Richard A. Rippe

Molecular mechanisms of hepatic fibrogenesis

Liver fibrosis, a wound‐healing response to a variety of chronic stimuli, is characterized by excessive deposition of extracellular matrix (ECM) proteins, of which type I collagen predominates. This alters the structure of the liver leading to organ dysfunction. The activated hepatic stellate cell (HSC) is primarily responsible for excess collagen deposition during liver fibrosis. Two important aspects are involved in mediating the fibrogenic response: first the HSC becomes directly fibrogenic by synthesizing ECM proteins; second, the activated HSC proliferates, effectively amplifying the fibrogenic response. Although the precise mechanisms responsible for HSC activation remain elusive, substantial insight is being gained into the molecular mechanisms responsible for ECM production and cell proliferation in the HSC. The activated HSC becomes responsive to both proliferative (platelet‐derived growth factor) and fibrogenic (transforming growth factor‐β[TGF‐β]) cytokines. It is becoming clear that these cytokines activate both mitogen‐activated protein kinase (MAPK) signaling, involving p38, and focal adhesion kinase–phosphatidylinositol 3‐kinase–Akt–p70 S6 kinase (FAK‐PI3K‐Akt‐p70S6K) signaling cascades. Together, these regulate the proliferative response, activating cell cycle progression as well as collagen gene expression. In addition, signaling by both TGF‐β, mediated by Smad proteins, and p38 MAPK influence collagen gene expression. Smad and p38 MAPK signaling have been found to independently and additively regulate α1(I) collagen gene expression by transcriptional activation while p38 MAPK, but not Smad signaling, increases α1(I) collagen mRNA stability, leading to increased synthesis and deposition of type I collagen. It is anticipated that by understanding the molecular mechanisms responsible for HSC proliferation and excess ECM production new therapeutic targets will be identified for the treatment of liver fibrosis.

New aspects of hepatic fibrosis.

Hepatic stellate cells are the major source of extracellular matrix proteins in hepatic fibrosis, including Type I collagen. In response to liver injury, the hepatic stellate cells change from a quiescent to an activated phenotype. This activation process includes a phenotypic change to a myofibroblast-like cell, increased proliferation rate, loss of retinoid stores, increased production of extracellular matrix proteins, chemokines, and cytokines, and contractility. Ongoing studies are characterizing the genes that are differentially expressed in the quiescent and activated hepatic stellate cells. We have also investigated the regulation of Type I collagen expression, the cleavage of collagen propeptides, and the formation of collagen cross-links. Understanding these pathways may provide new insights into the molecular pathogenesis of hepatic fibrosis.

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.)

Liver fibrosis: signals leading to the amplification of the fibrogenic hepatic stellate cell.

Liver fibrosis represents a major medical problem with significant morbidity and mortality. Worldwide hepatitis viral infections represent the major cause liver fibrosis; however, within the United States chronic ethanol consumption is the leading cause of hepatic fibrosis. Other known stimuli for liver fibrosis include helminthic infection, iron or copper overload and biliary obstruction. Fibrosis can be classified as a wound healing response to a variety of chronic stimuli that is characterized by an excessive deposition of extracellular matrix proteins of which type I collagen predominates. This excess deposition of extracellular matrix proteins disrupts the normal architecture of the liver resulting in pathophysiological damage to the organ. If left untreated fibrosis can progress to liver cirrhosis ultimately leading to organ failure and death if left untreated. This review will discuss the molecular events leading to liver fibrosis. The discussion will include collagen gene regulation and proliferative signals that contribute to the amplification of the hepatic stellate cell, the primary fibrogenic cell type that resides in the liver.

Accumulation of hedgehog-responsive progenitors parallels alcoholic liver disease severity in mice and humans.

