Robert Schoonhoven

NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen alpha1(I) mRNA expression, and secretion of TGF-beta1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox-/- mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox-/- mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle alpha-actin and expression of TGF-beta1 were reduced in p47phox-/- mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.

DNA Damage in Nasal and Brain Tissues of Canines Exposed to Air Pollutants Is Associated with Evidence of Chronic Brain Inflammation and Neurodegeneration

Acute, subchronic, or chronic exposures to particulate matter (PM) and pollutant gases affect people in urban areas and those exposed to fires, disasters, and wars. Respiratory tract inflammation, production of mediators of inflammation capable of reaching the brain, systemic circulation of PM, and disruption of the nasal respiratory and olfactory barriers are likely in these populations. DNA damage is crucial in aging and in age-associated diseases such as Alzheimers disease. We evaluated apurinic/apyrimidinic (AP) sites in nasal and brain genomic DNA, and explored by immunohistochemistry the expression of nuclear factor NFκB p65, inducible nitric oxide synthase (iNOS), cyclo-oxygenase 2 (COX2), metallothionein I and II, apolipoprotein E, amyloid precursor protein (APP), and beta-amyloid1-42 in healthy dogs naturally exposed to urban pollution in Mexico City. Nickel (Ni) and vanadium (V) were measured by inductively coupled plasma mass spectrometry (ICP-MS). Forty mongrel dogs, ages 7 days—10 years were studied (14 controls from Tlaxcala and 26 exposed to urban pollution in South West Metropolitan Mexico City (SWMMC)). Nasal respiratory and olfactory epithelium were found to be early pollutant targets. Olfactory bulb and hippocampal AP sites were significantly higher in exposed than in control age matched animals. Ni and V were present in a gradient from olfactory mucosa > olfactory bulb > frontal cortex. Exposed dogs had (a) nuclear neuronal NFκB p65, (b) endothelial, glial and neuronal iNOS, (c) endothelial and glial COX2, (d) ApoE in neuronal, glial and vascular cells, and (e) APP and β amyloid1-42 in neurons, diffuse plaques (the earliest at age 11 months), and in subarachnoid blood vessels. Increased AP sites and the inflammatory and stress protein brain responses were early and significant in dogs exposed to urban pollution. Oil combustion PM-associated metals Ni and V were detected in the brain. There was an acceleration of Alzheimers-type pathology in dogs chronically exposed to air pollutants. Respiratory tract inflammation and deteriorating olfactory and respiratory barriers may play a role in the observed neuropathology. These data suggest that Alzheimers disease may be the sequela of air pollutant exposures and the resulting systemic inflammation.

Decreasing fibrogenesis: an immunohistochemical study of paired liver biopsies following lamivudine therapy for chronic hepatitis B

BACKGROUND Activation of hepatic stellate cells is the earliest step in fibrogenesis. Alpha-smooth muscle actin (alpha-SMA), expressed by activated hepatic stellate cells, and C-terminal procollagen alpha1(III) propeptide (PIIICP) are early markers of fibrogenesis and should precede fibrosis. AIM Determine if suppression of hepatitis B virus replication with lamivudine would decrease fibrogenesis as measured by immunohistochemical markers. METHODS Paired liver biopsies from patients with hepatitis B before and after therapy with lamivudine (n=47) or placebo (n=33) were studied. alpha-SMA and PIIICP were detected in paraffin-embedded tissue by immunohistochemistry and quantified in a blinded manner by video imaging analysis. RESULTS Liver biopsies from patients treated with lamivudine showed a significant decrease in alpha-SMA expression (1.06+/-0.23 vs. 0.58+/-0.11, pre vs. post, P<0.05). Placebo recipients had increased levels of alpha-SMA (0.82+/-0.14 vs. 1.32+/-0.21, P<0.05). PIIICP was similarly decreased after lamivudine. Among subjects whose Histologic Activity Index fibrosis score was unchanged or worsened, the mean change in alpha-SMA expression was significantly decreased in the lamivudine group compared with placebo. CONCLUSIONS Lamivudine decreased markers of hepatic stellate cell activation and collagen synthesis. Immunohistochemical techniques are sensitive for assessing fibrogenesis and will be useful in trials of antiviral and antifibrotic agents.

