New therapeutic options for liver cirrhosis are needed. Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have emerged as a promising tool for delivering therapeutic factors in regenerative medicine. Our aim is to establish a new therapeutic tool that employs EVs derived from MSCs to deliver therapeutic factors for liver fibrosis. EVs were isolated from supernatants of adipose tissue MSCs, induced-pluripotent-stem-cell-derived MSCs, and umbilical cord perivascular cells (HUCPVC-EVs) by ion exchange chromatography (IEC). To produce engineered EVs, HUCPVCs were transduced with adenoviruses that code for insulin-like growth factor 1 (AdhIGF-I-HUCPVC-EVs) or green fluorescent protein. EVs were characterized by electron microscopy, flow cytometry, ELISA, and proteomic analysis. We evaluated EVs’ antifibrotic effect in thioacetamide-induced liver fibrosis in mice and on hepatic stellate cells in vitro. We found that IEC-isolated HUCPVC-EVs have an analogous phenotype and antifibrotic activity to those isolated by ultracentrifugation. EVs derived from the three MSCs sources showed a similar phenotype and antifibrotic potential. EVs derived from AdhIGF-I-HUCPVC carried IGF-1 and showed a higher therapeutic effect in vitro and in vivo. Remarkably, proteomic analysis revealed that HUCPVC-EVs carry key proteins involved in their antifibrotic process. This scalable MSC-derived EV manufacturing strategy is a promising therapeutic tool for liver fibrosis.

The severity of non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to steatohepatitis, and it is not yet clearly understood which patients will progress to liver fibrosis or cirrhosis. SPARC (Secreted Protein Acidic and Rich in Cysteine) has been involved in NAFLD pathogenesis in mice and humans. The aim of this study was to investigate the role of SPARC in inflammasome activation, and to evaluate the relationship between the hepatic expression of inflammasome genes and the biochemical and histological characteristics of NAFLD in obese patients. In vitro studies were conducted in a macrophage cell line and primary hepatocyte cultures to assess the effect of SPARC on inflammasome. A NAFLD model was established in SPARC knockout (SPARC^−/−) and SPARC^+/+ mice to explore inflammasome activation. A hepatic RNAseq database from NAFLD patients was analyzed to identify genes associated with SPARC expression. The results were validated in a prospective cohort of 59 morbidly obese patients with NAFLD undergoing bariatric surgery. Our results reveal that SPARC alone or in combination with saturated fatty acids promoted IL-1β expression in cell cultures. SPARC^−/− mice had reduced hepatic inflammasome activation during the progression of NAFLD. NAFLD patients showed increased expression of SPARC, NLRP3, CASP1, and IL-1β. Gene ontology analysis revealed that genes positively correlated with SPARC are linked to inflammasome-related pathways during the progression of the disease, enabling the differentiation of patients between steatosis and steatohepatitis. In conclusion, SPARC may play a role in hepatic inflammasome activation in NAFLD.

The Rho GTPase RAC1 regulates key processes in acute liver failure (ALF), including oxidative stress and inflammation. We aimed to evaluate the therapeutic potential of RAC1 inhibition in ALF. Ingenuity Pathway Analysis and Gene Ontology analysis were performed on transcriptomic datasets from patients with ALF (GSE38941 and GSE80751). ALF was induced in mice using concanavalin A, acetaminophen, or D-galactosamine/lipopolysaccharide (n = 10-21/group). The RAC1 pharmacological inhibitor 1D-142 was used in vivo and in vitro. Hepatocytes and macrophages, from primary cultures and cell lines, were analyzed. RNA-sequencing data from ALF mouse livers (n = 3/group) were integrated with human datasets. Human liver explants (n = 6) were treated in vitro with 1D-142. RAC1 emerged as an upstream regulator correlating with immune activation and oxidative stress responses (p <0.05) in human ALF samples. Administration of 1D-142 ameliorated liver injury in murine ALF models when administered at early or late stages post-injury (p <0.05). 1D-142 treatment diminished reactive oxygen species formation (p <0.01), inflammatory cell migration (p <0.001), cytokine production (p <0.05) and hepatocyte death (p <0.05). Liver transcriptomics revealed that RAC1 inhibition modulated key dysregulated pathways in ALF. Human ALF liver explants treated with 1D-142 showed reduced necrosis (p <0.05) and reduced expression of pro-inflammatory genes (p <0.01). RAC1 drives sterile inflammation and oxidative stress in ALF. Its pharmacological inhibition protects against liver injury in preclinical models and human explants, supporting RAC1 as a potential therapeutic target in ALF. Acute liver failure (ALF) is a life-threatening condition characterized by severe inflammation and oxidative stress for which there are limited treatment options. Our study provides strong scientific justification for targeting the RAC1 protein, demonstrating that its pharmacological inhibition with 1D-142 reduces liver injury, immune cell infiltration, and oxidative damage in murine models of ALF and in human liver explants. These findings identify RAC1 as a novel therapeutic target and provide translational support for its potential clinical application in ALF. RAC1-targeted therapy merits further studies in clinical settings.