Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition

Abstract

Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.

Keywords: ascorbic acid/vitamin C; collagen-I; hypoxia; liver fibrosis; myofibroblast.