Systemic infusion of angiotensin II exacerbates liver fibrosis in bile duct–ligated rats

Recent evidence indicates that the renin–angiotensin system (RAS) plays a major role in liver fibrosis. Here, we investigate whether the circulatory RAS, which is frequently activated in patients with chronic liver disease, contributes to fibrosis progression. To test this hypothesis, we increased circulatory angiotensin II (Ang II) levels in rats undergoing biliary fibrosis. Saline or Ang II (25 ng/kg/h) were infused into bile duct–ligated rats for 2 weeks through a subcutaneous pump. Ang II infusion increased serum levels of Ang II and augmented bile duct ligation–induced liver injury, as assessed by elevated liver serum enzymes. Moreover, it increased the hepatic concentration of inflammatory proteins (tumor necrosis factor α and interleukin 1β) and the infiltration of CD43‐positive inflammatory cells. Ang II infusion also favored the development of vascular thrombosis and increased the procoagulant activity of tissue factor in the liver. Livers from bile duct–ligated rats infused with Ang II showed increased transforming growth factor β1 content, collagen deposition, accumulation of smooth muscle α‐actin–positive cells, and lipid peroxidation products. Moreover, Ang II infusion stimulated phosphorylation of c‐Jun and p42/44 mitogen‐activated protein kinase and increased proliferation of bile duct cells. In cultured rat hepatic stellate cells (HSCs), Ang II (10−8 mol/L) increased intracellular calcium and stimulated reactive oxygen species formation, cellular proliferation and secretion of proinflammatory cytokines. Moreover, Ang II stimulated the procoagulant activity of HSCs, a newly described biological function for these cells. In conclusion, increased systemic Ang II augments hepatic fibrosis and promotes inflammation, oxidative stress, and thrombogenic events. (HEPATOLOGY 2005;41:1046–1055.)

Nuclear factor κB in proliferation, activation, and apoptosis in rat hepatic stellate cells

Abstract Background/Aims: Activation of the transcription factor NFκB has been demonstrated in activated hepatic stellate cells (HSCs). We investigated the role of NFκB in proliferation, in activation, and in TNFα-induced apoptosis of HSCs. Methods: NFκB activation was inhibited using an adenovirus expressing an IκB dominant negative protein (Ad5IκB) in both quiescent and activated HSCs. Quiescent HSCs were infected with Ad5IκB or an adenovirus expressing β-galactosidase (Ad5LacZ). The cells were cultured for 7 days. HSCs activation was determined by cell morphology, smooth muscle α-actin (α-sma) expression, and steady-state mRNA levels of α1(I) collagen as assessed by Western blot and RNase protection assay, respectively. Proliferation was determined in culture-activated HSCs by 3 H-thy-midine incorporation and direct cell counting. Apoptosis was analyzed by infecting quiescent or activated HSCs with Ad5IκB or Ad5LacZ, and then treating with TNFα. Apoptosis was demonstrated by determining cell number, assessing nuclear morphology, TUNEL assay and caspase 3 activity. Results: After 7 days in culture no differences were noted between the Ad5IκB- and the Ad5LacZ-in-fected cells in the morphology, α-sma expression or in α1(I) collagen mRNA levels. Ad5IκB infection did not modify proliferation in activated HSCs. TNFα induced apoptosis only in Ad5IκB-infected activated, but not quiescent HSCs. Apoptosis was initially demonstrated 12 h after exposure to TNFα. Twenty-four h after the TNFα treatment, 60% of the activated HSCs were apoptotic. Conclusion: NFκB activity is not required for proliferation or activation of HSCs; however, NFκB protects activated HSCs against TNFα-induced apoptosis.

Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation: Role of Kupffer cells

BACKGROUND The etiology of primary graft nonfunction and dysfunction is unknown but most likely involves Kupffer cell-dependent reperfusion injury. However, the donor operation and surgical technique may also have an effect on the outcome after transplantation. Because liver manipulation during harvest cannot be prevented completely with standard procedures, its effect on survival was assessed here. METHODS Donor livers were harvested from female Sprague-Dawley rats (200-230 g). Briefly, after minimal dissection during the first 12 min, livers were either manipulated gently or left alone for 13 subsequent minutes. At 25 min, all livers were perfused with cold University of Wisconsin solution via the portal vein, and transplantation was performed after cold storage (1 hr). In some rats, Kupffer cells were destroyed with gadolinium chloride or inactivated with dietary glycine before harvest. Survival, proteolytic activity in the rinse effluent, serum transaminases, trypan blue distribution to index microcirculation, and histology were compared. RESULTS In the nonmanipulated group, survival was 100% after transplantation; however, gentle manipulation decreased survival by 70%. Further, manipulation elevated transaminases fivefold and caused about 200% necrosis. At harvest, proteolytic activity and the time for trypan blue to distribute homogeneously were elevated three- to eightfold by manipulation. Gadolinium chloride and glycine prevented the effects of manipulation on all parameters studied. CONCLUSION These data indicate for the first time that brief, gentle manipulation of the donor liver has a marked detrimental effect on survival by priming or activating Kupffer cells. This may represent an important early event in pathogenesis, because Kupffer cells play an important role in primary graft nonfunction.

Early hepatic stellate cell activation is associated with advanced fibrosis after liver transplantation in recipients with hepatitis C

Recurrent hepatitis C after liver transplantation is a serious problem faced by liver transplant recipients. Activation of hepatic stellate cells is an early step in hepatic fibrogenesis. The aim of this study was to evaluate hepatic stellate cell activation, early after liver transplantation, as a predictor for the subsequent development of advanced fibrosis. Forty‐six patients who underwent liver transplantation for hepatitis C and protocol liver biopsies were divided into rapid fibrosers (n = 21), defined as recipients who developed bridging fibrosis or cirrhosis within 2 years of liver transplantation, and slow fibrosers (n = 25). The protocol liver biopsy obtained 4 months after transplantation was stained and quantitated for hepatic stellate cell activation with antibody to alpha smooth muscle actin. Hepatic stellate cell activity was independently associated with rapid fibrosis (odds ratio: 1.6 [95% CI: 1.1,2.2], P = 0.013). The c‐statistics for the receiver operating characteristic curve for stellate cell activity and fibrosis were 0.78 and 0.67, respectively, P = 0.36. The receiver operating characteristic curve for a model including stellate cell activity, histology activity index, and alanine aminotransferase. obtained at month 4 had the best c‐statistic (0.88). In recipients with stage 0 or 1 fibrosis on the month 4 liver biopsy who subsequently developed advanced fibrosis, the c‐statistic for the receiver operating characteristic curves was significantly better for stellate cell activity than for stage of fibrosis (0.77 and 0.51, respectively; P = 0.004). In conclusion, hepatic stellate cell activation early after liver transplantation complements traditional testing for identifying liver transplant recipients with hepatitis C at greatest risk for developing advanced fibrosis. (Liver Transpl 2005;11:1235–1241.)

Delivery of Cu/Zn-superoxide dismutase genes with a viral vector minimizes liver injury and improves survival after liver transplantation in the rat.

