Background: Hepatic stellate cells (HSCs) are the primary collagen-secreting cells in the liver. While HSCs are the major cell type involved in the pathogenesis of liver fibrosis, hepatic macrophages also play an important role in mediating fibrogenesis and fibrosis resolution. Previously, we observed a reduction in HSC activation, proliferation, and collagen synthesis following exposure to human amnion epithelial cells (hAEC) and hAEC-conditioned media (hAEC-CM). This suggested that specific factors secreted by hAEC might be effective in ameliorating liver fibrosis. hAEC-derived extracellular vesicles (hAEC-EVs), which are nanosized (40–100 nm) membrane bound vesicles, may act as novel cell–cell communicators. Accordingly, we evaluated the efficacy of hAEC-EV in modulating liver fibrosis in a mouse model of chronic liver fibrosis and in human HSC.Methods: The hAEC-EVs were isolated and characterized. C57BL/6 mice with CCl4-induced liver fibrosis were administered hAEC-EV, hAEC-CM, or hAEC-EV depleted medium (hAEC-EVDM). LX2 cells, a human HSC line, and bone marrow-derived mouse macrophages were exposed to hAEC-EV, hAEC-CM, and hAEC-EVDM. Mass spectrometry was used to examine the proteome profile of each preparation.Results: The extent of liver fibrosis and number of activated HSCs were reduced significantly in CCl4-treated mice given hAEC-EVs, hAEC-CM, and hAEC EVDM compared to untreated controls. Hepatic macrophages were significantly decreased in all treatment groups, where a predominant M2 phenotype was observed. Human HSCs cultured with hAEC-EV and hAEC-CM displayed a significant reduction in collagen synthesis and hAEC-EV, hAEC-CM, and hAEC-EVDM altered macrophage polarization in bone marrow-derived mouse macrophages. Proteome analysis showed that 164 proteins were unique to hAEC-EV in comparison to hAEC-CM and hAEC-EVDM, and 51 proteins were co-identified components with the hAEC-EV fraction.Conclusion: This study provides novel data showing that hAEC-derived EVs significantly reduced liver fibrosis and macrophage infiltration to an extent similar to hAEC-EVDM and hAEC-CM. hAEC-EV-based therapy may be a potential therapeutic option for liver fibrosis.

Background and Aim Non‐alcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma. Currently, lifestyle modification is the only effective treatment. We have shown that human amnion epithelial cells (hAECs) reduce inflammation and fibrosis in toxin‐induced liver injury models. We examined the effect of these cells and the soluble factors released by the cells into culture medium (hAEC conditioned medium [hAEC‐CM]) in a diet‐induced murine NASH model. Methods C57BL/6J male mice received a Western “fast food diet” for 42 weeks. Group 1 received an intraperitoneal injection of 2 × 106 hAECs at week 34, group 2 received an additional hAEC dose at week 38, and group 3 received thrice weekly hAEC‐CM injections intraperitoneal for 8 weeks from week 34. Liver fibrosis area, inflammation, and fibrosis regulators were measured by immunohistochemistry, qPCR, and gelatin zymography. Metabolic parameters were also assessed. Results Fast food diet‐fed mice demonstrated peri‐cellular hepatic fibrosis, inflammation, and steatosis typical of NASH. Liver fibrosis area was reduced by 40% in hAEC‐treated and hAEC‐CM‐treated mice. hAEC treatment significantly reduced pSMAD 2/3 signaling and the number of activated hepatic stellate cells and liver macrophages. Matrix metalloproteinase 2 and 9 gene and protein expression were variably affected. hAEC treatment did not alter the NASH activity score or metabolic parameters such as bodyweight, total cholesterol, or glucose tolerance. Conclusion Human amnion epithelial cell and hAEC‐CM significantly reduced hepatic inflammation and fibrosis in a diet‐induced non‐alcoholic fatty liver disease model. Although hAEC and hAEC‐CM did not affect the metabolic components of NASH, their therapeutic potential is promising and warrants further investigation.

Emerging evidence suggest sex-specific regulation of dopamine neurons may underlie susceptibility of males to disorders such as Parkinson's disease (PD). In healthy male dopamine neurons, the Y-chromosome gene product, the sex-determining region on the Y chromosome (SRY) modulates dopamine biosynthesis and motor function. We investigated the regulation and function of SRY in a model of dopamine cell injury. Treatment with the dopaminergic toxin, 6-hydroxydopamine (6-OHDA), significantly elevated SRY mRNA expression (9-fold) in human male dopamine M17 cells. SRY up-regulation occurred via the p-quinone pathway, associated with a 3.5-fold increase in expression of GADD45γ, a DNA damage inducible factor gene and known SRY regulator. In turn, a signaling cascade involving GADD45γ/p38-MAPK/GATA activated the SRY promoter. Knockdown of SRY mRNA in 6-OHDA-treated M17 cells was deleterious, increasing levels of reactive oxygen species (ROS), pro-apoptotic marker PUMA mRNA, and cell injury (+25%, +32% and +34%, respectively). Conversely, ectopic over-expression of SRY in 6-OHDA-treated female SH-SY5Y cells was protective, decreasing ROS, PUMA, and cell injury (-40%, -46%, and -30%, respectively). However, the 6-OHDA-induced increase in SRY expression was diminished with higher concentrations of toxins or with chronic exposure to 6-OHDA. We conclude that SRY upregulation after dopamine cell injury is initially a protective response in males, but diminishes with gradual loss in dopamine cells. We speculate that dysregulation of SRY may contribute the susceptibility of males to PD.