Retinoic acid signaling pathway in pancreatic stellate cells: Insight into the anti-fibrotic effect and mechanism.

Pancreatic stellate cells (PSCs) are activated following loss of cytoplasmic vitamin A (retinol)-containing lipid droplets, which is a key event in the process of fibrogenesis of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDCA). PSCs are the major source of cancer-associated fibroblasts (CAFs) that produce stroma to induce PDAC cancer cell growth, invasion, and metastasis. As an active metabolite of retinol, retinoic acid (RA) can regulate target gene expression in PSCs through its nuclear receptor complex (RAR/RXR or RXR/RXR) or transcriptional intermediary factor. Additionally, RA also has extranuclear and non-transcriptional effects. In vitro studies have shown that RA induces PSC deactivation which reduces extracellular matrix production through multiple modes of action, such as inhibiting TßRⅡ, PDGFRß, ß-catenin and Wnt production, downregulating ERK1/2 and JNK phosphorylation and suppressing active TGF-ß1 release. RA alone or in combination with other reagents have been demonstrated to have an effective anti-fibrotic effect on cerulein-induced mouse CP models in vivo studies. Clinical trial data have shown that repurposing all-trans retinoic acid (ATRA) as a stromal-targeting agent for human pancreatic cancer is safe and tolerable, suggesting the possibility of using RA for the treatment of CP and PDCA in humans. This review focuses on RA signaling pathways in PSCs and the effects and mechanisms of RA in PSC-mediated fibrogenesis as well as the anti-fibrotic and anti-tumor effects of RA targeting PSCs or CAFs in vitro and in vivo, highlighting the potential therapies of RA against CP and PDAC.

Production, Exacerbating Effect, and EV-Mediated Transcription of Hepatic CCN2 in NASH: Implications for Diagnosis and Therapy of NASH Fibrosis.

Non-alcoholic steatohepatitis (NASH) is characterized by steatosis, hepatocyte ballooning, and inflammation and may progress to include increasingly severe fibrosis, which portends more serious disease and is predictive of patient mortality. Diagnostic and therapeutic options for NASH fibrosis are limited, and the underlying fibrogenic pathways are under-explored. Cell communication network factor 2 (CCN2) is a well-characterized pro-fibrotic molecule, but its production in and contribution to NASH fibrosis requires further study. Hepatic CCN2 expression was significantly induced in NASH patients with F3-F4 fibrosis and was positively correlated with hepatic Col1A1, Col1A2, Col3A1, or αSMA expression. When wild-type (WT) or transgenic (TG) Swiss mice expressing enhanced green fluorescent protein (EGFP) under the control of the CCN2 promoter were fed up to 7 weeks with control or choline-deficient, amino-acid-defined diet with high (60%) fat (CDAA-HF), the resulting NASH-like hepatic pathology included a profound increase in CCN2 or EGFP immunoreactivity in activated hepatic stellate cells (HSC) and in fibroblasts and smooth muscle cells of the vasculature, with little or no induction of CCN2 in other liver cell types. In the context of CDAA-HF diet-induced NASH, Balb/c TG mice expressing human CCN2 under the control of the albumin promoter exhibited exacerbated deposition of interstitial hepatic collagen and activated HSC compared to WT mice. In vitro, palmitic acid-treated hepatocytes produced extracellular vesicles (EVs) that induced CCN2, Col1A1, and αSMA in HSC. Hepatic CCN2 may aid the assessment of NASH fibrosis severity and, together with pro-fibrogenic EVs, is a therapeutic target for reducing NASH fibrosis.

Correction: Activation of the Connective Tissue Growth Factor (CTGF)-Transforming Growth Factor ß 1 (TGF-ß 1) Axis in Hepatitis C Virus-Expressing Hepatocytes.

[This corrects the article DOI: 10.1371/journal.pone.0046526.].

Vitamin D3 analogue calcipotriol inhibits the profibrotic effects of transforming growth factor- ß1 on pancreatic stellate cells.

