Phenotype Of Hepatic Stellate Cells Research Articles

P0452 : Changes in DNA methylation and hydroxymethylation during HSC activation

Background and Aims: DNA methylation (5mC) is an epigenetic mark that is an established regulator of gene transcription with an important role in liver fibrosis. Currently, there is only very basic knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cells (HSC), the key cell type responsible for onset and progression of liver fibrosis. Evidence has shown that presence of higher levels of DNA methylation around gene promoters correlate with low or no transcriptional activity. However, recently discovered 5-hydroxymethylcytosine (5hmC), generated by oxidation of 5mC through action of TET enzymes, is actually involved in gene activation and its patterns are often altered in human diseases. In this study we investigated the role of 5mC and 5hmC in liver fibrosis. Methods: Levels of 5mC and 5hmC were assessed by slot blot in a range of animal liver fibrosis models and diseased human livers. Expression levels of TET enzymes and DNA methyltransferases (DNMTs) were measured by qRT-PCR and WB. Reduced representation bisulfide sequencing (RRBS) method was used to examine 5mC and 5hmC patterns in quiescent and in vivo activated rat HSC. Results: Expression of TET enzymes and therefore global levels of 5hmC, are both downregulated in activated rat HSC and in diseased human livers. Moreover, DNMTs expression is changed in human and rat samples suggesting changed levels of 5mC. Using RRBS, we examined the exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. Conclusions: We found significant differences in TET and DNMT protein expression between quiescent and activated rat HSC as well as in human samples, which in turn cause alteration in the HSC DNA methylome and hydroxymethylome. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.

P0454 : An additional heme oxygenase-1 knockout increases maturation of dendritic cells and liver inflammation in Mdr2 knockout mice

Background and Aims: DNA methylation (5mC) is an epigenetic mark that is an established regulator of gene transcription with an important role in liver fibrosis. Currently, there is only very basic knowledge available as to how DNA methylation controls the phenotype of hepatic stellate cells (HSC), the key cell type responsible for onset and progression of liver fibrosis. Evidence has shown that presence of higher levels of DNA methylation around gene promoters correlate with low or no transcriptional activity. However, recently discovered 5-hydroxymethylcytosine (5hmC), generated by oxidation of 5mC through action of TET enzymes, is actually involved in gene activation and its patterns are often altered in human diseases. In this study we investigated the role of 5mC and 5hmC in liver fibrosis. Methods: Levels of 5mC and 5hmC were assessed by slot blot in a range of animal liver fibrosis models and diseased human livers. Expression levels of TET enzymes and DNA methyltransferases (DNMTs) were measured by qRT-PCR and WB. Reduced representation bisulfide sequencing (RRBS) method was used to examine 5mC and 5hmC patterns in quiescent and in vivo activated rat HSC. Results: Expression of TET enzymes and therefore global levels of 5hmC, are both downregulated in activated rat HSC and in diseased human livers. Moreover, DNMTs expression is changed in human and rat samples suggesting changed levels of 5mC. Using RRBS, we examined the exact genomic positions of changed methylation patterns in quiescent and in vivo activated rat HSC. Conclusions: We found significant differences in TET and DNMT protein expression between quiescent and activated rat HSC as well as in human samples, which in turn cause alteration in the HSC DNA methylome and hydroxymethylome. Changes in DNA methylation during HSC activation may bring new insights into the molecular events underpinning fibrogenesis and may provide biomarkers for disease progression as well as potential new drug targets.

KLF2 exerts antifibrotic and vasoprotective effects in cirrhotic rat livers: behind the molecular mechanisms of statins

ObjectiveIn the liver, the transcription factor, Kruppel-like factor 2 (KLF2), is induced early during progression of cirrhosis to lessen the development of vascular dysfunction; nevertheless, its endogenous expression results insufficient...

