ABSTRACT
Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFß (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-ß-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.
Subject(s)
Extracellular Vesicles/metabolism , Hepatic Stellate Cells/metabolism , Induced Pluripotent Stem Cells/metabolism , Liver Cirrhosis/metabolism , Liver Cirrhosis/therapy , Animals , Apoptosis , Cell Movement , Cell Proliferation , Chemical and Drug Induced Liver Injury/therapy , Chemotaxis , Disease Models, Animal , Humans , Liver/metabolism , Liver/pathology , Liver Cirrhosis/pathology , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/metabolism , Transcriptome , Transforming Growth Factor beta/metabolismABSTRACT
BACKGROUND&AIMS: Hepatic stellate cells (HSCs) play a key role in liver fibrosis in various chronic liver disorders including nonalcoholic fatty liver disease (NAFLD). The development of liver fibrosis requires a phenotypic switch from quiescent to activated HSCs. The triggers for HSCs activation in NAFLD remain poorly understood. We investigated the role and molecular mechanism of extracellular vesicles (EVs) released by hepatocytes during lipotoxicity in modulation of HSC phenotype. METHODS: EVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSCs activation, migration and proliferation were assessed. Loss- and gain-of-functions studies were performed to explore the potential role of PPAR-γ-targeting miRNAs carried by EVs into HSC. RESULTS: Hepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of pro-fibrogenic genes (Collagen-I, α-SMA and TIMP-2), proliferation, chemotaxis and wound healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSC. Hepatocyte-derived EVs miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression with miR-128-3p being the most effectively transferred. Furthermore loss- and gain-of-function studies identified miR-128-3p as a central modulator of the effects of EVs on PPAR-γ inhibition and HSC activation. CONCLUSION: Our findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel anti-fibrotic targets for NAFLD and other fibrotic diseases.
ABSTRACT
BACKGROUND/AIMS: Transanal endoscopic microsurgery is a minimally invasive approach for rectal lesions. Superior exposure and access to the entire rectum result in lesser risk of compromised margin and lower recurrence rates compared to conventional transanal excision. It was aimed at describing a single institution's initial experience with transanal endoscopic microsurgery. METHODOLOGY: Retrospective review of a prospective database. Fifty-two procedures from March 2009 to November 2011 were analyzed. RESULTS: Fifty operations were completed. There were 23 men. Mean age was 67.5 (42-89). Mean follow-up was 23 (1-31) months. Average tumor size was 4.8 cm (1.5-14 cm). Mean distance from anal verge was 6 (3-15) cm. Mean operating time was 110 (86-170) min. Postoperative complication rate was 16%o. There were no re-admissions. Mortality was null. Operative pathology was adenoma in 25, in situ adenocarcinoma in eight, invasive adenocarcinoma in 13, neuroendocrine carcinoma in three and no residual lesion in one case. Recurrence was 4% for benign and 8% for malignant tumors. CONCLUSIONS: TEM is a minimally invasive procedure with low postoperative morbidity during initial experience. TEM is curative for benign lesions and for selected early cancers. It is useful after neoadjuvant therapy for strictly selected cancers while the results of multi-institutional trials are awaited.