OBJECTIVE: To evaluate the inhibitory effect of vitamin D3 analogue calcipotriol (Cal) on the fibrosis of pancreatic stellate cells (PSCs) induced by TGF-ß1 and the rationality of Cal use in alcoholic chronic pancreatitis (ACP). MATERIAL AND METHODS: Double-labeling immunofluorescence was used for the identification of VDR+PSCs in the pancreas of healthy controls (HC) and ACP patients. Van Gieson staining for examination of collagen fibers. RT-qPCR and Western Blot for determining the mRNAs and proteins of VDR, TGF-ß1 and COL1A1 in the pancreas of ACP or in vitro PSCs. ELISA or LC-MS/MS for detection of serum TGF-ß1 and COL1A1 or 25(OH)D3. The PSC line (RP-2 cell) was used for the determination of proteomic alterations in Cal plus TGF-ß1 versus TGF-ß1 and to examine the effect of VDR gene knockdown. RESULTS: Enhanced expression of VDR was detected in RP-2 cells stimulated with alcohol (ALC) plus Cal versus Cal alone and in PSCs in the pancreas of ACP versus HC. The increased VDR+PSCs were positively correlated with the levels of COL1A1 mRNAs or areas of collagen deposition in the pancreas of ACP. TGF-ß1 was overexpressed in the pancreas of ACP and ALC-treated RP-2 cells while 25(OH)D3 level in serum was significantly decreased in ACP versus HC. Through a VDR-dependent mechanism, Cal antagonized 16 profibrotic proteins in TGF-ß1-induced RP-2 cells that included 7 extracellular matrix components, 2 cytoskeletal proteins, 2 fibrosis-associated factors (RUNX1 and TRAF2), TIMP-1, CCN1, integrin α11, an adhesion scaffold protein (TGFB1i1) and an enzyme mediating TGF-ß1-induced fibrogenesis (ENPP1). CONCLUSION: This study suggests that Cal administration may be a potential antifibrotic strategy via inhibiting TGF-ß1-mediated PSC action during the development of ACP.

Graves' disease overlapping with chronic hepatitis B and methimazole-induced liver injury and autoimmune hepatitis: a case report.

BACKGROUND: Liver injury related to Graves' Disease (GD) includes hepatotoxicity of thyroid hormone excess, drug-induced liver injury, and changes resulting from concomitant liver disease. Methimazole (MMI) has been shown to induce several patterns of liver injury. However, the diagnosis and treatment of autoimmune hepatitis (AIH) overlapping with either GD or chronic hepatitis B are challenging. CASE PRESENTATION: A 35-year-old man from China presented with a two-year history of GD and a 10-day history of progressive jaundice. He had taken MMI for two months and discontinuing treatment due to liver toxicity 1 year ago and for another 6 days 20 days prior to hospitalization. The patient was diagnosed with GD overlapping with chronic hepatitis B and MMI-induced liver injury with early stage of acute-on-chronic liver failure on admission. However, the elevated aminotransferase and bilirubin levels could not be controlled after correction of liver failure and effective control of HBV replication and hyperthyroidism by daily oral entecavir and one-time oral administration of 131-iodine. The patient underwent liver biopsy on the 43rd day of hospitalization, showing HBsAg expression on the membrane of hepatocytes and typical histopathological characteristics of AIH. He was finally diagnosed with GD overlapping with chronic hepatitis B and MMI-induced liver injury and AIH. The elevated aminotransferase and bilirubin completely returned to normal by 3-month glucocorticoid therapy and continuous entecavir treatment and there was no recurrence during a 6-month follow-up, suggesting that AIH in this patient is different from classical AIH or GD-associated AIH. CONCLUSIONS: GD together with AIH is a complex and difficult subject. It needs to be clarified whether MMI or HBV can act as a trigger for AIH in this patient.

Extracellular Vesicles in Organ Fibrosis: Mechanisms, Therapies, and Diagnostics.

Fibrosis is the unrelenting deposition of excessively large amounts of insoluble interstitial collagen due to profound matrigenic activities of wound-associated myofibroblasts during chronic injury in diverse tissues and organs. It is a highly debilitating pathology that affects millions of people globally and leads to decreased function of vital organs and increased risk of cancer and end-stage organ disease. Extracellular vesicles (EVs) produced within the chronic wound environment have emerged as important vehicles for conveying pro-fibrotic signals between many of the cell types involved in driving the fibrotic response. On the other hand, EVs from sources such as stem cells, uninjured parenchymal cells, and circulation have in vitro and in vivo anti-fibrotic activities that have provided novel and much-needed therapeutic options. Finally, EVs in body fluids of fibrotic individuals contain cargo components that may have utility as fibrosis biomarkers, which could circumvent current obstacles to fibrosis measurement in the clinic, allowing fibrosis stage, progression, or regression to be determined in a manner that is accurate, safe, minimally-invasive, and conducive to repetitive testing. This review highlights the rapid and recent progress in our understanding of EV-mediated fibrotic pathogenesis, anti-fibrotic therapy, and fibrosis staging in the lung, kidney, heart, liver, pancreas, and skin.