Adenoviral overexpression of Lhx2 attenuates cell viability but does not preserve the stem cell like phenotype of hepatic stellate cells

Hepatic stellate cells (HSC) are well known initiators of hepatic fibrosis. After liver cell damage, HSC transdifferentiate into proliferative myofibroblasts, representing the major source of extracellular matrix in the fibrotic organ. Recent studies also demonstrate a role of HSC as progenitor or stem cell like cells in liver regeneration. Lhx2 is described as stem cell maintaining factor in different organs and as an inhibitory transcription factor in HSC activation. Here we examined whether a continuous expression of Lhx2 in HSC could attenuate their activation and whether Lhx2 could serve as a potential target for antifibrotic gene therapy. Therefore, we evaluated an adenoviral mediated overexpression of Lhx2 in primary HSC and investigated mRNA expression patterns by qRT-PCR as well as the activation status by different in vitro assays. HSC revealed a marked increase in activation markers like smooth muscle actin alpha (αSMA) and collagen 1α independent from adenoviral transduction. Lhx2 overexpression resulted in attenuated cell viability as shown by a slightly hampered migratory and contractile phenotype of HSC. Expression of stem cell factors or signaling components was also unaffected by Lhx2. Summarizing these results, we found no antifibrotic or stem cell maintaining effect of Lhx2 overexpression in primary HSC.

Upregulation of cannabinoid receptor‐1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: role of NADPH oxidase (693.13)

The endocannabinoid system (CS) has been implicated in schistosomiasis‐associated liver fibrosis (SSLF). However, the molecular mechanisms mediating the action of the CS in SSLF are largely unknown. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor (CB) signaling via activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). A SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin. HSCs from uninfected (control) or Schistosoma J.‐infected (SSLF) mice were isolated, cultured and identified by analyzing HSC markers α‐SMA and desmin. In HSCs isolated from control or SSLF mice, both CB1 and CB2 receptors were detected, however, only the CB1 significantly increased in HSCs from SSLF mice compared to that control mice. Such increase of CB1 in HSCs was accompanied by enhanced expression of fibrotic markers α‐SMA, collagen I, and TIMP‐1. Similarly, CB1 upregulation and enhanced fibrotic changes were observed in HSCs isolated from control mice when these cells were treated with soluble egg antigen (SEA) from Schistosoma J. Interestingly, HSCs from SSLF mice or SEA‐treated HSCs from control mice exhibited enhanced superoxide (O2.‐) production as analyzed by electron spin resonance. Importantly, inhibition of O2.‐ production by knocking down NADPH oxidase genes (Nox1 or Nox4) prevented SEA‐induced upregulation of CB1 and fibrotic markers in HSCs. Further, CB1 gene silencing blocked SEA‐induced fibrotic changes in HSCs but had no effect on SEA‐induced O2.‐ production in these cells indicating a triggering role of O2.‐ production in CB1‐mediated fibrotic signaling. Taken together, these data suggest that the fibrotic activation of HSCs upon Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase‐mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316

Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J. infection: Role of NADPH oxidase

The endocannabinoid system (CS) has been implicated in the development of hepatic fibrosis such as schistosomiasis-associated liver fibrosis (SSLF). However, the mechanisms mediating the action of the CS in hepatic fibrosis are unclear. The present study hypothesized that Schistosoma J. infection upregulates cannabinoid receptor 1 (CB1) due to activation of NADPH oxidase leading to a fibrotic phenotype in hepatic stellate cells (HSCs). The SSLF model was developed by infecting mice with Schistosoma J. cercariae in the skin, and HSCs from control and infected mice were then isolated, cultured, and confirmed by analysis of HSC markers α-SMA and desmin. CB1 significantly increased in HSCs isolated from mice with SSLF, which was accompanied by a greater expression of fibrotic markers α-SMA, collagen I, and TIMP-1. CB1 upregulation and enhanced fibrotic changes were also observed in normal HSCs treated with soluble egg antigen (SEA) from Schistosoma J. Electron spin resonance (ESR) analysis further demonstrated that superoxide (O2−) production was increased in infected HSCs or normal HSCs stimulated with SEA. Both Nox4 and Nox1 siRNA prevented SEA-induced upregulation of CB1, α-SMA, collagen I, and TIMP-1 by inhibition of O2− production, while CB1 siRNA blocked SEA-induced fibrotic changes without effect on O2− production in these HSCs. Taken together, these data suggest that the fibrotic activation of HSCs on Schistosoma J. infection or SEA stimulation is associated with NADPH oxidase-mediated redox regulation of CB1 expression, which may be a triggering mechanism for SSLF.