BACKGROUND & AIMS Improving outcomes in alcoholic liver disease (ALD) necessitates better understanding of how habitual ethanol (EtOH) consumption alters normal regenerative mechanisms within the liver. Hedgehog (Hh) pathway activation promotes expansion of progenitor populations in other tissues. We evaluated the hypothesis that chronic EtOH exposure activates Hh signaling in liver. METHODS Hh signaling, liver progenitors, transforming growth factor (TGF)-beta induction, and liver damage were compared in mice fed chow, high-fat diets (HF), or HF + EtOH for 4 weeks. Susceptibility to TGF-beta-mediated apoptosis was compared in Hh-responsive liver cells (eg, immature cholangiocytes and oval cells) and mature hepatocytes (which are unresponsive to Hh). Hepatic accumulation of Hh-responsive cells were compared in controls and ALD patients and correlated with a discriminant function (DF) that predicts subacute mortality. RESULTS Hh signaling and numbers of Hh-responsive cells were increased in HF mice and greatest in HF+EtOH mice. In both, progenitor and stromal cell populations harbored Hh-responsive cells. More ductular-type progenitors and fibrosis markers were noted in HF+EtOH mice than in HF mice. The former also expressed more TGF-beta-1. TGF-beta-1 treatment selectively promoted the viability of Hh-responsive immature liver cells and caused mature hepatocytes that survived to produce Hh ligands. Hh-responsive cells were increased in ALD patients. Lobular accumulation of Hh-responsive immature ductular cells was greater in those with a DF >32 than those with a DF <32. CONCLUSIONS Hh signaling is increased in ALD and may influence ALD outcomes by promoting hepatic accumulation of immature ductular cells.

NF-kappaB Inhibits Expression of the alpha1(I) Collagen Gene

Fibrosis results from an increase in the synthesis and deposition of type I collagen. Fibrosis is frequently associated with inflammation, which is accompanied by increased levels of tumor necrosis factor-alpha (TNFalpha) and activation of the transcription factor NF-kappaB. However, several agents known to activate NF-kappaB, such as phorbol 12-myristate 13-acetate (PMA) and TNFalpha, result in decreased expression of type I collagen. Therefore, we directly examined the effects of NF-kappaB on alpha1(I) collagen gene expression in two collagen-producing cells, NIH 3T3 fibroblasts and hepatic stellate cells (HSCs). Transient transfections of NIH 3T3 cells or HSCs using NF-kappaB p50, p65, and c-Rel expression plasmids with collagen reporter gene plasmids demonstrated a strong inhibitory effect on transcription of the collagen gene promoter. Dose-response curves showed that p65 was a stronger inhibitor of collagen gene expression than was NF-kappaB p50 or c-Rel (maximum inhibition 90%). Transient transfections with reporter gene plasmids containing one or two Spl binding sites demonstrated similar inhibitory effects of NF-kappaB p65 on the activity of these reporter genes, suggesting that the inhibitory effects of NF-kappaB p65 are mediated through the critical Spl binding sites in the alpha1(I) collagen gene promoter. Cotransfection experiments using either a super-repressor I[ke]B or Spl partially blocked the inhibitory effects of p65 on collagen reporter gene activity. Coimmunoprecipitation experiments demonstrated that NF-kappaB and Spl do interact in vivo. Nuclear run-on assays showed that NF-kappaB p65 inhibited transcription of the endogenous alpha1(I) collagen gene. Together, these results demonstrate that NF-kappaB decreases transcription of the alpha1(I) collagen gene.