Structural and Functional Characterization of Fibronectin in Extracellular Vesicles From Hepatocytes.

Extracellular vesicles (EVs) are membrane-limited nanoparticles that are liberated by cells and contain a complex molecular payload comprising proteins, microRNA, RNAs, and lipids. EVs may be taken up by other cells resulting in their phenotypic or functional reprogramming. In the liver, EVs produced by non-injured hepatocytes are involved in the maintenance of hepatic homeostasis or therapeutic outcomes following injury while EVs produced by damaged hepatocytes may drive or exacerbate liver injury. In this study, we examined the contribution of EV fibronectin (FN1) to the biogenesis, release, uptake, and action of hepatocyte-derived EVs. While FN1 is classically viewed as a component of the extracellular matrix that regulates processes such as cell adhesion, differentiation, and wound healing and can exist in cell-associated or soluble plasma forms, we report that FN1 is also a constituent of hepatocyte EVs that functions in EV uptake by target cells such as hepatocytes and hepatic stellate cells (HSC). FN1 co-purified with EVs when EVs were enriched from conditioned medium of human or mouse hepatocytes and a direct association between FN1 and hepatocyte EVs was established by immunoprecipitation and proteinase protection. FN1 ablation in mouse hepatocytes using CRISPR-Cas9 did not alter EV biogenesis but EV uptake by HSC was significantly reduced for FN1 knockout EVs (EVΔFN1 ) as compared to EVs from wild type hepatocytes (EVWT). The uptake by hepatocytes or HSC of either EVWT or EVΔFN1 required clathrin- and caveolin-mediated endocytosis, cholesterol, lysosomal acidic lipase activity, and low pH, while macropinocytosis was also involved in EVΔFN1 uptake in HSC. Despite their differences in rate and mechanisms of uptake, EVΔFN1 functioned comparably to EVWT in ameliorating CCl4-induced hepatic fibrosis in mice. In conclusion, FN1 is a constituent of hepatocyte EVs that facilitates EV uptake by target cells but is dispensable for EV-mediated anti-fibrotic activity in vivo.

Interleukin-6 participates in human pancreatic stellate cell activation and collagen I production via TGF-ß1/Smad pathway.

Pancreatic stellate cells (PSCs) play a key role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor-ß1 (TGF-ß1) is a major regulator of PSC activation and extracellular matrix production. Interleukin-6 (IL-6) has shown to participate in TGF-ß1 production and rat PSC activation. This study aimed to investigate whether IL-6 promotes human PSC activation and collagen 1(Col1) production through the TGF-ß1/Smad pathway. Our results showed that the expression of IL-6 and IL-6R in activated PSCs and macrophages (Mφs) were enhanced in the pancreas of ACP compared to healthy controls and that the mRNA expression of IL-6, IL-6R, TGF-ß1, α-SMA or Col1a1 were significantly increased in the pancreas of ACP, showing positive correlations between elevated IL-6 levels and either TGF-ß1 or α-SMA or Col1a1 levels and between elevated TGF-ß1 levels and α-SMA or Col1a1 levels. In in vitro studies, we identified that IL-6R expression or IL-6 and TGF-ß1 secretions were significantly increased in, respectively, Mφs and PSCs by ethanol (EtOH) or lipopolysaccharide (LPS) stimulation while EtOH- or LPS-induced α-SMA or Col1a1 mRNA and protein production in PSCs were partially blocked by IL-6 antibody. IL-6-induced TGF-ß1 production in PSCs was antagonized by si-IL-6R RNA or by an inhibitor of STAT3. Additionally, IL-6-promoted α-SMA or Col1a1 protein production was blocked by TGF-ß1 antibody and IL-6-induced phosphorylation of Smad2/3 and transcription of α-SMA and Col1a1 mRNA were antagonized by si-TGF-ß1 RNA. Our findings indicate that IL-6 contributes to PSC activation and Col1 production through up-regulation of TGF-ß1/Smad2/3 pathway.

