Prevention of cultured rat stellate cell transformation and endothelin-B receptor upregulation by retinoic acid.

Abstract

1 Physiologically, perisinusoidal hepatic stellate cells (HSC) are quiescent and store retinoids. During liver injury and in cell culture, HSC transform into proliferating myofibroblast-like cells that express alpha-smooth muscle actin (alpha-sma) and produce excessive amounts of extracellular matrix. During transformation (also known as activation), HSC are depleted of the retinoid stores, and their expression of the endothelin-1 (ET-1) system is increased. ET-1 causes contraction of transformed HSC and is implicated in their proliferation and fibrogenic activity. In order to understand the association between retinoids, ET-1 and the activation of HSC, we investigated the effect of 13-cis-retinoic acid on the transformation of cultured HSC and the expression of ET-1 system. 2 HSC derived from normal rat liver were maintained for 10-12 days in a medium supplemented with 5% serum and containing 2.5 micro M retinoic acid without or with 50 nM ET-1 (ETA+ETB agonist) or sarafotoxin S6c (ETB agonist). In another set of experiments, cells treated for 10-12 days with vehicle (ethanol) or retinoic acid were challenged with ET-1 or sarafotoxin S6c, and various determinations were made at 24 h. 3 Retinoic acid inhibited transformation and proliferation of HSC as assessed by morphological characteristics, expression of alpha-sma, bromodeoxyuridine incorporation and cell count. Retinoic acid also prevented upregulation of ETB receptors without affecting ET-1 or ETA expression. Total protein synthesis ([(3)H]leucine incorporation), collagen alpha types I mRNA expression and collagen synthesis ([(3)H]proline incorporation) were lower in retinoic acid-treated cells. Although ET-1-treated cells were morphologically similar to the control cells, their expression of alpha-smooth muscle actin was significantly inhibited. The presence of retinoic acid in the medium during treatment with ET-1 caused further reduction in the expression of alpha-smooth muscle actin. ET-1 and sarafotoxin S6c stimulated total protein synthesis in vehicle- and retinoic acid-treated cells, but collagen synthesis only in the latter. 4 These results showing prevention of HSC activation and negative regulation of ETB receptor expression in them by retinoic acid may have important pathophysiologic implications.