Karine Hellemans

Differential modulation of rat hepatic stellate phenotype by natural and synthetic retinoids

Activation of hepatic stellate cells (HSC) is a central event in the pathogenesis of liver fibrosis during chronic liver injury. We examined the expression of retinoic acid (RAR) and retinoid X receptors (RXR) during HSC activation and evaluated the influence of natural and synthetic retinoic acids (RA) on the phenotype of culture‐activated HSC. The expression of the major RAR/RXR subtypes and isoforms was analyzed by Northern hybridization. Presence of functional receptor proteins was established by gel shift analysis. Retinoic acids, RAR, and RXR selective agonists and an RAR antagonist were used to evaluate the effects of retinoid signalling on matrix synthesis by Northern blotting and immunoprecipitation, and on cell proliferation by BrdU incorporation. The 9‐cisRA and synthetic RXR agonists reduced HSC proliferation and synthesis of collagen I and fibronectin. All‐trans RA and RAR agonists both reduced the synthesis of collagen I, collagen III, and fibronectin, but showed a different effect on cell proliferation. Synthetic RAR agonists did not affect HSC proliferation, indicating that ATRA inhibits cell growth independent of its interaction with RARs. In contrast, RAR specific antagonists enhance HSC proliferation and demonstrate that RARs control proliferation in a negative way. In conclusion, natural RAs and synthetic RAR or RXR specific ligands exert differential effects on activated HSC. Our observations may explain prior divergent results obtained following retinoid administration to cultured stellate cells or to animals subjected to fibrogenic stimuli. (HEPATOLOGY 2004;39:97–108.)

PPARbeta regulates vitamin A metabolism-related gene expression in hepatic stellate cells undergoing activation.

Activation of cultured hepatic stellate cells correlated with an enhanced expression of proteins involved in uptake and storage of fatty acids (FA translocase CD36, Acyl-CoA synthetase 2) and retinol (cellular retinol binding protein type I, CRBP-I; lecithin:retinol acyltransferases, LRAT). The increased expression of CRBP-I and LRAT during hepatic stellate cells activation, both involved in retinol esterification, was in contrast with the simultaneous depletion of their typical lipid-vitamin A (vitA) reserves. Since hepatic stellate cells express high levels of peroxisome proliferator activated receptor β (PPARβ), which become further induced during transition into the activated phenotype, we investigated the potential role of PPARβ in the regulation of these changes. Administration of L165041, a PPARβ-specific agonist, further induced the expression of CD36, B-FABP, CRBP-I, and LRAT, whereas their expression was inhibited by antisense PPARβ mRNA. PPARβ-RXR dimers bound to CRBP-I promoter sequences. Our observations suggest that PPARβ regulates the expression of these genes, and thus could play an important role in vitA storage. In vivo, we observed a striking association between the enhanced expression of PPARβ and CRBP-I in activated myofibroblast-like hepatic stellate cells and the manifestation of vitA autofluorescent droplets in the fibrotic septa after injury with CCl4 or CCl4 in combination with retinol.

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.

Peroxisome proliferator-activated receptor alpha-retinoid X receptor agonists induce beta-cell protection against palmitate toxicity

Fatty acids can stimulate the secretory activity of insulin‐producing beta‐cells. At elevated concentrations, they can also be toxic to isolated beta‐cells. This toxicity varies inversely with the cellular ability to accumulate neutral lipids in the cytoplasm. To further examine whether cytoprotection can be achieved by decreasing cytoplasmic levels of free acyl moieties, we investigated whether palmitate toxicity is also lowered by stimulating its β‐oxidation. Lower rates of palmitate‐induced beta‐cell death were measured in the presence of l‐carnitine as well as after addition of peroxisome proliferator‐activated receptor α (PPARα) agonists, conditions leading to increased palmitate oxidation. In contrast, inhibition of mitochondrial β‐oxidation by etomoxir increased palmitate toxicity. A combination of PPARα and retinoid X receptor (RXR) agonists acted synergistically and led to complete protection; this was associated with enhanced expression levels of genes involved in mitochondrial and peroxisomal β‐oxidation, lipid metabolism, and peroxisome proliferation. PPARα–RXR protection was abolished by the carnitine palmitoyl transferase 1 inhibitor etomoxir. These observations indicate that PPARα and RXR regulate beta‐cell susceptibility to long‐chain fatty acid toxicity by increasing the rates of β‐oxidation and by involving peroxisomes in fatty acid metabolism.