Dynamic Changes in Function and Proteomic Composition of Extracellular Vesicles from Hepatic Stellate Cells during Cellular Activation.

: During chronic liver injury, hepatic stellate cells (HSC) undergo activation and are the principal cellular source of collagenous scar. In this study, we found that activation of mouse HSC (mHSC) was associated with a 4.5-fold increase in extracellular vesicle (EV) production and that fibrogenic gene expression (CCN2, Col1a1) was suppressed in Passage 1 (P1; activated) mHSC exposed to EVs from Day 4 (D4; relatively quiescent) mHSC but not to EVs from P1 mHSC. Conversely, gene expression (CCN2, Col1a1, αSMA) in D4 mHSC was stimulated by EVs from P1 mHSC but not by EVs from D4 mHSC. EVs from Day 4 mHSC contained only 46 proteins in which histones and keratins predominated, while EVs from P1 mHSC contained 337 proteins and these were principally associated with extracellular spaces or matrix, proteasome, collagens, vesicular transport, metabolic enzymes, ribosomes and chaperones. EVs from the activated LX-2 human HSC (hHSC) line also promoted fibrogenic gene expression in D4 mHSC in vitro and contained 524 proteins, many of which shared identity or had functional overlap with those in P1 mHSC EVs. The activation-associated changes in production, function and protein content of EVs from HSC likely contribute to the regulation of HSC function in vivo and to the fine-tuning of fibrogenic pathways in the liver.

Biological and Proteomic Characteristics of an Immortalized Human Pancreatic Stellate Cell Line.

Human pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during chronic pancreatitis (CP). However, primary PSCs have a short lifespan in vitro, which seriously affects their use in various applications. We have established a stable immortalized human PSC line (HP-1) by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated human PSCs. HP-1 cells express cytoskeleton proteins including glial fibrillary acidic protein (GFAP), α-smooth muscle actin (α-SMA), vimentin and desmin, and are typical of PSCs, which are high transfeciability and viable in 0.5% serum. The cells express receptors such as TGFßR2, PDGFR, TGF-ß pseudoreceptor Bambi and PPRPγ that are commonly found in PSCs. HP-1 cells are similar to activated human PSCs in that they have enhanced expression of α-SMA, CTGF, Col1 and TIMP-2 mRNAs or proteins, as well as decreased expression of MMP-1/2 mRNAs or proteins in response to TGF-ß1 stimulation. Comparative proteomics revealed 4,537 shared proteins between HP-1 cells and PSCs and no single protein in HP-1 cells versus PSCs. Statistical analysis reveals no significantly difference between HP-1 cells and PSCs in their expression of proteins associated with matrix and matrix remodeling. The similarity between HP-1 cell and PSC is further shown by the finding that only 9 proteins are differentially up-regulated > ± 2-fold in HP-1 cells and 13 proteins are up-regulated > ± 2-fold in PSCs and none of these proteins include ECM proteins, cytokines, growth factors or matrix remodeling regulatory proteins. Therefore, HP-1 cells can be used as an effective tool for the study of PSC-mediated pancreatic fibrosis.

Human Breast Milk-Derived Extracellular Vesicles in the Protection Against Experimental Necrotizing Enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) is a leading cause of death in premature infants. Breast feeding decreases the incidence of NEC but, even with aggressive promotion of nursing in Neonatal Intensive Care Units, morbidity and mortality remain high. Previous studies from our laboratory have demonstrated that extracellular vesicles (EVs) purified from mouse and rat stem cells can protect the intestines from NEC. The aim of this study was to determine whether human breast milk (BM)-derived EVs could prevent NEC. METHODS: EVs were purified from human donor breast milk. NEC was induced in premature rat pups by exposure to asphyxia/hypothermia/hypercaloric feeds. Pups were randomized to: (1) breast fed, no injury, (2) NEC, (3) NEC + BM-derived EVs once intraperitoneally (IP), (4) NEC + BM-derived EVs enterally (PO) with each feed. Intestinal tracts were examined for histologic damage. Additionally, the effect of BM-derived EVs on intestinal epithelial cells (IEC) subjected to hypoxia/reoxygenation injury in vitro was examined. RESULTS: NEC incidence was 0% in breast-fed pups and 62% in pups subjected to NEC. IP administration of BM-derived EVs decreased NEC incidence to 29% and enteral administration further decreased NEC incidence to 11.9%. (p < 0.05). BM-derived EVs significantly increased cell proliferation and decreased apoptosis in IEC in vitro. CONCLUSION: Breast milk-derived EVs delivered either IP or enterally significantly decrease the incidence and severity of experimental NEC, protect IEC from injury in vitro, and may represent an innovative therapeutic option for NEC in the future. TYPE OF STUDY: Basic science study. LEVEL OF EVIDENCE: N/A.