BACKGROUND Oxygen-derived free radicals play a central role in pathomechanisms of reperfusion injury after organ transplantation. Endogenous radical scavenger systems such as superoxide dismutase (SOD) degrade toxic radicals; however, SOD is degraded rapidly when given exogenously. Therefore, the hypothesis that treatment of the donor liver with an adenoviral vector encoding the Cu/Zn-SOD gene (Ad-SOD1) would lead to permanent gene expression and therefore protect the organ against injury and increase survival in a rat model of liver transplantation was tested. METHODS Some donors were infected with Ad-SOD1, whereas untreated grafts and livers infected with the indicator gene lacZ encoding bacterial beta-galactosidase (Ad-lacZ) served as controls. After orthotopic liver transplantation, survival, serum transaminases, and histopathology were evaluated. RESULTS Approximately 80% of hepatocytes expressed beta-galactosidase 72 hr after injection of Ad-lacZ. Moreover, SOD1 gene expression and activity were increased 3- and 10-fold in the Ad-SOD1 group, respectively. After transplantation, 20-25% of rats treated with Ad-lacZ survived. In contrast, all SOD1-treated animals survived. Transaminases measured 8 hr after transplantation in Ad-SOD1 rats were only 40% of those in controls, which increased 40-fold above normal values. Approximately 20% of hepatocytes in untreated and Ad-lacZ-infected organs were necrotic 8 hr after reperfusion, whereas necrosis was nearly undetectable in grafts from rats treated with Ad-SOD1. CONCLUSIONS This study provides clear evidence for the first time that gene therapy with Ad-SOD1 increases survival and decreases hepatic injury after liver transplantation. Genetic modification of the liver represents a future approach to protect organs against injury where oxygen-derived free radicals are involved.

Ski Promotes Tumor Growth Through Abrogation of Transforming Growth Factor-β Signaling in Pancreatic Cancer

Objective:We hypothesized that human pancreatic cancer resists TGF-β signaling and cell death through increased Ski expression. Summary Background Data:Ski is an oncogenic protein that acts as a TGF-β repressor and prevents related gene transcription. Previous work suggests that Ski acts as an oncoprotein in melanoma and esophageal cancer. Ski expression and function have not been determined in human pancreatic cancer. Methods:Immunohistochemistry and immunoblots assessed Ski expression in human pancreatic cancer. Panc-1 cells were treated with or without Ski siRNA, and Ski and Smad protein expression, transcriptional reporter activation, and growth assays were determined. Panc-1 cells were inoculated in the flank of nude mice and tumor volume and histology assessed after administration of Ski siRNA or control vector. Results:Ski was abundantly expressed in human pancreatic cancer specimens assessed by immunohistochemistry (91%) and immunoblot analysis (67%). Panc-1 cells exhibited nascent Ski expression that was maximally inhibited 48 hours after transfection with Ski siRNA. TGF-β transcriptional activity was increased 2.5-fold in Ski siRNA-treated cells compared with control (P < 0.05). Ski siRNA increased TGF-β-induced Smad2 phosphorylation and p21 expression. Panc-1 growth in culture was decreased 2-fold at 72 hours. A Ski siRNA expression vector injected into nude mice resulted in a 5-fold decrease in growth. Conclusion:Inhibition of Ski through RNA interference restored TGF-β signaling and growth inhibition in vitro, and decreased tumor growth in vivo.

Induction of Transitional Cell Hyperplasia in the Urinary Bladder and Aberrant Crypt Foci in the Colon of Rats Treated with Individual and a Mixture of Drinking Water Disinfection By-Products

Cancer of the urinary bladder and colon are significant human health concerns. Epidemiological studies have suggested a correlation between these cancers and the chronic consumption of chlorinated surface water containing disinfection by-products (DBPs). The present study was designed to determine if exposure to DBPs would cause preneoplastic or neoplastic lesions in the urinary bladder and colon of rats, and what effect a mixture of DBPs would have on these lesions. Male and female Eker rats were treated via drinking water with low and high concentrations of potassium bromate, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), chloroform, or bromodichloromethane individually or in a mixture for 10 months. The urinary bladders and colons were examined for the presence of preneoplastic lesions. Cell proliferation in the urothelium was examined using immunohistochemical staining for bromodeoxyuridine. Aberrant crypt foci (ACF), as well as the number of individual crypts in each ACF, were identified and counted microscopically after staining with 0.2% methylene blue. Colon crypt cell proliferation and mitotic index were determined using immunohistochemical staining for proliferating cell nuclear antigen. Labeling indexes for the urinary bladder and colon were calculated based on the percentage of positively labeled cells. Treatment with the high dose of MX caused transitional epithelial hyperplasia and cell proliferation in the rat urinary bladder, and this effect was diminished in the high dose mixture animals. Treatment with 4 individual DBPs, as well as a mixture of them, caused the development of ACF, the putative preneoplastic lesion of colon cancer.