Krista Rombouts

Expression of somatostatin receptors in normal and cirrhotic human liver and in hepatocellular carcinoma

Background: Somatostatin analogues have been used with conflicting results to treat advanced hepatocellular carcinoma (HCC). The aim of this study was to investigate expression of somatostatin receptor (SSTR) subtypes in human liver, and to examine the effect of selective SSTR agonists on proliferation, apoptosis, and migration of hepatoma cells (HepG2, HuH7) and hepatic stellate cells (HSCs). Methods: Expression of SSTRs in cell lines, normal and cirrhotic liver, and HCC was examined by immunohistochemistry and reverse transcription-polymerase chain reaction. Effects of SSTR agonists on proliferation and apoptosis of tumour cells and HSCs were assessed by the 5-bromo-2′ deoxyuridine and TUNEL methods, respectively. The influence of SSTR agonists on migration was investigated using Boyden chambers. Results: In normal liver, both hepatocytes and HSCs were negative for all five SSTRs. Cirrhotic liver and HCC as well as cultured hepatoma cells and HSCs expressed all five SSTRs, both at the protein and mRNA levels, except for HuH7 cells which did not immunoreact with SSTR3. None of the agonists influenced proliferation or apoptosis. However, compared with untreated cells, L-797,591, an SSTR1 agonist, reduced migration of HepG2, HuH7, and HSCs significantly to 88 (7)% (p<0.05), 83 (11)% (p<0.05), and 67 (13)% (p<0.01), respectively. Conclusions: Cirrhotic liver and HCC express SSTRs. Although the somatostatin analogues used in this study did not affect proliferation and apoptosis, stimulation of SSTR1 may decrease invasiveness of HCC by reducing migration of hepatoma cells and/or HSCs. Clinical trials evaluating somatostatin analogues for the treatment of HCC should take these findings into account.

All-trans and 9-cis retinoic acid alter rat hepatic stellate cell phenotype differentially.

BACKGROUND Hepatic stellate cells exert specific functions in the liver: storage of large amounts of retinyl esters, synthesis and breakdown of hepatic extracellular matrix, secretion of a variety of cytokines, and control of the diameter of the sinusoids. AIMS To examine the influence of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9RA) on extracellular matrix production and proliferation of activated hepatic stellate cells. METHODS Cells were isolated using collagenase/pronase, purified by centrifugation in nycodenz, and cultured for two weeks. At this time point the cells exhibited the activated phenotype. Cells were exposed to various concentrations of ATRA and 9RA. The expression of procollagens I, III, and IV, of fibronectin and of laminin were analysed by immunoprecipitation and northern hybridisation. RESULTS ATRA exerted a significant inhibitory effect on the synthesis of procollagens type I, III, and IV, fibronectin, and laminin, but did not influence stellate cell proliferation, whereas 9RA showed a clear but late effect on proliferation. 9RA increased procollagen I mRNA 1.9-fold, but did not affect the expression of other matrix proteins. CONCLUSION Results showed that ATRA and 9RA exert different, often contrary effects on activated stellate cells. These observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or in animals subjected to fibrogenic stimuli.

Actin filament formation, reorganization and migration are impaired in hepatic stellate cells under influence of trichostatin A, a histone deacetylase inhibitor

BACKGROUND/AIMS Previously, trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to exhibit strong antifibrotic characteristics in hepatic stellate cells (HSC), which are known to play a central role in chronic liver diseases. TSA retained a more quiescent phenotype in spite of culture conditions that favor transdifferentiation into activated HSC. METHODS To identify TSA-sensitive genes, differential mRNA display, Northern and Western blot analysis were used and genes were functionally validated by using contraction and motility assays. RESULTS TSA prevented new actin filament formation by down-regulation of two nucleating proteins, actin related protein 2 (Arp2) and Arp3, and by up-regulation of adducin like protein 70 (ADDL70) and gelsolin, two capping proteins. RhoA, a key mediator in the development of the actin cytoskeleton, decreased following TSA exposure. Expression of proteins of Class III intermediate filaments was affected by TSA. Furthermore, F-actin and G-actin were expressed heterogeneously under influence of TSA. Functionally, TSA treatment abrogated migration of quiescent HSC, while migration was reduced in transitional HSC. The endothelin-1-induced contractility properties of HSC was not affected by TSA. CONCLUSIONS These data indicate that TSA affects the development of the actin cytoskeleton in quiescent HSC and thereby abrogates the process of HSC transdifferentiation.

Influence of aldosterone on collagen synthesis and proliferation of rat cardiac fibroblasts.