Extracellular Vesicles From Hepatocytes Are Therapeutic for Toxin-Mediated Fibrosis and Gene Expression in the Liver.

Extracellular vesicles (EVs) are nano-sized membrane-limited organelles that are liberated from their producer cells, traverse the intercellular space, and may interact with other cells resulting in the uptake of the EV molecular payload by the recipient cells which may become functionally reprogramed as a result. Previous in vitro studies showed that EVs purified from normal mouse AML12 hepatocytes ("EVNorm") attenuate the pro-fibrogenic activities of activated hepatic stellate cells (HSCs), a principal fibrosis-producing cell type in the liver. In a 10-day CCl4 injury model, liver fibrogenesis, expression of hepatic cellular communication network factor 2 [CCN2, also known as connective tissue growth factor (CTGF)] or alpha smooth muscle actin (αSMA) was dose-dependently blocked during concurrent administration of EVNorm. Hepatic inflammation and expression of inflammatory cytokines were also reduced by EVNorm. In a 5-week CCl4 fibrosis model in mice, interstitial collagen deposition and mRNA and/or protein for collagen 1a1, αSMA or CCN2 were suppressed following administration of EVNorm over the last 2 weeks. RNA sequencing (RNA-seq) revealed that EVNorm therapy of mice receiving CCl4 for 5 weeks resulted in significant differences [false discovery rate (FDR) <0.05] in expression of 233 CCl4-regulated hepatic genes and these were principally associated with fibrosis, cell cycle, cell division, signal transduction, extracellular matrix (ECM), heat shock, cytochromes, drug detoxification, adaptive immunity, and membrane trafficking. Selected gene candidates from these groups were verified by qRT-PCR as targets of EVNorm in CCl4-injured livers. Additionally, EVNorm administration resulted in reduced activation of p53, a predicted upstream regulator of 40% of the genes for which expression was altered by EVNorm following CCl4 liver injury. In vitro, EVs from human HepG2 hepatocytes suppressed fibrogenic gene expression in activated mouse HSC and reversed the reduced viability or proliferation of HepG2 cells or AML12 cells exposed to CCl4. Similarly, EVs produced by primary human hepatocytes (PHH) protected PHH or human LX2 HSC from CCl4-mediated changes in cell number or gene expression in vitro. These findings show that EVs from human or mouse hepatocytes regulate toxin-associated gene expression leading to therapeutic outcomes including suppression of fibrogenesis, hepatocyte damage, and/or inflammation.

Therapeutic effects of serum extracellular vesicles in liver fibrosis.

