Liver myofibroblasts activate protein C and respond to activated protein C

AIM:To study the protein C activation system in human liver myofibroblasts,and the effects of activated protein C(APC)on these cells.METHODS:Human liver myofibroblasts were obtained by outgrowth.Expression of protease activated receptor 1(PAR-1),endothelial protein C receptor(EPCR) and thrombomodulin(TM)was analyzed by flow cytometry.Extracellular signal-regulated kinase(ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies.Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction(RT-PCR).Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate. RESULTS:Primary cultures of human liver myofibroblasts expressed EPCR on their surface,together with PAR-1 and TM.This receptor system was functional since exposure of myofibroblasts to APC inducedERK1/2 phosphorylation in a dose-and time-dependent manner.Furthermore,APC significantly upregulated the expression of collagen mRNA,as shown by real-time RT-PCR.Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059.Finally,using a cell-based colorimetric assay,we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin.CONCLUSION:These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.

A red wine polyphenolic extract reduces the activation phenotype of cultured human liver myofibroblasts

AIM: To test the effect of a standardized red wine polyphenolic extract (RWPE) on the phenotype of human liver myofibroblasts in culture. METHODS: Human myofibroblasts grown from liver explants were used in this study. Cell proliferation was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Signaling events were analyzed by western blot with phosphospecific antibodies. Matrix-metalloproteinase activity was measured with gel zymography. RESULTS: We found that cell proliferation was dose-dependently decreased by up to 90% by RWPE while cell viability was not affected. Exposure to RWPE also greatly decreased the phosphorylation of ERK1/ERK2 and Akt in response to stimulation by the mitogenic factor platelet-derived growth factor BB (PDGF-BB). Finally, RWPE affected extracellular matrix remodeling by decreasing the secretion by myofibroblasts of matrix-metalloproteinase-2 and of tissue inhibitor of matrix- metalloproteinases-1.CONCLUSION: Altogether, RWPE decreases the activation state of liver myofibroblasts. The identification of the active compounds in RWPE could offer new therapeutic strategies against liver fibrosis.