Previous in vivo studies in men and experimental animal models have shown that hyperaldosteronemia is correlated with cardiac fibrosis due to increased total collagen synthesis. As yet, it is unclear whether aldosterone has direct pro‐fibrogenic effect on cardiac fibroblasts, the fibrogenic effector cell in the myocardium, and if so which procollagens specifically are synthesized at higher rates. The present study aims at establishing whether de novo collagen synthesis by cardiac fibroblasts is enhanced following exposure for 2×24 h to pharmacological (10−7 – 10−8 M), near‐physiological (10−9 M) or physiological (10−10 – 10−11 M) aldosterone concentrations. During the last 24 h, cells were metabolically labelled with [35S]‐methionine/[35S]‐cysteine. Labelled procollagens were immunoprecipitated quantitatively using antibodies against specific procollagens. Contrary to expectations, 10−7 M aldosterone inhibited significantly de novo synthesis of procollagens type I and IV (−35% and −42%, respectively). For procollagen type III, only a tendency towards inhibition was observed. At lower concentrations of aldosterone (10−8 – 10−10 M), synthesis of procollagens type I, III or IV was unaffected. Cellular DNA synthesis under influence of aldosterone was evaluated by measuring BrdU incorporation. Cells were treated with aldosterone, while BrdU was added during the last 16 h of treatment. Aldosterone had no demonstrable effect on cellular proliferation. Reverse transcription‐polymerase chain reaction (RT – PCR) clearly demonstrated the presence of mineralocorticoid receptor mRNA in cardiac fibroblasts. In spite of the expression of the mineralocorticoid receptor by cultured cardiac fibroblasts, the pro‐fibrogenic effect of aldosterone as observed in vivo, is not likely to be due to a direct effect of this hormone in cardiac fibroblasts.

Effect of aldosterone on collagen steady state levels in primary and subcultured rat hepatic stellate cells

BACKGROUND/AIMS Activation of the renin-angiotensin-aldosterone system can lead to collagen accumulation and reactive myocardial fibrosis. This study aims at evaluating the effect of aldosterone on extracellular matrix synthesis by rat hepatic stellate cells. METHODS Cultured cells were treated with different concentrations of aldosterone (10(-6)-10(-10) M) and metabolically labeled with 35S-methionine/35S-cysteine. Procollagen types I, III and IV, laminin and fibronectin were specifically immunoprecipitated and quantified by phosphor imaging. Using the reverse transcription-polymerase chain reaction, we investigated the expression of the mineralocorticoid receptor in hepatic stellate cells. RESULTS Quantitation showed that 10(-6) M aldosterone induced procollagen type I synthesis significantly, whereas procollagen type IV expression was significantly affected by 10(-9) and 10(-10) M aldosterone, both in primary hepatic stellate cells. RT-PCR experiments clearly demonstrated a lack of expression of the mineralocorticoid receptor in hepatic stellate cells. CONCLUSION We demonstrated that aldosterone altered moderately procollagen type I and IV synthesis by primary hepatic stellate cells, but not by activated stellate cells which are the principal cellular sources of extracellular matrix proteins in chronic liver disease. Moreover, hepatic stellate cells do not express the mineralocorticoid receptor, suggesting that the observed modest changes of extracellular matrix synthesis are probably due to mineralocorticoid receptor unrelated mechanisms.

Somatostatin at nanomolar concentration reduces collagen I and III synthesis by, but not proliferation of activated rat hepatic stellate cells

Previous studies have shown antifibrotic effects of somatostatin. Since hepatic stellate cells (HSC) express somatostatin receptors and play a key role in hepatic fibrogenesis, we investigated the in vitro antifibrotic effect of somatostatin on rat HSC. At day 12 after isolation, cells were exposed to different concentrations of somatostatin (10−6–10−9 mol l−1). mRNA expression of collagen types I and III, and of smooth muscle α‐actin (α‐SMA) was analysed by Northern blotting. At 10−9 mol l−1, somatostatin significantly reduced mRNA expression of collagen I (72.3±10.7%; 95% confidence interval (95% CI): 45.5–99.0), collagen III (79.0±4.5%; 95% CI: 67.6–90.4) and α‐SMA (65.7±5.9%; 95% CI: 51.1–80.2), as compared to control normalized at 100%. These results were confirmed by quantitative RT–PCR. Cycloheximide experiments indicated that somatostatin has no direct transcriptional effect. Using immunoprecipitation, we demonstrated that somatostatin also decreased de novo synthesis of collagen I (73±10%; 95% CI: 48–98%), collagen III (65±13%; 95% CI: 33–97%) and α‐SMA (47±9%; 95% CI: 25–69%). Remarkably, at higher concentrations, somatostatin did not suppress collagen mRNA expression nor de novo protein synthesis. We ascribe this observation to desensitization of the cells for somatostatin. Cell proliferation, as measured by 5‐bromo‐2′‐deoxyuridine labelling, was not altered by somatostatin. No significant effect on the intermediate and actin cytoskeleton were detected by immunohistochemistry and Western blotting. Our findings imply that in vivo antifibrotic effects of somatostatin could result partially from a direct action of somatostatin on HSC, but other, in vivo effects are probably also involved.

Role of Histone Deacetylases in Transcriptional Control of the Hepatic Stellate Cell Phenotype

Publisher Summary This chapter studies eukaryotic gene expression in the context of trans-acting transcription factors and their interaction with regulatory cis elements. It also establishes a role for histone modifications in transcription processes and the remodeling of chromatin structure. Hepatic stellate cells (HSC) are the major cellular sources of extracellular matrix synthesis in chronic liver diseases leading to fibrosis. This chapter presents a study that explores the antifibrogenic effect of Trichostatin A (TSA) on HSC in vitro. In the experiment, primary and fully activated HSC were exposed to 10–7–10–9 M TSA. Collagens type I and III and smooth muscle α-actin (α-SMA) were investigated on the protein and mRNA steady-state level by performing Northern hybridization and de novo immunoprecipitation. The antiproliferative effect was examined by [3H] thymidine incorporation and cell counting. Differential mRNA display, Northern hybridization, and Western blotting were performed to identify TSA sensitive genes. The ultimate goal of fibrosis research is the development of a rational basis for effective antifibrotic therapy.