The lack of approved therapies for hepatic fibrosis seriously limits medical management of patients with chronic liver disease. Since extracellular vesicles (EVs) function as conduits for intercellular molecular transfer, we investigated if EVs from healthy individuals have anti-fibrotic properties. Hepatic fibrogenesis or fibrosis in carbon tetrachloride (CCl4)- or thioacetic acid-induced liver injury models in male or female mice were suppressed by serum EVs from normal mice (EVN) but not from fibrotic mice (EVF). CCl4-treated mice undergoing EVN therapy also exhibited reduced levels of hepatocyte death, inflammatory infiltration, circulating AST/ALT levels and hepatic or circulating pro-inflammatory cytokines. Hepatic histology, liver function tests or circulating proinflammatory cytokine levels were unaltered in control mice receiving EVN. As determined using PKH26-labelled EVN, principal target cells included hepatic stellate cells (HSC; a normally quiescent fibroblastic cell that undergoes injury-induced activation and produces fibrosis during chronic injury) or hepatocytes which showed increased EVN binding after, respectively, activation or exposure to CCl4. In vitro, EVN decreased proliferation and fibrosis-associated molecule expression in activated HSC, while reversing the inhibitory effects of CCl4 or ethanol on hepatocyte proliferation. In mice, microRNA-34c, -151-3p, -483-5p, -532-5p and -687 were more highly expressed in EVN than EVF and mimics of these microRNAs (miRs) individually suppressed fibrogenic gene expression in activated HSC. A role for these miRs in contributing to EVN actions was shown by the ability of their corresponding antagomirs to individually and/or collectively block the therapeutic effects of EVN on activated HSC or injured hepatocytes. Similarly, the activated phenotype of human LX-2 HSC was attenuated by serum EVs from healthy human subjects and contained higher miR-34c, -151-3p, -483-5p or -532-5p than EVs from hepatic fibrosis patients. In conclusion, serum EVs from normal healthy individuals are inherently anti-fibrogenic and anti-fibrotic, and contain microRNAs that have therapeutic actions in activated HSC or injured hepatocytes. Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCl4: carbon tetrachloride; CCN2: connective tissue growth factor; E: eosin; EGFP: enhanced green fluorescent protein; EVs: extracellular vesicles; EVF: serum EVs from mice with experimental hepatic fibrosis; EVN: serum EVs from normal mice; H: hematoxylin; HSC: hepatic stellate cell; IHC: immunohistochemistry; IL: interleukin; MCP-1: monocyte chemotactic protein-1; miR: microRNA; mRNA: messenger RNA; NTA: nanoparticle tracking analysis; PCNA: proliferating cell nuclear antigen; qRT-PCR: quantitative real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulphate - polyacrylamide gel electrophoresis; αSMA: alpha smooth muscle actin; TAA: thioacetic acid; TG: transgenic; TGF-ß: transforming growth factor beta; TEM: transmission electron microscopy; TNFα: tumour necrosis factor alpha.

CD4+Foxp3+CD25+/- Tregs characterize liver tissue specimens of patients suffering from drug-induced autoimmune hepatitis: A clinical-pathological study.

BACKGROUND: The diagnosis of drug-induced autoimmune hepatitis (DIAIH) and its differentiation from idiopathic autoimmune hepatitis (AIH) is challenging. This study aimed to differentiate DIAIH from AIH by comparing the biochemical changes, histological features, and frequencies of CD4+Foxp3+CD25+/- regulatory T cells (Tregs) in liver tissues or peripheral blood lymphocytes. METHODS: A total of 15 DIAIH patients and 24 AIH patients who underwent liver biopsies at initial presentation were enrolled in this study. The liver histological changes were assessed by HE staining. The phenotypic recognition and distribution of CD4+Foxp3+CD25+/- Tregs in liver tissues were evaluated by single/double immunostains in serial sections. The CD4+Foxp3+CD25+/- Tregs in peripheral blood were analyzed by flow cytometry. RESULTS: The median values of ALT and AST were 404.50 U/L and 454.10 U/L in DIAIH patients and 309.50 U/L and 315.00 U/L in AIH patients, respectively. More importantly, for the first time we found that patients with DIAIH had higher levels of serum ALT and AST, more severe degree of lobular inflammation, higher frequencies of zone 3 necrosis and higher number of lobular CD4+Foxp3+CD25-Tregs compared with AIH (P < 0.05). Furthermore, there were positive correlations in DIAIH between the degree of lobular inflammation and either the AST/ALT level or the number of lobular CD4+Foxp3+CD25- Tregs (P < 0.05). However, the frequency of peripheral blood CD4+Foxp3+CD25+/- Tregs were not significantly different between DIAIH and AIH. CONCLUSIONS: The differences of ALT, AST and the number of lobular CD4+Foxp3+CD25- Tregs between patients with DIAIH and those with AIH are clinically helpful in differentiating these two diseases in their early stage.

Lipopolysaccharide enhances TGF-ß1 signalling pathway and rat pancreatic fibrosis.

Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor-beta1 (TGF-ß1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF-ß1 signalling and pancreatic fibrosis. Sprague-Dawley rats fed with a Lieber-DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP-2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF-ß1 which was paralleled by an increased number of TLR4-positive or TGF-ß1-positive Mφs or PSCs in ALC-fed rats. In vitro, TLR4 or TGF-ß1 production in Mφs or RP-2 cells was up-regulated by LPS. LPS alone or in combination with TGF-ß1 significantly increased type I collagen and α-SMA production and Smad2 and 3 phosphorylation in serum-starved RP-2 cells. TGF-ß pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si-TLR4 RNA or by inhibitors of MyD88/NF-kB. Additionally, knockdown of Bambi with Si-Bambi RNA significantly increased TGF-ß1 signalling in RP-2 cells. These findings indicate that LPS increases TGF-ß1 production through paracrine and autocrine mechanisms and that LPS enhances TGF-ß1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF-kB activation.