Specificity in Beta Cell Expression of l-3-Hydroxyacyl-CoA Dehydrogenase, Short Chain, and Potential Role in Down-regulating Insulin Release

A loss-of-function mutation of the mitochondrial β-oxidation enzyme l-3-hydroxyacyl-CoA dehydrogenase, short chain (HADHSC), has been associated with hyperinsulinemic hypoglycemia in man. It is still unclear whether loss of glucose homeostasis in these patients (partly) results from a dysregulation of beta cells. This study examines HADHSC expression in purified rat beta cells and investigates whether its selective suppression elevates insulin release. Beta cells expressed the highest levels of HADHSC mRNA and protein of all examined tissues, including those with high rates of mitochondrial β-oxidation. On the other hand, beta cells expressed relatively low levels of other β-oxidation enzymes (acyl-CoA dehydrogenase short, medium, and long chain and acetyl-coenzyme A acyltransferase 2). HADHSC expression was sequence-specifically silenced by RNA interference, and the effects were examined on glucose-stimulated insulin secretion following 48–72 h of suppression. In both rat beta cells and in the beta cell line INS1 832-13, HADHSC silencing resulted in elevated insulin release at low and at high glucose concentrations, which appeared not to be caused by increased rates of glucose metabolism or an inhibition in fatty acid oxidation. These data indicate that the normal beta cell phenotype is characterized by a high expression of HADHSC and a low expression of other β-oxidation enzymes. Down-regulation of HADHSC causes an elevated secretory activity suggesting that this enzyme protects against inappropriately high insulin levels and hypoglycemia.

Protein Markers for Insulin-Producing Beta Cells with Higher Glucose Sensitivity

Background and Methodology Pancreatic beta cells show intercellular differences in their metabolic glucose sensitivity and associated activation of insulin production. To identify protein markers for these variations in functional glucose sensitivity, rat beta cell subpopulations were flow-sorted for their level of glucose-induced NAD(P)H and their proteomes were quantified by label-free data independent alternate scanning LC-MS. Beta cell-selective proteins were also identified through comparison with rat brain and liver tissue and with purified islet alpha cells, after geometrical normalization using 6 stably expressed reference proteins. Principal Findings All tissues combined, 943 proteins were reliably quantified. In beta cells, 93 out of 467 quantifiable proteins were uniquely detected in this cell type; several other proteins presented a high molar abundance in beta cells. The proteome of the beta cell subpopulation with high metabolic and biosynthetic responsiveness to 7.5 mM glucose was characterized by (i) an on average 50% higher expression of protein biosynthesis regulators such as 40S and 60S ribosomal constituents, NADPH-dependent protein folding factors and translation elongation factors; (ii) 50% higher levels of enzymes involved in glycolysis and in the cytosolic arm of the malate/aspartate-NADH-shuttle. No differences were noticed in mitochondrial enzymes of the Krebs cycle, beta-oxidation or respiratory chain. Conclusions Quantification of subtle variations in the proteome using alternate scanning LC-MS shows that beta cell metabolic glucose responsiveness is mostly associated with higher levels of glycolytic but not of mitochondrial enzymes.

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.

Renal antioxidant enzymes and fibrosis-related markers in the rat adriamycin model

Excessive generation of reactive oxygen intermediates can induce changes in the cellular antioxidant defence system. In this study we examine the antioxidant enzyme status and the expression of fibrosis-related marker proteins in the Adriamycin model of chronic renal failure in the rat. Twenty weeks after Adriamycin treatment, rats have overt nephrotic syndrome and renal failure with development of tubulo-interstitial fibrosis and glomerulosclerosis. Lipids accumulate in blood and in both glomeruli and tubulo-interstitial tissue. Desmin and α-smooth muscle actin expression increases in glomeruli and in the tubulo-interstitial area. Renal cortex antioxidant enzyme activities are decreased 20 weeks after Adriamycin injection (to 41% for catalase, to 56% for total superoxide dismutase and to 69% for glutathione peroxidase). The mRNA levels of catalase, Cu/Zn-superoxide dismutase and glutathione peroxidase-1 evaluated by Northern blot are decreased by more than 50% for catalase, Cu/Zn-superoxide dismutase and glutathione peroxidase-1. We conclude that in the rat Adriamycin-induced model of chronic renal failure with fibrosis, the combination of decreased antioxidant enzyme status in renal cortex with high concentrations of lipids in blood and renal tissue facilitates oxidative damage. Development of fibrosis is paralleled by increased expression of desmin and α-smooth muscle actin.