Direct analysis of hepatic stellate cells with flow cytometry in specimens derived from the human liver.

Gene Expression Profiles During Hepatic Stellate Cell Activation in Culture and In Vivo

Fibrogenic Signaling Is Suppressed in Hepatic Stellate Cells through Targeting of Connective Tissue Growth Factor (CCN2) by Cellular or Exosomal MicroRNA-199a-5p

TLR4 signaling induces down-regulation of the bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI), which enhances TGF-β signaling during hepatic stellate cell (HSC) activation. We investigated the mechanism by which TLR4 signaling down-regulates BAMBI expression in HSCs and found that TLR4- and TNF-α-mediated BAMBI down-regulation is dependent on regulation of BAMBI promoter activity through the interaction with NF-κBp50 and HDAC1 in HSCs. Bambi was predominantly expressed in HSCs, at high levels in quiescent HSCs but at low levels in in vivo-activated and LPS-stimulated HSCs. In human HSCs, BAMBI expression was down-regulated in response to LPS and TNF-α. A BAMBI reporter assay demonstrated that the regulatory element to repress BAMBI transcription is located between 3384 and 1560 bp upstream from the transcription start site. LPS stimulation down-regulated BAMBI expression in cells with NF-κBp65 knockdown. However, it failed to down-regulate BAMBI in cells with inactivation of NF-κB or NF-κBp50 silencing, indicating that NF-κBp50 is a factor for BAMBI down-regulation. ChIP analysis revealed that LPS and TNF-α induced binding of the NF-κBp50/p50 homodimer to the BAMBI promoter region. We also found that HDAC1 is bound to this region as part of the NF-κBp50-HDAC1 complex, repressing transcriptional activity of the BAMBI promoter. Finally, we confirmed that LPS does not repress BAMBI reporter activity using a BAMBI reporter construct with a mutation at 3166 bp upstream of the coding region. In summary, our study demonstrates that LPS- and TNF-α-induced NF-κBp50-HDAC1 interaction represses BAMBI transcriptional activity, which contributes to TLR4-mediated enhancement of TGF-β signaling in HSCs during liver fibrosis.

Hepatic stellate cells (HSCs), vitamin A-storing liver pericytes, undergo myofibroblastic trans-differentiation or "activation" to participate in liver wound healing. This cellular process involves loss of regulation by adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ). Necdin, a melanoma antigen family protein, promotes neuronal and myogenic differentiation while inhibiting adipogenesis. The present study demonstrates that necdin is selectively expressed in HSCs among different liver cell types and induced during their activation in vitro and in vivo. Silencing of necdin with adenovirally expressed shRNA, reverses activated HSCs to quiescent cells in a manner dependent on PPARγ and suppressed canonical Wnt signaling. Promoter analysis, site-directed mutagenesis, and chromatin immunoprecipitation demonstrate that Wnt10b, a canonical Wnt induced in activated HSCs, is a direct target of necdin. Necdin silencing abrogates three epigenetic signatures implicated in repression of PPARγ: increased MeCP2 (methyl CpG binding protein 2) and HP-1α co-repressor recruitments to Pparγ promoter and enhanced H3K27 dimethylation at the exon 5 locus, again in a manner dependent on suppressed canonical Wnt. These epigenetic effects are reproduced by antagonism of canonical Wnt signaling with Dikkopf-1. Our results demonstrate a novel necdin-Wnt pathway, which serves to mediate antiadipogenic HSC trans-differentiation via epigenetic repression of PPARγ.

Non-heat-stressed Method to Isolate Hepatic Stellate Cells From Highly Steatotic Tumor-bearing Liver Using CD49a.

Upon liver injury, excessive deposition of collagen from activated hepatic stellate cells (HSCs) is a leading cause of liver fibrosis. An understanding of the mechanism by which collagen biosynthesis is regulated in HSCs will provide important clues for practical anti-fibrotic therapy. Endoplasmic reticulum oxidase 1α (ERO1α) functions as an oxidative enzyme of protein disulfide isomerase, which forms intramolecular disulfide bonds of membrane and secreted proteins. However, the role of ERO1α in HSCs remains unclear. Here, we show that ERO1α is expressed and mainly localized in the endoplasmic reticulum in human HSCs. When HSCs were transfected with ERO1α siRNA or an ERO1α shRNA-expressing plasmid, expression of ERO1α was completely silenced. Silencing of ERO1α expression in HSCs markedly suppressed their proliferation but did not induce apoptosis, which was accompanied by impaired secretion of collagen type 1. Silencing of ERO1α expression induced impaired disulfide bond formation and inhibited autophagy via activation of the Akt/mammalian target of rapamycin signaling pathway, resulting in intracellular accumulation of collagen type 1 in HSCs. Furthermore, silencing of ERO1α expression also promoted proteasome-dependent degradation of membrane type 1-matrix metalloproteinase (MT1-MMP), which stimulates cell proliferation through cleavage of secreted collagens. The inhibition of HSC proliferation was reversed by treatment with MT1-MMP-cleaved collagen type 1. The results suggest that ERO1α plays a crucial role in HSC proliferation via posttranslational modification of collagen and MT1-MMP and, therefore, may be a suitable therapeutic target for managing liver fibrosis.