Cellular senescence and chronic inflammation

It has recently become apparent that obesity is associated with chronic inflammation and several common types of cancer development. Although several events were proposed to be involved in these pathologies, the precise mechanisms underlying obesity-associated inflammation and cancer largely remain unclear. Here, we show that senescence-associated secretory phenotype (SASP) plays crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of a bacterial metabolite that cause DNA damage. The enterohepatic circulation of the bacterial metabolites provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn, secretes various inflammatory and tumour promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Importantly, reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the induction of SASP by the gut bacterial metabolite in HSCs plays key roles in obesity-associated HCC development. Interestingly, moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with nonalcoholic steatohepatitis (NASH), implying that a similar pathway may contribute to at least certain aspects of obesity-associated HCC development in humans as well. These findings provide valuable new insights into the development of obesity-associated cancer.

Caffeic acid phenethyl ester attenuates pro-inflammatory and fibrogenic phenotypes of LPS-stimulated hepatic stellate cells through the inhibition of NF-κB signaling

Hepatic stellate cells (HSCs) are the major cell type involved in liver fibrosis. Lipopolysaccharide (LPS)-mediated signaling through Τoll-like receptor 4 (TLR4) in HSCs has been identified as a key event in liver fibrosis, and as the molecular link between inflammation and liver fibrosis. In this study, we investigated the effects of caffeic acid phenethyl ester (CAPE), one of the main medicinal components of propolis, on the pro-inflammatory and fibrogenic phenotypes of LPS-stimulated HSCs. HSCs from rats were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM). Following treatment with LPS, HSCs showed a strong pro-inflammatory phenotype with an upregulation of pro-inflammatory mediators, and a fibrogenic phenotype with enhanced collagen synthesis, mediated by transforming growth factor-β1 (TGF-β1). CAPE significantly and dose-dependently reduced LPS-induced nitrite production, as well as the transcription and protein synthesis of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting and enzyme-linked immunosorbent assays (ELISA). CAPE further reduced the TGF-β1-induced transcription and translation (protein synthesis) of the gene coding for collagen type I α1 (col1A1), in LPS-stimulated HSCs. Following LPS stimulation, the phosphorylation of the nuclear factor-κB (NF-κB) inhibitor IκBα and consequently, the nuclear translocation of NF-κB, were markedly increased in the HSCs, and these changes were reversed by pre-treatment with CAPE. In conclusion, CAPE attenuates the pro-inflammatory phenotype of LPS-stimulated HSCs, as well as the LPS-induced sensitization of HSCs to fibrogenic cytokines by inhibiting NF-κB signaling. Our results provide new insight into the treatment of hepatic fibrosis through regulation of the TLR4 signaling pathway.

Fructose-1,6-bisphosphate induces phenotypic reversion of activated hepatic stellate cell

Hepatic stellate cells (HSC) play a key role in liver fibrogenesis. Activation of PPARγ and inhibition of fibrogenic molecules are potential strategies to block HSC activation and differentiation. Aware that the process of hepatic fibrosis involves inflammatory mediators, various anti-inflammatory substances have been studied in an attempt to revert fibrosis. The purpose of this study was to investigate the in vitro effects of fructose-1,6-bisphosphate (FBP) on HSC phenotype reversion. The results demonstrated that FBP induced quiescent phenotype in GRX cells via PPARγ activation. Significant decrease in type I collagen mRNA expression was observed in the first 24h of treatment. These events preceded the reduction of TGF-β1 and total collagen secretion. Thus, FBP promoted downregulation of HSC activation by its antifibrotic action. These findings demonstrate that FBP may have potential as a novel therapeutic agent for the treatment of liver fibrosis.

The effect of lipid metabolism on biological characteristics of hepatic stellate cell

Hepatic stellate cells(HSCs) are a kind of fat-storing cells, the lipid droplets are rich in the Cytoplasm, in which retinyl ester accounts for 42%, triglyceride occupies 28%, cholesterol (total) occupies 13%, phospholipids occupies 4% respectively. Studies have confirmed that thetransforms of HSC phenotype follows the changing of the cell lipid. After the activation of HSC, with HSC phenotype changing from fat-storing cells into myofibroblast, the lipid droplets decreased or disappeared gradually, which means HSCs are under the differentiating process of removing adipose, meawhile triglyceride, and the main content of lipid droplets, also obviously reduced. It was ever declined that during the process of HSC re-fating, the activated HSC would turn into quiescent state. Therefore this shows HSCs fat metabolism is closely related to the biological activity.

