J. Herrmann

Dominant-negative soluble PDGF- β receptor inhibits hepatic stellate cell activation and attenuates liver fibrosis

Hepatic fibrogenesis is a consequence of hepatic stellate cells that become activated and transdifferentiate into a myofibroblastic phenotype with the ability to proliferate and synthesize large quantities of extracellular matrix components. In this process, platelet-derived growth factor (PDGF) is the most potent stimulus for hepatic stellate cell proliferation and migration, and is overexpressed during active hepatic fibrogenesis. This cytokine binds to the PDGF receptor type β, activates Ras and sequentially propagates the stimulatory signal sequentially via phosphorylation of Raf-1, MEK and the extracellular-signal regulated kinases ERK1/ERK2. Hepatic injury is associated with both increased autocrine PDGF signaling and upregulation of PDGF receptor. In this study, we report that a dominant-negative soluble PDGF-β receptor consisting of a chimeric IgG containing the extracellular portion of the PDGF receptor type β blocks HSC activation and attenuates fibrogenesis induced by ligation of the common bile duct in rats. In culture-activated hepatic stellate cells, the soluble receptor blocks phosphorylation of endogenous PDGF receptor, phosphorylation of the ERK1/EKR2 signal and reduces proliferative activities of HSC. In vivo, both the delivery of the purified soluble PDGF antagonist and the administration of adenoviruses expressing the artificial transgene were able to reduce significantly the expression of collagen and α-smooth muscle actin. Our results demonstrate that PDGF plays a critical role in the progression and initiation of experimental liver fibrogenesis, and suggest that early anti-PDGF intervention should have a therapeutical impact on the treatment of liver fibrogenesis.

Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function?

•  Introduction •  General aspects of immortal HSC •  Immortalized murine HSC lines ‐  GRX ‐  SV68 c‐IS ‐  A640‐IS ‐  M1–4HSC ‐  A7 •  Immortalized rat HSC lines ‐  HSC‐T6 ‐  NFSC, CFSC and derivatives ‐  PAV‐1 ‐  HSC‐PQ ‐  BSC ‐  MFBY2 ‐  T‐HSC/Cl‐6 ‐  Other rat HSC lines •  Immortalized human HSC lines ‐  LI90 and derivatives ‐  GREF‐X ‐  hTERT‐HSC ‐  LX‐1 and LX‐2 ‐  HSC 180 •  Qualities of primary and immortalized HSC ‐  Culture and growth characteristics ‐  Differences in cellular morphology ‐  Differences in gene expression ‐  Most immortal HSC cells are highly transfectable ‐  Response to growth factors •  Conclusions

Activation of TGF‐β within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor

Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV‐family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up‐regulated by exogenous TGF‐β. In this study TGF‐β‐dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF‐β‐free conditions was analysed by Western‐blots, metabolic labelling, and CTGF‐reporter gene assays. In alkaline phosphatase monoclonal anti‐alkaline phosphatase complex (APAAP)‐staining of cultured hepatocytes it was demonstrated that latent TGF‐β within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF‐β antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p‐Smad2, whereas p‐Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF‐β, but calpain inhibitors partially suppressed intracellular TGF‐β activation and subsequently CTGF up‐regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF‐β signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4‐/Alk 5‐specific inhibitor SB‐431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF‐β is activated by calpains during culture‐induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF‐β. In conclusion, these data are the first to suggest the possibility of intracrine TGF‐β signalling due to calpain‐dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF‐β‐sensitive reporter gene. This mechanism might be deleterious for keeping long‐term hepatocyte cultures due to TGF‐β‐induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial‐mesenchymal transition of hepatocytes in injured liver.

CSRP2, TIMP-1, and SM22alpha promoter fragments direct hepatic stellate cell-specific transgene expression in vitro, but not in vivo.

Background/Aims: The activation of hepatic stellate cells (HSC) and their transdifferentiation into myofibroblasts (MFB) is the key step for development of liver fibrosis. Over the past several years, significant progress has been made in the understanding of the critical pathways involved incells undergoing activation. Cellular activation in the course of transdifferentiation involves, among other biochemical modifications, functionally relevant changes in the control of gene expression. These include the up‐regulation of transcription factors, different extracellular matrix proteins, cell adhesion molecules, smooth muscle specific genes, and proteins involved in matrix remodelling, or cytoskeletal organization. The corresponding regulatory elements of these genes have afforded us the opportunity to express transgenes with antifibrotic potential in a cell type‐ and/or transdifferentiation‐dependent manner.

Comparative analysis of adenoviral transgene delivery via tail or portal vein into rat liver.

Summary.The potential indications for gene therapy are expanding continuously. Currently, hepatotropic adenoviruses are useful vector systems for targeting liver in experimental animal models. Although this gene delivery technique is widely distributed, there is no common sense about how these viruses should be applied. In general, the local delivery into portal vein and the systemic application via tail vein induces above all substantial transgene expression. We here comparatively analysed both methods and found that the systemic administration of an adeno-virus expressing the green fluorescent protein resulted in a stronger infiltration, a more homogenous distribution, and a higher inter-individual reproducibility of reporter gene expression in rat liver than organ-specific administration via the portal vein.

Global analysis of host tissue gene expression in the invasive front of colorectal liver metastases

Host cell reactions are a crucial determinant for tumor invasion. We analyzed on a genomewide scale gene expression differences between microdissected tissues taken from unaffected liver tissue of a human colorectal tumor (LS174) growing in the livers of nude mice and tissue from the host part of the invasive front. Due to the low degree of interspecies cross‐hybridization of 15% as determined on Affymetrix microarrays, our xenograft model allowed for the distinction of genes of murine versus human origin even if the respective tissues could not be isolated separately. Using the gene ontology (GO) classification, we were able to determine patterns of up‐ and downregulated genes in the liver part of the invasive front. We observed a pronounced overrepresentation, e.g., of the GO terms “extracellular matrix,” “cell communication,” “response to biotic stimulus,” “structural molecule activity” and “cell growth,” indicating a very pronounced host cell response to tumor invasion. On the single gene level, hepatic stellate cell (HSC) activation markers were overrepresented in the liver part of the invasion front. Immunohistochemistry and qPCR confirmed an activation of HSC as well as an increased number of HSC in the invasive front as compared to the noninvaded liver tissue. In summary, our data demonstrate the feasibility of an interspecies differential gene expression approach on a genomewide scale.