Biliary fibrosis and resultant cirrhosis are among the most common outcomes of chronic liver diseases. Currently, liver transplantation remains the only effective treatment. In seeking alternative therapeutic approaches, we focused on the potential use of the human amniotic membrane (AM). Indeed, AM has gained increasing importance for its antiscarring, anti-inflammatory, and wound-healing properties, as well as for the multipotent differentiation ability and immunomodulatory features of AM-derived cells. Intriguingly, we have recently demonstrated that placenta-derived cells reduce lung fibrosis in bleomycin-treated mice, and that AM patches reduce postischemic cardiac injury in rats. Hence, we have now investigated the effects of human AM on biliary fibrosis induced in rats through the bile duct ligation (BDL) procedure. A fragment of human AM was applied onto the liver surface after BDL and the effects on fibrosis establishment and progression were evaluated at different time points in comparison with fibrosis progression in control BDL rats. The degree of liver fibrosis was first assessed by the semiquantitative Knodell scoring system and, thereafter, by digital image morphometric analysis to quantify the area occupied by ductular reaction, activated myofibroblasts, and collagen deposition. We demonstrated a significant reduction in the severity of BDL-induced fibrosis in AM-treated rats. Indeed, while fibrosis progressed rapidly in control BDL rats, leading to cirrhosis within 6 weeks, AM-treated rats showed confined fibrosis at the portal/periportal area with no signs of cirrhosis, and a reduction in collagen deposition to about 50% of levels observed in control BDL rats. In addition, the AM was able to significantly slow the gradual progression of the ductular reaction and reduce, at all time points, the area occupied by activated myofibroblasts. These findings suggest that human AM, when applied as a patch onto the liver surface, might inhibit fibrosis progression in BDL-injured livers, and could protect against hepatic damage associated with fibrotic degeneration.

The human amniotic membrane (hAM), thanks to its favorable properties, including anti-inflammatory, anti-fibrotic and pro-regenerative effects, is a well-known surgical material for many clinical applications, when used both freshly after isolation and after preservation. We have shown previously that hAM patching is a potential approach to counteract liver fibrosis. Indeed, when fresh hAM was used to cover the liver surface of rats with liver fibrosis induced by the bile duct ligation (BDL) procedure, the progression and severity of fibrosis were significantly reduced. Since cryopreservation enables safety and long-term storage of hAM but may influence its functional properties, here we compared the anti-fibrotic effects of fresh and cryopreserved hAM in rats with BDL-induced liver fibrosis. After BDL, the rat liver was covered with a piece of fresh or cryopreserved hAM, or left untreated. Six weeks later, the degree of liver fibrosis was assessed histologically using the Knodell and the METAVIR scoring systems. Digital image analysis was used to quantify the percentage of the areas of each liver section displaying ductular reaction, extracellular matrix (ECM) deposition, activated myofibroblasts and hepatic stellate cells (HSCs). Liver collagen content was also determined by spectrophotometric technique. The degree of liver fibrosis, ductular reaction, ECM deposition, and the number of activated myofibroblasts and HSCs were all significantly reduced in hAM-treated rats compared to control animals. Fresh and cryopreserved hAM produced the same anti-fibrotic effects. These findings indicate that cryopreservation maintains the anti-fibrotic properties of hAM when used as a patch to reduce the severity of liver fibrosis.

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-b, the main fibrogenic growth factor, through avb6 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 avb6 integrin expression, with a consequent decrease in TGF-b 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.

The epicardial ablation procedure using the thoracoscopic approach is feasible, safe, and reproducible.

No abstract