Fsp27 gene inhibits the proliferation and activation of hepatic stellate cells in vitro

Objective To investigate the Fsp27 gene's influence on the regulation of hepatic stellate cells (HSCs) in vitro.Methods HSCs were isolated from rat liver,the Fsp27 gene was detected in primary HSCs,and activated HSCs were detected by RT-qPCR.After 72 h of Fsp27 transduction through a lentivirus expressing Fsp27 (pLV-Fsp27),the proliferation of HSCs was tested by the CCK-8 test kit,smooth muscle α-actin (α-SMA) expression of HSCs was tested by Western blot,and the fibrosis-related genes were tested by RT-qPCR.Results The HSCs were isolated and cultured successfully,and the Fsp27 genetic difference between primary and activated HSCs was significant (P＜0.01).After coculture for 72 h,Fsp27 inhibited the proliferation and activation of HSCs (P＜0.05).Fsp27 can enhance expression of the MMP-2 gene and down-regulate expression of the TIMP-1 and TGF-β1 gene in activated HSCs (P＜0.05).Conclusion The Fsp27 gene can inhibit the proliferation and activation of HSCs,regulate the expression of fibrosis-related genes,and may play an important role in maintaining the quiescent phenotype of HSCs. Key words: Fat-specific protein 27; Genes; Hepatic stellate cells; Proliferation; Activation

3,3′‐Diindolylmethane ameliorates experimental hepatic fibrosis via inhibiting miR‐21 expression

Hepatic fibrosis is a type of liver disease characterized by excessive collagen deposition produced by activated hepatic stellate cells (HSCs), and no appropriate drug treatment is available clinically. The microRNA, miR-21 exhibits an important role in the pathogenesis and progression of hepatic fibrosis. 3,3'-Diindolylmethane (DIM) is a natural autolytic product in plants and can down-regulate miR-21 expression. Here we have assessed the therapeutic effects of DIM against hepatic fibrosis and investigated the underlying mechanisms. The effects of DIM on HSC activation were measured by analysing the expression of α-smooth muscle actin and collagen I in both HSC-T6 cell line and primary HSCs. Expression of miR-21 was also measured after DIM treatment and the therapeutic effect of DIM was further studied in vivo, using the model of hepatic fibrosis induced by thioacetamide in mice. The antagonist oligonucleotide, antagomir-21, was also used to suppress the effects of miR-21. DIM suppressed the central TGF-β signalling pathway underlying HSC activation by down-regulating the expression of miR-21. The decreased miR-21 expression was achieved by inhibiting the activity of the transcription factor, AP-1. Moreover, DIM blunted the activation phenotype of primary HSCs. Administration of DIM in vivo attenuated liver fibrosis induced by thioacetamide, as assessed by collagen deposition and profiles of profibrogenic markers. DIM shows potential as a therapeutic agent for the treatment of hepatic fibrosis.

Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome

Obesity has become more prevalent in most developed countries over the past few decades, and is increasingly recognized as a major risk factor for several common types of cancer. As the worldwide obesity epidemic has shown no signs of abating, better understanding of the mechanisms underlying obesity-associated cancer is urgently needed. Although several events were proposed to be involved in obesity-associated cancer, the exact molecular mechanisms that integrate these events have remained largely unclear. Here we show that senescence-associated secretory phenotype (SASP) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage. The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn secretes various inflammatory and tumour-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the DCA-SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis, indicating that a similar pathway may contribute to at least certain aspects of obesity-associated HCC development in humans as well. These findings provide valuable new insights into the development of obesity-associated cancer and open up new possibilities for its control.

Knockdown of N-Acetylglucosaminyl Transferase V Ameliorates Hepatotoxin-Induced Liver Fibrosis in Mice

Aberrant N-glycosylation caused by altered N-acetyl glucosaminyltransferase V (GnT-V) expression is known to regulate tumor invasion and metastasis by modulating multiple cytokine signaling pathways. However, the exact role of GnT-V in the development of liver fibrosis has not been clearly defined. Here, we induced mouse liver fibrosis by ip injections of carbon tetrachloride (CCl4) or thioacetamide (TAA) and observed significant increase of hepatic GnT-V during the processes of liver fibrogenesis. Meanwhile, upregulations of GnT-V were detected in the activated hepatic stellate cells (HSCs) and injured hepatocytes. To knock down hepatic GnT-V expression, adenovirus that expressed the GnT-V siRNA was injected via the tail vein. Adenovirus-mediated delivery of GnT-V siRNA dramatically reduced the GnT-V expression in fibrotic liver and activated HSC in vivo and consequently alleviated CCl4- or TAA-induced liver fibrosis as assessed through collagen deposition and profiles of profibrogenic markers. Furthermore, knockdown of GnT-V in HSCs reduced transforming growth factor beta (TGF-β)/Smad signaling and blunted the activated HSC phenotype. The suppression of TGF-β/Smad signaling in HSCs correlated with the decrease of GnT-V-modified β1,6-branched N-glycan on TGF-β receptors. Knockdown of GnT-V also suppressed platelet-derived growth factor (PDGF)-induced HSC proliferation and migration through inhibiting PDGF/Erk signaling. Finally, we demonstrated that knockdown of GnT-V profoundly suppressed TGF-β1-induced epithelial-mesenchymal transition (EMT) in hepatocytes by morphological assessment and reversal of EMT markers. In conclusion, this study demonstrates that GnT-V is implicated in hepatotoxin-induced liver fibrosis, and targeting GnT-V may be a feasible and promising approach for treating liver fibrosis.

