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.

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.

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.

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.

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.

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.

Fibrogenic Signaling Is Suppressed in Hepatic Stellate Cells through Targeting of Connective Tissue Growth Factor (CCN2) by Cellular or Exosomal MicroRNA-199a-5p.

Pathways of liver fibrosis are controlled by connective tissue growth factor (CCN2). In this study, CCN2 was identified as a target of miR-199a-5p, which was principally expressed in quiescent mouse hepatic stellate cells (HSCs) and directly suppressed production of CCN2. Up-regulated CCN2 expression in fibrotic mouse livers or in activated primary mouse HSCs was associated with miR-199a-5p down-regulation. MiR-199a-5p in quiescent mouse HSCs inhibited the activity of a wild-type CCN2 3' untranslated region (3'-UTR) but not of a mutant CCN2 3'-UTR lacking the miR-199a-5p-binding site. In activated mouse HSCs, CCN2, α-smooth muscle actin, and collagen 1(α1) were suppressed by a miR-199a-5p mimic, whereas in quiescent mouse HSCs, the inhibited CCN2 3'-UTR activity was blocked by a miR-199a-5p antagomir. CCN2 3'-UTR activity in human HSCs was reduced by a miR-199a-5p mimic. MiR-199a-5p was present at higher levels in exosomes from quiescent versus activated HSCs. MiR-199a-5p-containing exosomes were shuttled from quiescent mouse HSCs to activated mouse HSCs in which CCN2 3'-UTR activity was then suppressed. Exosomes from quiescent HSCs caused miR-199a-5p-dependent inhibition of CCN2, α-smooth muscle actin, or collagen 1(α1) in activated HSCs in vitro and bound to activated HSCs in vivo. Thus, CCN2 suppression by miR-199a-5p accounts, in part, for low-level fibrogenic gene expression in quiescent HSCs and causes dampened gene expression in activated HSCs after horizontal transfer of miR-199a-5p in exosomes from quiescent HSCs.

Enhanced Steatosis and Fibrosis in Liver of Adult Offspring Exposed to Maternal High-Fat Diet.

Early life exposures can increase the risk of developing chronic diseases including nonalcoholic fatty liver disease. Maternal high-fat diet increases susceptibility to development of steatosis in the offspring. We determined the effect of maternal high-fat diet exposure in utero and during lactation on offspring liver histopathology, particularly fibrosis. Female C57Bl/6J mice were fed a control or high-fat diet (HFD) for 8 weeks and bred with lean males. Nursing dams were continued on the same diet with offspring sacrificed during the perinatal period or maintained on either control or high-fat diet for 12 weeks. Increased hepatocyte proliferation and stellate cell activation were observed in the liver of HFD-exposed pups. Offspring exposed to perinatal high-fat diet and high-fat diet postweaning showed extensive hepatosteatosis compared to offspring on high-fat diet after perinatal control diet. Offspring exposed to perinatal high-fat diet and then placed on control diet for 12 weeks developed steatosis and pericellular fibrosis. Importantly, we found that exposure to perinatal high-fat diet unexpectedly promotes more rapid disease progression of nonalcoholic fatty liver disease, with a sustained fibrotic phenotype, only in adult offspring fed a postweaning control diet.

Suppression of fibrogenic signaling in hepatic stellate cells by Twist1-dependent microRNA-214 expression: Role of exosomes in horizontal transfer of Twist1.

A hallmark of liver fibrosis is the activation of hepatic stellate cells (HSC), which results in their production of fibrotic molecules, a process that is largely regulated by connective tissue growth factor (CCN2). CCN2 is increasingly expressed during HSC activation because of diminished expression of microRNA-214 (miR-214), a product of dynamin 3 opposite strand (DNM3os) that directly suppresses CCN2 mRNA. We show that an E-box in the miR-214 promoter binds the basic helix-loop-helix transcription factor, Twist1, which drives miR-214 expression and results in CCN2 suppression. Twist1 expression was suppressed in HSC of fibrotic livers or in cultured HSC undergoing activation in vitro or after treatment with ethanol. Furthermore, Twist1 decreasingly interacted with DNM3os as HSC underwent activation in vitro. Nanovesicular exosomes secreted by quiescent but not activated HSC contained high levels of Twist1, thus reflecting the suppression of cellular Twist1 during HSC activation. Exosomal Twist1 was intercellularly shuttled between HSC and stimulated expression of miR-214 in the recipient cells, causing expression of CCN2 and its downstream effectors to be suppressed. Additionally, the miR-214 E-box in HSC was also regulated by hepatocyte-derived exosomes, showing that functional transfer of exosomal Twist1 occurs between different cell types. Finally, the levels of Twist1, miR-214, or CCN2 in circulating exosomes from fibrotic mice reflected fibrosis-induced changes in the liver itself, highlighting the potential utility of these and other constituents in serum exosomes as novel circulating biomarkers for liver fibrosis. These findings reveal a unique function for cellular or exosomal Twist1 in CCN2-dependent fibrogenesis.

Epigenetic regulation of connective tissue growth factor by MicroRNA-214 delivery in exosomes from mouse or human hepatic stellate cells.

