Cellular Retinol-Binding Protein-1 Expression and Modulation during In Vivo and In Vitro Myofibroblastic Differentiation of Rat Hepatic Stellate Cells and Portal Fibroblasts

Abstract

SUMMARY:Cellular retinol-binding protein-1 (CRBP-1) is involved in vitamin A metabolism because it mediates both retinol esterification to retinyl esters and retinol oxidation to retinal and retinoic acid. CRBP-1 is highly expressed in the liver, particularly in hepatic stellate cells (HSC). In this study, we investigated the liver expression of CRBP-1 during experimental fibrogenesis. We also studied the regulation of CRBP-1 expression in cultured HSC and portal fibroblasts, two fibroblastic cell types involved in liver fibrogenesis. Fibrosis was induced in rats by carbon tetrachloride (CCl 4 ) or bile duct ligation. Immunohistochemical staining was performed for CRBP-1 and ␣-smooth muscle (SM) actin, an activation marker of fibrogenic cells. CRBP-1 and ␣-SM actin expression was studied by Western blotting and/or Northern blot in primary cultures of HSC isolated by conventional methods and in portal fibroblasts that were obtained by outgrowth from the biliary tree after enzymatic digestion. In normal liver, contrary to HSC, portal fibroblasts did not express CRBP-1. After CCl 4 injury, CRBP-1 expression was maintained in myofibroblastic ␣-SM actin-positive HSC. After bile duct ligation, portal fibroblasts (which proliferated around ductular structures) acquired expression of both CRBP-1 and ␣-SM actin. During HSC activation in culture, CRBP-1 expression gradually increased until Day 5 when ␣-SM actin expression was obvious. Cultured portal fibroblasts developed both CRBP-1 and ␣-SM actin expression. In both cell populations, transforming growth factor-␤1 treatment increased CRBP-1 expression. Thus, in normal liver, CRBP-1 expression was different among fibroblastic cells, a finding that adds to the concept of heterogeneity of liver fibrogenic cells. Furthermore, during myofibroblastic differentiation, HSC that lost their stores of retinol maintained a high level of CRBP-1 expression, whereas portal fibroblasts acquired CRBP1 expression. Together, these data suggest a correlation between CRBP-1 expression and myofibroblastic differentiation. (Lab Invest 2002, 82:619 -628).

Transforming growth factor-β and substrate stiffness regulate portal fibroblast activation in culture

“…41 However, our findings differ from published reports that showed that PFs express CRBP-1 only after myofibroblastic differentiation. 7,16 We cannot explain this difference. Nonetheless, although PFs and HSCs in our study showed similar CRBP-1 profiles, PFs did not show the changes in PPAR-␥ expression seen in HSCs and failed to form lipid droplets in response to retinol treatment.…”

“…10 These cells have been studied in culture, although generally after outgrowth from fragments of the biliary tree or late after isolation 16,17 ; the characteristics of PF myofibroblastic differentiation have not been studied. We suggest that myofibroblastic PFs are fibrogenic and that PFs in culture can be studied in much the same way as HSCs.…”

“…15 PFs have also been isolated by outgrowth from dissected, HSC-free segments of bile duct. 16 Cells isolated by this technique and cultured by standard methods rapidly expressed ␣-SMA and type I collagen although they were clearly distinct from HSCs by marker analysis. 17 Isolation of PFs from rat liver by perfusion and size selection has been described, and the ecto-ATPase nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) has been shown to be a specific marker for PFs before myofibroblastic differentiation, distinguishing them from quiescent HSCs.…”

Effects of aging on liver microcirculatory function and sinusoidal phenotype

“…Underlying mechanisms explaining HSC activation in aging revealed no modification in different proinflammatory mediators including TLR4, NFκB, and TGFβ (data not shown), but interestingly suggested alterations in retinoid metabolism and breakdown. In fact, aged livers exhibited down‐regulated CRBP‐1 together with over‐expressed PNPLA3, which have been associated with HSC activation and susceptibility to develop steatohepatitis (Bruschi et al, 2017; Uchio et al, 2002). …”

Portal fibroblasts marked by the surface antigen Thy1 contribute to fibrosis in mouse models of cholestatic liver injury

“…PFs are defined as a non‐HSC fibroblast population that can be found in the periportal mesenchyme surrounding the bile ducts; they are considered to be a heterogeneous population 11. However, studies on PFs have depended on isolation methods based on outgrowth from dissected bile segments,12 size selection,13 and purification of HSC marker‐negative, non‐HSC‐derived myofibroblasts by fluorescence‐activated cell sorting 14. None of these methods identify or isolate PFs by positive selection, thus hampering accurate evaluation of the cell population of interest.…”