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Link to original content: https://pubmed.ncbi.nlm.nih.gov/10369717/
All-trans and 9-cis retinoic acid alter rat hepatic stellate cell phenotype differentially - PubMed Skip to main page content
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. 1999 Jul;45(1):134-42.
doi: 10.1136/gut.45.1.134.

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

K Hellemans  1 , I Grinko, K Rombouts, D Schuppan, A Geerts
Affiliations

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

K Hellemans et al. Gut. 1999 Jul.

Abstract

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.

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Figures

Figure 1
Figure 1
Immunocytochemistry of stellate cells in culture. The stellate cell origin of cultured cells was established by immunocytochemistry for desmin (A), αSMA (B), and GFAP (C). The insert of (A) shows a negative control using non-immune IgG for desmin, counterstained using haematoxylin to show nuclei. Cultured stellate cells still contained small lipid droplets that showed up when cells were exposed to excitation light of 320 nm. Under these conditions, autofluorescence became apparent (D). Original magnification × 110.
Figure 2
Figure 2
Representative immunoprecipitation of connective tissue proteins from media and cell lysates. Cultured stellate cells were exposed to ATRA or 9RA for 48 hours and were metabolically labelled for the last 24 hours with trans 35S-label (50 µCi/ml). The arrows show the positions of the 205 kDa molecular weight marker (myosin). Procollagens α1(I), α1(III), and α1(IV), as well as fibronectin (FN) and laminin could be specifically immunoprecipitated.
Figure 3
Figure 3
Quantitative assessment of immunoprecipitated bands by phosphor imaging. The ratios of treated versus control samples were calculated for 1 µM ATRA (A) and 9RA (B). Data are expressed as mean (SD). The number of observations was four for ATRA and three for 9RA. Administration of ATRA or 9RA did not significantly affect the cellular pool of translabel (C). *p<0.05.
Figure 4
Figure 4
Representative autoradiographs of northern hybridisation experiments. Cultured stellate cells were exposed to 1 µM ATRA or 9RA for 48 hours. Hybridisation was carried out with probes for procollagen α1(I), α1(IIII), and α1(IV), fibronectin, laminin B1, and 18S ribosomal RNA. In accordance with previous reports,62 all hybridisations provided specific signals.
Figure 5
Figure 5
Northern hybridisation analysis of connective tissue protein transcripts. After normalising the signal for 18S ribosomal RNA, the ratios of treated versus control were calculated and expressed as mean (SD).
Figure 6
Figure 6
Effect 1 µM 9RA on BrdU incorporation by stellate cells after incubation times varying between 48 and 120 hours; representative time course experiment. Significant reduction of proliferation was found from 72 hours of treatment onward. Values are expressed as mean (SD). The number of observations was four in all cases.
Figure 7
Figure 7
Effect of different concentrations of ATRA or 9RA on BrdU incorporation. Cultured stellate cells were treated for 72 hours with 1, 0.1, and 0.01 µM ATRA or 9RA. Cells were labelled for the last 24 hours with 10 µM BrdU solution in the presence of ATRA or 9RA. Values are expressed as mean (SD). The number of observations was four in all cases. *p<0.025.
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