The semisynthetic plant alkaloid halofuginone (HAL) was reported to prevent and partly reverse experimental liver fibrosis. However, its mechanisms of action are poorly understood. We therefore aimed to determine the antifibrotic potential of HAL and to characterize involved signal transduction pathways in hepatic stellate cells (HSCs). Results were compared with its in vivo effects in a rat model of reversal of established liver fibrosis induced by thioacetamide. In vitro HAL inhibited HSC proliferation and migration dose dependently at submicromolar concentrations. HAL (200 nm) up-regulated matrix metalloproteinase (MMP)-3 and MMP-13 expression between 10- and 50-fold, resulting in a 2- to 3-fold increase of interstitial collagenase activity. Procollagen alpha1(I) and MMP-2 transcript levels were suppressed 2- to 3-fold, whereas expression of other profibrogenic mRNAs remained unaffected. p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor kappaB(NFkappaB) pathways were activated by HAL, and specific inhibitors of p38 MAPK and NFkappaB dose dependently inhibited MMP-13 induction. Treatment with HAL did not affect HSC viability, and observed effects were reversible after its removal. In vivo HAL up-regulated MMP-3 and -13 mRNA expression 1.5- and 2-fold, respectively, in cirrhotic rats, whereas tissue inhibitor of metalloproteinase-1 was suppressed by 50%. In conclusion, submicromolar concentrations of HAL inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NFkappaB. The remarkable induction of MMP-3 and -13 makes HAL a promising agent for antifibrotic combination therapies.

Dysregulated activation of hepatic stellate cells (HSCs) is a critical event in the development of liver fibrosis/cirrhosis. We previously reported that astaxanthin (ASTX), a xanthophyll carotenoid, prevents the activation of HSCs and facilitates the reversal of activated HSCs to a quiescent phenotype using primary HSCs from C57BL/6J mice. We further evaluated the underlying mechanismsand their human relevance. As histone deacetylases (HDACs) are known to play important roles in HSC activation, we examined changes in the expression of 11 classical HDAC isoforms and the effect of ASTX on their expression during HSC activation. HDAC9 mRNA and protein were significantly higher in activated HSCs than quiescent HSCs. The presence of ASTX during HSC activation attenuated the induction by ~70-80%. The expression of myocyte enhancer factor 2 (MEF2), a known transcriptional activator of HDAC9, was induced by HSC activation, but diminished by ASTX. Activated HSCs incubated with ASTX displayed a quiescent phenotype with concomitant decreases in HDAC9 and MEF2 expression. In the livers of patients with primary biliary cirrhosis, HDAC9 expression was also significantly higher than in normal livers. Taken together, the inhibitory effect of ASTX on HSC activation is attributable at least partly to the repression of MEF2-mediated expression of HDAC9. The findings suggest that ASTX may be developed as a potential preventive/therapeutic agent for liver fibrosis/cirrhosis. (Funded by USDA AFRI 2012-67018-19290 and Multi-state Hatch)

Early activation of hepatic stellate cells induces rapid initiation of retinyl ester breakdown while maintaining lecithin:retinol acyltransferase (LRAT) activity

To identify and characterize the function of nonmuscle myosin II (NMM II) isoforms in primary rat hepatic stellate cells (HSCs). Primary HSCs were isolated from male Sprague-Dawley rats by pronase/collagenase digestion. Total RNA and protein were harvested from quiescent and culture-activated HSCs. NMM II isoform (II-A, II-B and II-C) gene and protein expression were measured by RealTime polymerase chain reaction and Western blot analyses respectively. NMM II protein localization was visualized in vitro using immunocytochemical analysis. For in vivo assessment, liver tissue was harvested from bile duct-ligated (BDL) rats and NMM IIisoform expression determined by immunohistochemistry. Using a selective myosin II inhibitor and siRNA-mediated knockdown of each isoform, NMM II functionality in primary rat HSCs was determined by contraction and migration assays. NMM II-A and II-B mRNA expression was increased in culture-activated HSCs (Day 14) with significant increases seen in all pair-wise comparisons (II-A: 12.67 ± 0.99 (quiescent) vs 17.36 ± 0.78 (Day 14), P < 0.05; II-B: 4.94 ± 0.62 (quiescent) vs 13.90 ±0.85 (Day 14), P < 0.001). Protein expression exhibited similar expression patterns (II-A: 1.87 ± 2.50 (quiescent) vs 58.64 ± 8.76 (Day 14), P < 0.05; II-B: 1.17 ± 1.93 (quiescent) vs 103.71 ± 21.73 (Day 14), P < 0.05). No significant differences were observed in NMM II-C mRNA and protein expression between quiescent and activated HSCs. In culture-activated HSCs, NMM II-A and II-B merged with F-actin at the cellular periphery and throughout cytoplasm respectively. In vitro studies showed increased expression of NMM II-B in HSCs activated by BDL compared to sham-operated animals. There were no apparent increases of NMM II-A and II-C protein expression in HSCs during hepatic BDL injury. To determine the contribution of NMM II-A and II-B to migration and contraction, NMM II-A and II-B expression were downregulated with siRNA. NMM II-A and/or II-B siRNA inhibited HSC migration by approximately 25% compared to scramble siRNA-treated cells. Conversely, siRNA-mediated NMM II-A and II-B inhibition had no significant effect on HSC contraction; however, contraction was inhibited with the myosin II inhibitor, blebbistatin (38.7% ± 1.9%). Increased expression of NMM II-A and II-B regulates HSC migration, while other myosin IIclasses likely modulate contraction, contributing to development and severity of liver fibrosis.