Role of Gut-Derived Endotoxin on Type I Collagen Production in the Rat Pancreas After Chronic Alcohol Exposure.

BACKGROUND: Pancreatic fibrosis is a key pathological feature of alcoholic chronic pancreatitis (ACP). Bacterial endotoxin lipopolysaccharide (LPS) is considered as an important cofactor in the fibrogenesis of ACP. However, there are limitations in the use of exogenous LPS for evaluating the role of endotoxin in ACP pathogenesis. In this study, we determined the relationship between the concentration of LPS in the portal vein and pancreatic type I collagen (Col1) content in chronic alcohol-fed rats. METHODS: Male Sprague Dawley rats were divided into 2 groups and fed with Lieber-DeCarli isocaloric control (CON) liquid diet or ethanol (EtOH) (15 g/kg/d) liquid diet. Eleven CON or EtOH rats were euthanized at the end of week 8, 9, or 10. The plasma LPS from portal vein was determined. Pancreatic inflammatory injury and fibrosis were assessed. Pancreatic stellate cells (PSCs) and macrophages were identified; pancreatic type I collagen alpha 1 (Col1A1) and Toll-like receptor (TLR4) mRNA and protein were examined; pancreatic chemokines and transforming growth factor-beta1 (TGF-ß1) were determined. RESULTS: Pancreatic inflammatory scores were increased in 10-week EtOH rats compared with CON rats, but there was no significant difference in collagen deposition between 2 groups. The levels of portal vein LPS and pancreatic TLR4 and Col1A1 mRNA and protein were increased in a time-dependent fashion in EtOH rats, with the highest levels occurring at 10 weeks. Additionally, by 8 weeks, pancreatic TLR4 and Col1A1 mRNA in EtOH rats were statistically increased as compared to CON rats, whereas portal vein LPS remained unchanged. The number of PSCs and macrophages and expression of chemokines (MCP-1, MIP-1α, and RANTES), TGF-ß1, or Col1A1 were significantly increased, each of which was positively correlated with the level of portal vein LPS in 10-week EtOH rats. CONCLUSIONS: These results suggest that LPS is associated with alcohol-induced fibrosis in pancreatitis and targeting of bacterial endotoxin may be a promising therapeutic strategy for ACP.

Pathways of production and delivery of hepatocyte exosomes.

Hepatocyte exosomes (ExoHep) are proposed to mediate physiological or pathophysiological signaling in a variety of hepatic target cells. ExoHep were purified from the medium of primary mouse hepatocytes or AML12 cells and characterized as ~100 nm nanovesicles that were positive for proteins commonly found in exosomes (CD9, CD81, flotillin) or hepatocytes (asialoglycoprotein receptor). Ethanol treatment of hepatocytes caused increased ExoHep release and increased cellular mRNA expression of components involved in intracellular vesicle trafficking (Rab 5a,b,c, Rab 7a, Rab 27a,b) or exosome biogenesis via the ESCRT (HGS, Alix, STAM1, TSG101, VTA1, YKT6) or ceramide (nSmase2) pathways. RNA interference of HGS, Alix, TSG101 or nSmase 2 caused exosome production by normal or ethanol-treated hepatocytes to be reduced. In mice, in vivo administration of fluorescently-labeled ExoHep resulted in their accumulation in the liver and preferential localization to hepatic stellate cells (HSC) or hepatocytes, the latter of which showed enhanced ExoHep binding when isolated from fibrotic mice. In cell co-cultures, the intercellular transfer of RNA from hepatocytes to hepatocytes or HSC was blocked by the exosome inhibitor GW4869. ExoHep binding to HSC or hepatocytes occurred via mechanisms that involved heparin-like molecules and cellular integrin αv or ß1 subunits , and resulted in a reversal of fibrosis-associated gene expression in HSC and of ethanol-induced damage in hepatocytes. These studies provide insight regarding the regulation and/or participation of exosome biogenesis or trafficking components in hepatocytes and show that ExoHep can mediate therapeutic changes in activated HSC or injured hepatocytes that occur downstream of heparin- or integrin-dependent binding interactions.

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.