Resistin mediates the hepatic stellate cell phenotype

To describe the role of resistin in liver fibrosis. For the in vivo animal study, Sprague Dawley rats were subjected to bile duct ligation (BDL) for 4 wk. Rat liver, adipose tissue (epididymal fat) and serum were analyzed for resistin expression. For the in vitro experiment, rat primary hepatic stellate cells (HSCs) and Kupffer cells (KCs) were used. HSCs were exposed to recombinant resistin, and collagen I, transforming growth factor β1, α smooth muscle actin, tissue inhibitor of metalloproteinase 1 and connective tissue growth factor expression were analyzed. Resistin gene and protein expression was quantified as was the expression of pro-inflammatory cytokines including tumor necrosis factor α (TNFα), interleukin (IL)-1, IL-6, IL-8 and monocyte chemotactic protein-1 (MCP-1). The effects of resistin on HSC proliferation, migration and apoptosis were determined. The effects of resistin on KCs were also investigated. Following BDL, rat epididymal fat and serum rather than liver showed higher resistin expression compared to control rats. In liver, resistin was expressed in quiescent HSCs and KCs. Resistin treatment resulted in enhancement of TNFα, IL-6, IL-8 and MCP-1 gene expression and increased IL-6 and MCP-1 protein in HSCs. Resistin activated HSC phospho-MAPK/p38, and p38 inhibition diminished IL-6 and MCP-1 expression. Furthermore, resistin facilitated HSC proliferation and migration, but decreased apoptosis which was via an IL-6 and MCP-1 mechanism. Finally, resistin-induced transforming growth factor β1 from KCs enhanced HSC collagen I expression. Resistin directly and indirectly modulates HSC behavior towards a more pro-fibrogenic phenotype.

Selective activation of Toll-like receptor 7 in activated hepatic stellate cells may modulate their profibrogenic phenotype

Cholestatic liver injury may activate HSCs (hepatic stellate cells) to a profibrogenic phenotype, contributing to liver fibrogenesis. We have previously demonstrated the involvement of TLR (Toll-like receptor) 7 in the pathogenesis of biliary atresia. In the present study we investigated the ability of TLR7 to modulate the profibrogenic phenotype in HSCs. Obstructive jaundice was associated with significant down-regulation of TLR7. Primary HSCs isolated from BDL (bile duct ligation) rats with obstructive jaundice exhibited reduced expression of TLR7 and increased expression of α-SMA (α-smooth muscle actin) and collagen-α1 compared with sham rats, reflecting HSC-mediated changes. Treatment of primary activated rat HSCs and rat T6 cells with CL075, a TLR7 and TLR8 ligand, significantly decreased expression of MCP-1 (monocyte chemotactic protein-1), TGF-β1 (transforming growth factor-β1), collagen-α1 and MMP-2 (matrix metalloproteinase-2), and inhibited cell proliferation and migration. In contrast, silencing TLR7 expression with shRNA (short hairpin RNA) in T6 cells effectively blocked the effects of CL075 stimulation, reversing the changes in MCP-1, TGF-β1 and collagen-α1 expression and accelerating cell migration. Our results indicate that obstructive jaundice is associated with down-regulation of TLR7 and up-regulation of profibrogenic gene expression in HSCs. Selective activation of TLR7 may modulate the profibrogenic phenotype in activated HSCs associated with cholestatic liver injury.

Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-β). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-β signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-β signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-β signaling components demonstrated by decreased TGF-β receptor II (TGF-βRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3' untranslated region of TGF-βRII was validated by luciferase reporter assay. Inhibition of TGF-β signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-β-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle α-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers. miR 19b is a novel regulator of TGF-β signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis.