SMAD and p38 MAPK Signaling Pathways Independently Regulate α1(I) Collagen Gene Expression in Unstimulated and Transforming Growth Factor-β-stimulated Hepatic Stellate Cells

The hepatic stellate cell (HSC) is the predominant cell type responsible for excess collagen deposition during liver fibrosis. Both transforming growth factor-β (TGF-β), the most potent fibrogenic cytokine for HSCs, which classically activates Smad signaling, and p38 MAPK signaling have been shown to influence collagen gene expression; however, the relative contribution and mechanisms that these two signaling pathways have in regulating collagen gene expression have not been investigated. The aim of this study was to investigate the relative roles and mechanisms of both Smad and p38 MAPK signaling in α1(I) collagen gene expression in HSCs. Inhibiting either p38 MAPK or Smad signaling reduced α1(I) collagen mRNA expression in untreated or TGF-β-treated HSCs, and when both signaling pathways were simultaneously inhibited, α1(I) collagen gene expression was essentially blocked. Both signaling pathways were found to independently and additively increase α1(I) collagen gene expression by transcriptional mechanisms. TGF-β treatment increased α1(I) collagen mRNA half-life, mediated by increased stability of α1(I) collagen mRNA through p38 MAPK signaling but not through Smad signaling. In conclusion, both p38 MAPK and Smad signaling independently and additively regulate α1(I) collagen gene expression by transcriptional activation, whereas p38 MAPK and not Smad signaling increased α1(I) collagen mRNA stability.

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.

Roles of oxidative stress in activation of Kupffer and Ito cells in liver fibrogenesis

An increasing body of experimental evidence is emerging to incriminate oxidative stress as a pivotal signal for liver fibrogenesis. This paper reviews the results from our studies testing this hypothesis. In the rat model of alcoholic liver disease, the importance of oxidative stress was supported by marked accentuation of liver fibrosis by dietary supplementation of iron, a pro‐oxidant, and the significant correlation of the liver malondialdehyde (MDA) and 4‐hydroxynonenal (4HNE) levels with the hepatic collagen accumulation. Both MDA and 4HNE adduct epitopes were detected intensely and diffusely in close association with collagen deposition. The direct cause and effect relationship between MDA/4HNE and Ito cell stimulation was indicated by the demonstration of Ito cell collagen gene induction by these aldehydes in culture. In primary cultures of rat Kupffer cells (KC), addition of antioxidants such as α‐tocopherol acetate and succinate suppressed mRNA expression and the release of interleukin (IL)‐6 and tumour necrosis factor alpha (TNFα). In rats with biliary fibrosis, an increase in the liver MDA level was accompanied by enhanced mRNA expression of procollagen α 1 (I) and transforming growth factor β 1 in Ito cells; and that of TNFα and IL‐6 in KC. Furthermore, the gel shift assay of KC nuclear extracts showed enhanced NF‐kB DNA binding activity. These results support the proposal that enhanced oxidative stress constitutes an important signal for activation of Kupffer and Ito cells in experimental liver fibrogenesis.

SMAD and p38 MAPK signaling pathways independently regulate α1() collagen gene expression in unstimulated and TGF-β stimulated hepatic stellate cells

The hepatic stellate cell (HSC) is the predominant cell type responsible for excess collagen deposition during liver fibrosis. Both transforming growth factor-β (TGF-β), the most potent fibrogenic cytokine for HSCs, which classically activates Smad signaling, and p38 MAPK signaling have been shown to influence collagen gene expression; however, the relative contribution and mechanisms that these two signaling pathways have in regulating collagen gene expression have not been investigated. The aim of this study was to investigate the relative roles and mechanisms of both Smad and p38 MAPK signaling in α1(I) collagen gene expression in HSCs. Inhibiting either p38 MAPK or Smad signaling reduced α1(I) collagen mRNA expression in untreated or TGF-β-treated HSCs, and when both signaling pathways were simultaneously inhibited, α1(I) collagen gene expression was essentially blocked. Both signaling pathways were found to independently and additively increase α1(I) collagen gene expression by transcriptional mechanisms. TGF-β treatment increased α1(I) collagen mRNA half-life, mediated by increased stability of α1(I) collagen mRNA through p38 MAPK signaling but not through Smad signaling. In conclusion, both p38 MAPK and Smad signaling independently and additively regulate α1(I) collagen gene expression by transcriptional activation, whereas p38 MAPK and not Smad signaling increased α1(I) collagen mRNA stability.