Effect of human amniotic epithelial cells on pro-fibrogenic resident hepatic cells in a rat model of liver fibrosis

Abstract

Myofibroblasts are key fibrogenic cells responsible for excessive extracellular matrix synthesis characterizing the fibrotic lesion. In liver fibrosis, myofibroblasts derive either from activation of hepatic stellate cells (HSC) and portal fibroblasts (PF), or from the activation of fibroblasts that originate from ductular epithelial cells undergoing epithelial-mesenchymal transition. Ductular cells can also indirectly promote myofibroblast generation by activating TGF-β, the main fibrogenic growth factor, through αvβ6 integrin. In addition, after liver injury, liver sinusoidal cells can lose their ability to maintain HSC quiescence, thus favouring HSC differentiation towards myofibroblasts. The amniotic membrane and epithelial cells (hAEC) derived thereof have been shown to decrease hepatic myofibroblast levels in rodents with liver fibrosis. In this study, in a rat model of liver fibrosis, we investigated the effects of hAEC on resident hepatic cells contributing to myofibroblast generation. Our data show that hAEC reduce myofibroblast numbers with a consequent reduction in fibronectin and collagen deposition. Interestingly, we show that hAEC strongly act on specific myofibroblast precursors. Specifically, hAEC reduce the activation of PF rather than HSC. In addition, hAEC target reactive ductular cells by inhibiting their proliferation and αvβ6 integrin expression, with a consequent decrease in TGF-β activation. Moreover, hAEC counteract the transition of ductular cells towards fibroblasts, while it does not affect injury-induced and fibrosis-promoting sinusoidal alterations. In conclusion, among the emerging therapeutic applications of hAEC in liver diseases, their specific action on PF and ductular cells strongly suggests their application in liver injuries involving the expansion and activation of the portal compartment.

Keywords: bile duct ligation; biliary liver fibrosis; ductular epithelial cells; human amniotic cells; human amniotic epithelial cells; human amniotic membrane; myofibroblasts; placenta-derived cells.

Figure 1

Liver structure and hepatic fibrogenic cells. The liver consists of lobules, representing the hepatic functional unit (panel A). Each lobule is intersected by a central vein (CV), from which hepatocyte cords radiate towards portal triads (PT) containing three different structures: bile ducts (BD), hepatic artery (HA) and portal vein (PV). Hepatocyte cords are separated by sinusoids, blood vessels lined by specialized fenestrated endothelial cells (liver sinusoidal cells). Hepatic stellate cells (HSC) are located in the space of Disse, and portal fibroblasts (PF) in the portal triad areas. Under injury conditions, both HSC and PF can be activated to myofibroblasts (ECM‐producing cells). Ductular epithelial cells, another cell type involved during liver injury, line the bile ducts.

Figure 2

hAEC treatment reduces extracellular matrix deposition. Livers extracted from control (BDL + PBS) and treated (BDL + hAEC) rats, 6 weeks after bile duct ligation (BDL), were evaluated for extracellular matrix protein (fibronectin and collagen) deposition and the degree of fibrosis. Representative microphotographs of fibronectin immuno‐staining and collagen staining (PicroSirius Red: PSR) are reported in panels (A) and (B). hAEC‐treated rats showed lower hepatic areas, quantified by computer‐assisted image analysis, positive for fibronectin (A) and PSR (B), and reduced liver fibrosis in terms of Knodell's score units (C). Similar results were obtained for fibronectin (D) and collagen 1A1 (E) mRNA liver expression. Scale bars = 100 μm (A), 200 μm (B). *P < 0.05 and **P < 0.01, versus BDL + PBS group.

Figure 3

hAEC treatment decreases hepatic fibroblast activation. Immunohistochemical and quantitative image analysis of rat liver sections from control (BDL + PBS) and treated (BDL + hAEC) rats, 6 weeks after BDL. In hAEC‐treated rats, the percentages of liver areas occupied by activated myofibroblasts (α‐SMA‐positive cells) (A), by myofibroblasts derived from portal fibroblasts (PF) (collagen 15A1‐positive liver areas) (B) and by myofibroblasts derived from hepatic stellate cells (HSC) (desmin‐positive liver areas) (C), are reduced compared to the control (BDL + PBS) group. Similar results were obtained from the analysis of mRNA expression (panels A, B and C). Scale bars = 100 μm. *P < 0.05 and **P < 0.01 versus BDL + PBS group.

Figure 4

hAEC treatment reduces the ductular reaction and EMT. Immunohistochemical and quantitative image analysis of rat liver sections from control (BDL + PBS) and treated (BDL + hAEC) rats, 6 weeks after BDL. Livers from hAEC‐treated rats exhibited a significant decrease in liver area occupied by ductular epithelial cells (positive for CK19) (A) and a lower number of cells undergoing EMT (positive for S100A4) (B). The treatment did not affect the number of positive cells located in parenchymal and ductular areas, but rather those close to ductular structures (periductular cells) (C and D). Scale bars = 100 μm (A and B) and 50 μm (C). *P < 0.05 versus BDL + PBS group.

Figure 5

hAEC treatment decreases TGF‐β pathway activation. Immunohistochemical and quantitative image analysis of rat liver sections from control (BDL + PBS) and treated (BDL + hAEC) rats, 6 weeks after BDL. Representative microphotographs related to: (A) αvβ6 integrin ductular expression—up‐regulated under BDL conditions—and (B) phosphorylated Smad2/3 expression—identifying cells with activated TGF‐β pathway. hAEC‐treated rats show a lower hepatic area occupied by αvβ6 integrin (A) associated with a strong reduction in p‐Smad2/3‐positive area (B). p‐Smad2/3 signals are located in perinuclear and nuclear areas indicating both ongoing and completed translocations of p‐Smad2/3 to the nucleus (in box C, 1000× magnification). Scale bars = 100 μm (A) and 50 μm (B). *P < 0.05 and **P < 0.01 versus BDL + PBS group.

Figure 6

hAEC treatment does not act on liver sinusoidal endothelial cell defenestration. Immunohistochemical and quantitative image analysis of rat liver sections from control (BDL + PBS) and treated (BDL + hAEC) rats, 6 weeks after BDL. Representative microphotographs of: (A) CD32b expression in sinusoidal cells—the expression of this protein correlates with the presence of fenestrations in these cells—and (B) CD34 expressing cells—shown to be possibly increased during sinusoidal defenestration/capillarization. hAEC treatment does not affect BDL‐induced sinusoidal defenestration, but modestly decreases the number of CD34‐expressing cells. Scale bars = 100 μm (A) and 50 μm (B).