest an important role for OPN in signaling the onset of liver injury and fibrosis in experimental NASH.

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We examined the effect of hypoxia on proliferation and osteopontin (OPN) expression in cultured rat aortic vascular smooth muscle (VSM) cells. In addition, we determined whether hypoxia-induced increases in OPN and cell proliferation are altered under hyperglycemic conditions. Quiescent cultures of VSM cells were exposed to hypoxia (3% O 2 ) or normoxia (18% O 2 ) in a serum-free medium, and cell proliferation as well as the expression of OPN was assessed. Cells exposed to hypoxia for 24 h exhibited a significant increase in [ 3 H]thymidine incorporation followed by a significant increase in cell number at 48 h in comparison with respective normoxic controls. Exposure to hypoxia produced significant increases in OPN protein and mRNA expression at 2 h followed by a gradual decline at 6 and 12 h, with subsequent significant increases at 24 h. Neutralizing antibodies to either OPN or its receptor ␤3 integrin but not neutralizing antibodies to ␤5 integrin prevented the hypoxia-induced increase in T he first descriptions of a link between systemic hypoxia and pulmonary artery smooth muscle cell proliferation came from both in vivo and in vitro models of pulmonary hypertension (1-3). Increased proliferation of aortic vascular smooth muscle (VSM) cells is also a key feature in the progression of atherosclerosis (4). Both systemic and local hypoxia contributes to the development of atherosclerotic lesions (5-11). Recent in vivo studies found a direct correlation of local arterial wall hypoxia, VSM cell proliferation, and atherosclerosis (12,13). However, the underlying signaling mechanisms whereby hypoxia induces VSM cell proliferation and subsequent atherosclerotic lesions remain poorly defined. Part of the problem has been in the demonstration of a mitogenic effect of hypoxia in cultured VSM cells in vitro. Hypoxia has only been shown to induce the proliferation of bovine pulmonary artery smooth muscle cells in culture when they are costimulated either with an activator of protein kinase C (PKC) or serum (3,14). To examine the effect of local hypoxia on cell proliferation, our laboratory has developed an appropriate cell culture model system in which cultured cells exhibited differentiated morphology and function by improved oxygenation (15). Using this culture model, we reported that hypoxia induces proliferation, dedifferentiation, and/or extracellular matrix synthesis in cultured renal tubular epithelial and glomerular mesangial cells (16 -18). In the present study, we examined the effect of hypoxia on the proliferation of cultured rat aortic VSM cells to determine whether hypoxia directly alters their proliferative behavior.Enhanced proliferation of VSM cells has also been demonstrated in both human and experimental models of diabetes (19,20). In addition, cultured VSM cells grown in high media glucose concentration (to mimic hyperglycemia of diabetes) have exhibited increased cell proliferation (21). The pathophysiological mechanisms responsible for accelerated VSM cell proliferation and progression into diabetic...

The effect of hypoxia on the proliferation and collagen synthesis of cultured rat mesangial cells was examined under normal-glucose (NG, 5 mM) and high-glucose (HG, 25 mM)-media conditions. In addition, a role for osteopontin (OPN) in mediating these processes was assessed. Quiescent cultures were exposed to hypoxia (3% O(2)) and normoxia (18% O(2)) in a serum-free medium with NG or HG, and cell proliferation, collagen synthesis, and OPN expression were assessed. Cells exposed to hypoxia in NG medium resulted in significant increases in [(3)H]thymidine incorporation, cell number, and [(3)H]proline incorporation, respectively. HG incubations also produced significant stimulation of these parameters under normoxic conditions, which were markedly enhanced in cells exposed to hypoxia in HG medium. In addition, hypoxia and HG stimulated the mRNA levels of type IV collagen, and the combination of hypoxia and HG resulted in additive increases in type IV collagen expression. Hypoxia and HG also stimulated OPN mRNA and protein levels in an additive fashion. A neutralizing antibody to OPN or its beta(3)-integrin receptor significantly blocked the effect of hypoxia and HG on proliferation and collagen synthesis. In conclusion, these results demonstrate for the first time that hypoxia in HG medium produces exaggerated mesangial cell growth and type IV collagen synthesis. In addition, OPN appears to play a role in mediating the accelerated mesangial cell growth and collagen synthesis found in a hyperglycemic and hypoxic environment.

Na+/H+ exchange activity and NHE-3 protein abundance in renal proximal tubules from the SHR are increased while NHE-3 mRNA is not. A post-transcriptional event(s) best explains the increase in NHE-3 protein expression since mRNA levels were not increased. The alterations in the SHR antedate the development of hypertension and fail to decrease as blood pressure increases with age in the SHR, which likely results in inappropriate renal sodium retention in the face of a chronic rise in blood pressure.

The acquired or perinatal form of biliary atresia is a Th1 fibro-inflammatory disease affecting both the extrahepatic and intrahepatic bile ducts. Osteopontin (OPN) is a Th1 cytokine implicated in several fibro-inflammatory and autoimmune diseases. We examined the expression of OPN in acquired biliary atresia in comparison to normal liver and several pediatric cholestatic liver diseases. We also assessed OPN expression by cultured human bile duct epithelial cells. We found that liver OPN mRNA and protein expression were significantly increased in biliary atresia versus normal and other cholestatic diseases. OPN expression in biliary atresia was localized to epithelium of proliferating biliary structures (ductules and/or ducts) and bile plugs contained therein. No portal biliary OPN expression could be demonstrated in normal liver, syndromic biliary atresia, biliary obstruction not due to biliary atresia, and idiopathic neonatal hepatitis. OPN expression by human bile duct epithelial cells in culture was responsive to IL-2 and TNF-␣. Our results demonstrate an up-regulation of OPN expression by interlobular biliary epithelium in biliary atresia, which correlates with biliary proliferation and portal fibrosis. These findings suggest a role for OPN in the pathogenesis of biliary atresia. Biliary atresia is the most common liver disease of infancy that leads to cirrhosis, end-stage liver disease and the need for liver transplantation (1). A small proportion of cases are thought to be congenital in that they are associated with other developmental anomalies, whereas most cases are diagnosed at 4 -8 wk of age and appear to represent a disease acquired in perinatal or neonatal life. The etiology and pathogenesis of biliary atresia remain unknown. A current working unifying hypothesis that can explain the clinical and pathologic features of the acquired form of biliary atresia is that some injury to the bile ducts, likely a perinatal viral infection, leads to an autonomous immune response that results in progressive bile duct injury and fibrosis (2). This progressive, inflammatory cholangiopathy results in extrahepatic bile duct obstruction (1,3). The extrahepatic bile duct is partially or entirely obliterated by fibrosis in association with inflammation within the fibrous remnant. The intrahepatic biliary system also appears to be affected by the primary disease process. Portal areas are expanded with fibrosis and exhibit marked proliferation of biliary elements (ductules and/or ducts) (4 -8). These duct structures often contain bile plugs. Biliary proliferation may still be a feature of the disease even at end-stage, but in some cases all duct structures disappear.Previous studies have shown that the mononuclear inflammatory infiltrate in the portal tracts comprises macrophages, CD4ϩ T-cells and NK cells (9 -13). We have shown that the inflammation is typically Th1 in character, in that it consists mainly of macrophages, CD4ϩ T-cells and CD8 ϩ T-cells with production of IL-2, IFN-␥, . The inciting agent leading to thi...