Functional aspects on the pathophysiology of portal hypertension in cirrhosis

Hepatic Hemodynamic Laboratory, Liver Unit, IMDIM, Hospital Clinic, Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS)and CIBERehd, SpainPortal hypertension is a severe and frequent complication ofchronic liver disease. Its consequences, bleeding from gastro-esophageal varices and portal hypertensive gastropathy, ascites,spontaneous bacterial peritonitis, hepatorenal syndrome, hepato-pulmonary/portopulmonary syndromes, and hepatic encephalop-athy, represent the first cause of death and liver transplantationin patients with cirrhosis. The primary factor in the developmentof portal hypertension is a marked increase in hepatic vascularresistance (HVR) to portal blood flow, which was classicallyattributed to distortion of the liver architecture inherent to cir-rhosis. However, over the past 20 years, a better understandingof the liver microcirculation has demonstrated that a dynamiccomponent due to an increased hepatic vascular tone furthercontributes to augment HVR. Secondarily to the increased HVR,there is a progressive splanchnic vasodilatation that incrementsportal blood flow, which aggravates and perpetuates the portalhypertension syndrome [1] (Fig. 1).Intrahepatic vascular regulationThe analysis of the structural factors (fibrosis, vascular remodel-ing, vascular occlusion, nodule formation) contributing toincrease HVR is out of the scope of this review that will focuson the dynamic component of the increased HVR.During progression to cirrhosis, sinusoidal endothelial cells(SEC) become dysfunctional and among other features acquire avasoconstrictor phenotype, characterized by elevated productionof vasoconstrictors and reduced release of vasodilators. Theresulting imbalance promotes the contraction of different cellsof the cirrhotic liver, such as hepatic stellate cells (HSC), portalmyofibroblasts and vascular smooth muscle cells that lead toincreased hepatic vascular tone and portal pressure. HSC in turnalso experience a profound phenotypical transformation, withmorphologic and functional consequences: loss of vitamin Adroplets, alpha-smooth muscle actin overexpression, hyper-response to vasoconstrictors and enhanced proliferative and fibr-ogenic activity. Interestingly, in experimental models of cirrhosis,strategies aimed at improving the hepatic vascular tone by tar-geting SEC have also been shown to improve liver fibrosis. Thismay be due to the fact that cellular phenotype alterations sharepathophysiological mechanisms, but also because SEC phenotypeamelioration positively affects HSC phenotype [2].Increase of vasoconstrictors in the liverIncreasedactivityof several endogenous vasoconstrictors,such asendothelin, norepinephrine, angiotensin II, vasopressin, leukotri-enes, and thromboxane A

Ablation of stellate cells during liver regeneration blocks mitosis and induces a switch in stellate cell phenotype

Hepatic stellate cells (HSCs) synthesize new hepatocyte growth factor (HGF) during liver regeneration (LR), and also play an important function in matrix production; thus, we hypothesize that HSCs are the master regulators of LR. Rats were injected with either gliotoxin (which induces HSC apoptosis) or DMSO one day before and simultaneous with partial hepatectomy (PH). There were more apoptotic cells in the gliotoxin livers as compared to DMSO livers, as assessed by TUNEL staining. Ki67 staining show the presence of many proliferating cells in the control, while in the gliotoxin‐treated rats, mitosis was absent. Although HGF mRNA expression was unchanged, HGF protein expression was significantly decreased in the gliotoxin livers. Chronic elimination of stellate cells over a period of 5 days after PH also causes decreased proliferation and increased apoptosis. Intriguingly, this treatment induced a switch in HSC phenotype, with higher expression of alpha‐SMA in control rats compared to gliotoxin rats, which had more desmin. Finally, there was a paradoxical increase in both hepatocyte proliferation and apoptosis when HSCs were ablated during the later stages of LR. Thus, gliotoxin treatment at early time points after PH causes a block in proliferation and an increase in apoptosis, while treatment during the latter stages of LR causes a phenotypic change in HSC marker expression and an increase in proliferation.

When GLP-1 hits the liver: a novel approach for insulin resistance and NASH

nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum ranging from simple steatosis to steatohepatitis (NASH), increasing fibrosis and eventually, cirrhosis ([22][1]). Importantly, NASH accompanied by fibrosis and severe inflammation is the most relevant predictor for disease progression