Downregulation of the Wnt antagonist Dkk2 links the loss of Sept4 and myofibroblastic transformation of hepatic stellate cells

Oxidative stress: does it 'initiate' hepatic stellate cell activation or only 'perpetuate' the process?

Connective tissue growth factor (CTGF/CCN2) drives hepatic fibrosis in vivo by stimulating fibrogenic pathways in hepatic stellate cells (HSC). Under normal conditions, CTGF/CCN2 expression is very low in quiescent HSC but CTGF/CCN2 expression is dramatically increased as HSC become activated during liver injury and adopt a fibrogenic phenotype. We have investigated the molecular basis for the suppressed CTGF/CCN2 levels in quiescent HSC. CTGF/CCN2 up‐regulation in fibrotic or steatotic livers, or in culture‐activated or ethanol‐treated primary mouse HSC was associated with a reciprocal down‐regulation of microRNA‐214 (miR‐214). By using protector or reporter assays to investigate the activity of the 3’‐untranslated region (UTR) of CTGF/CCN2 mRNA, induction of CTGF/CCN2 expression in HSC by fibrosis‐inducing stimuli was due to reduced expression of miR‐214 which otherwise inhibited CTGF/CCN2 expression by directly binding to the CTGF/CCN2 3’‐UTR. In primary mouse HSC, miR‐214 was co‐expressed with the transcription factor Twist‐1 which is a predicted target of an E box in the miR‐214 promoter. Expression of miR‐214 and Twist‐1 in HSC was dose‐dependently inhibited by ethanol or culture‐induced activation in a manner reciprocal to that of CTGF/CCN2. Transfection of primary D6 HSC (high endogenous Twist‐1 levels) with Twist‐1 siRNA reduced expression of miR‐214, but increased CTGF/CCN2 production. An opposite effect was shown by transfecting P6 HSC (low endogenous Twist‐1 levels) with Twist‐1 expression plasmids. Twist‐1 stimulated luciferase activity in HSC transfected with a wild‐type miR‐214 promoter but not with a mutant miR‐214 promoter lacking the E‐box site. We conclude that CTGF/CCN2 expression in HSC is dynamically regulated by an inhibitory Twist‐1‐miR214 axis. This regulatory mechanism may have utility for therapeutic intervention in pathways of CTGF/CCN2‐dependent fibrosis.Grant Funding Source: NIH 5 R01 AA021276

TGFβ1 in liver fibrosis: time to change paradigms?

Bile acids have been reported to induce epidermal growth factor receptor (EGFR) activation and subsequent proliferation of activated hepatic stellate cells (HSC), but the underlying mechanisms and whether quiescent HSC are also a target for bile acid-induced proliferation or apoptosis remained unclear. Therefore, primary rat HSC were cultured for up to 48 h and analyzed for their proliferative/apoptotic responses toward bile acids. Hydrophobic bile acids, i.e. taurolithocholate 3-sulfate, taurochenodeoxycholate, and glycochenodeoxycholate, but not taurocholate or tauroursodeoxycholate, induced Yes-dependent EGFR phosphorylation. Simultaneously, hydrophobic bile acids induced phosphorylation of the NADPH oxidase subunit p47(phox) and formation of reactive oxygen species (ROS). ROS production was sensitive to inhibition of acidic sphingomyelinase, protein kinase Czeta, and NADPH oxidases. All maneuvers which prevented bile acid-induced ROS formation also prevented Yes and subsequent EGFR phosphorylation. Taurolithocholate 3-sulfate-induced EGFR activation was followed by extracellular signal-regulated kinase 1/2, but not c-Jun N-terminal kinase (JNK) activation, and stimulated HSC proliferation. When, however, a JNK signal was induced by coadministration of cycloheximide or hydrogen peroxide (H2O2), activated EGFR associated with CD95 and triggered EGFR-mediated CD95-tyrosine phosphorylation and subsequent formation of the death-inducing signaling complex. In conclusion, hydrophobic bile acids lead to a NADPH oxidase-driven ROS generation followed by a Yes-mediated EGFR activation in quiescent primary rat HSC. This proliferative signal shifts to an apoptotic signal when a JNK signal simultaneously comes into play.