Human amniotic epithelial cell conditioned media reduces hepatic stellate cell activation and collagen partially through increased apoptosis.

Abstract

Background Hepatic fibrosis is the driving force behind the progression of chronic liver disease and the key effector is the hepatic stellate cell (HSC). Cell therapy with human amniotic epithelial cells (hAEC) has been shown to ameliorate fibrosis in murine models. Aims This study investigated whether factors secreted by hAEC reduce fibrosis by inhibiting HSC and whether soluble HLA-G (sHLA-G) is partly responsible. Methods The effect of conditioned media (CM) from hAEC was studied on LX2 cells, a human HSC cell line. Confluent HSC were treated with CM for 48 hours and mRNA expression of alpha smooth muscle actin (alpha-SMA) and platelet derived growth factor receptor (betaPDGF-R) assessed by RT-PCR. Protein levels of transforming growth factor beta1 (TGF-beta1) in the supernatant were measured by ELISA. Intracellular collagen production and supernatant collagen content were determined by 3H-proline incorporation and Sircol collagen assay respectively. HSC apoptosis was determined by FACS for Annexin V and proliferation by BrdU incorporation. Expression of genes responsible for collagen degradation; matrix metalloproteinases 2 and 9 (MMP2, MMP9) and tissue inhibitor of metalloproteinase 2 (TIMP2) were determined by RT-PCR. Anti-HLA-G neutralizing antibody was added to CM experiments to block the effects of sHLA-G in the hAEC CM. Results HSC treated with hAEC CM displayed reduced activation markers; alpha-SMA and betaPDGF-R mRNA expression were reduced by 56% (P = 0.0023) and 12% (P = 0.045), respectively. TGF-beta1 protein levels were reduced by 59% (P = 0.0009). In separate experiments in which TGF-beta was added to HSC, hAEC CM ameliorated alpha-SMA and betaPDGF-R mRNA expression to levels similar to controls (P < 0.001). Intracellular collagen production was reduced by 54% (P < 0.0001) in hAEC CM treated cells. Addition of hAEC CM to HSC cultures reduced collagen in the supernatant to levels below that in control media (P = 0.0025). Expression of MMP2 and MMP9 increased two-fold compared to controls (P = 0.0015; P = 0.0004) while TIMP2 was reduced by a third (P = 0.0024). Proliferation of HSC in sub-confluent cultures was reduced by 12% (P = 0.0047) in cells treated with hAEC CM. There was a 2.34 fold increase in early apoptosis in HSC treated with hAEC CM. Antibody blocking of sHLA-G in hAEC CM resulted in a partial reversal of diminished collagen production and TGF-beta1 suppression (P < 0.05). Conclusion hAEC CM treatment of activated hepatic stellate cells results in the suppression of markers of activation, proliferation and collagen production, likely through induction of HSC apoptosis. In addition active collagen degradation is enhanced by increasing MMP and decreasing TIMP gene expression. hAEC produce soluble factors, including sHLA-G, that inhibit human hepatic stellate cell collagen expression and therefore may be effective in ameliorating liver fibrosis.