UNLABELLED: Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC, is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3'-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214, which otherwise inhibited CCN2 expression by directly binding to the CCN2 3'-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50-150 nm in diameter, negatively charged (-26 mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pretreatment of the cells with the exosome inhibitor, GW4869. Coculture of either quiescent HSC or miR-214-transfected activated HSC with CCN2 3'-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild-type CCN2 3'-UTR reporter but not of a mutant CCN2 3'-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3'-UTR. MiR-214 in LX-2 cells was shuttled by way of exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3'-UTR activity or expression of CCN2 downstream targets, including alpha smooth muscle actin or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels. CONCLUSION: Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of intercellular regulation by exosomal miRs.

An immortalized rat pancreatic stellate cell line RP-2 as a new cell model for evaluating pancreatic fibrosis, inflammation and immunity.

BACKGROUND: Pancreatic stellate cells (PSCs) play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells, immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis, inflammation and immunity. METHODS: The presence of cytoskeletal proteins, integrin α5ß1 or TLR4, was determined by immunocytochemistry while the production of desmin, collagen I, MMP-1, MMP-2, TIMP-2, or CD14 was evaluated by Western blotting. The levels of desmin, collagen I, IL-1 and IL-6 mRNA were determined by real-time quantitative PCR. The secretion of cytokines was detected by ELISA. Cell function was assessed using adhesion, migration, or proliferation assays. RESULTS: A stable activated rat PSC cell line (designated as RP-2) was established by RSV promoter/enhancer-driven SV40 large T antigen expression. RP-2 cells retained typical PSC properties, exhibited a myofibroblast-like phenotype and persistently produced desmin. The cells produced collagen I protein, matrix metalloproteinases and inhibitors thereof. RP-2 cells demonstrated typical PSC functions, including proliferation, adherence, and migration, the latter two of which occurred in response to fibronectin and were mediated by integrin α5ß1. TLR4 and its response genes including proinflammatory cytokines (IL-1, IL-6, TNF-alpha) and chemotactic cytokines (MCP-1, MIP-1α, Rantes) were produced by RP-2 cells and activated by LPS. LPS-induced IL-1 or IL-6 mRNA expression in this cell line was fully blocked with MyD88 inhibitor. CONCLUSION: RP-2 cells provide a novel tool for analyzing the properties and functions of PSCs in the pathogenesis of fibrosis, inflammation and immunity in the pancreas.

Regulation of pancreatic inflammation by connective tissue growth factor (CTGF/CCN2).

Pancreatitis is caused by long-term heavy alcohol consumption, which results in injury and death of pancreatic acinar cells (PAC). The PAC play a pivotal role in mediating early inflammatory responses but the underlying mechanisms remain poorly understood. Treatment of C57BL/6 mice with ethanol and cerulein resulted in increased staining for acinar interleukin- 1b (IL-1b), chemokine (C-C motif) ligand 3 (CCL3), or connective tissue growth factor (CTGF/CCN2) by Day 16 and this was associated with increased infiltration of F4/80-positive macrophages and increased expression of pancreatic CTGF/CCN2 mRNA. Compared with wild-type Swiss Webster mice, ethanol treatment of pan-green fluorescent protein (GFP)-CTGF/CCN2 transgenic mice caused enhanced acinar staining for GFP or CTGF/CCN2 and a significant increase in pancreatic infiltration of F4/80-positive macrophages or NIMP-R14-positive neutrophils. Treatment of primary mouse PAC or the rat AR42J PAC line with ethanol or CTGF/CCN2 resulted in enhanced expression of IL-1b or CCL3. Conditioned medium from CTGF/CCN2-treated AR42J cells induced chemotaxis in NR8383 macrophages and this response was abrogated in a dose dependent manner by addition of BX471, an inhibitor of chemokine (C-C motif) receptor 1. These results reveal that acinar CTGF/CCN2 plays a novel role in alcohol-induced inflammatory processes in the pancreas by increasing infiltration of macrophages and neutrophils and increasing acinar production of inflammatory mediators such as IL-1b or CCL3. The early production of CTGF/CCN2 by PAC to drive inflammation is distinct from its previously reported production by pancreatic stellate cells to drive fibrosis at later stages of pancreatic injury.

Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis.

Hematopoiesis occurs in a complex bone marrow microenvironment in which bone marrow stromal cells provide critical support to the process through direct cell contact and indirectly through the secretion of cytokines and growth factors. We report that connective tissue growth factor (Ctgf, also known as Ccn2) is highly expressed in murine bone marrow stromal cells. In contrast, connective tissue growth factor is barely detectable in unfractionated adult bone marrow cells. While connective tissue growth factor has been implicated in hematopoietic malignancies, and is known to play critical roles in skeletogenesis and regulation of bone marrow stromal cells, its role in hematopoiesis has not been described. Here we demonstrate that the absence of connective tissue growth factor in mice results in impaired hematopoiesis. Using a chimeric fetal liver transplantation model, we show that absence of connective tissue growth factor has an impact on B-cell development, in particular from pro-B to more mature stages, which is linked to a requirement for connective tissue growth factor in bone marrow stromal cells. Using in vitro culture systems, we demonstrate that connective tissue growth factor potentiates B-cell proliferation and promotes pro-B to pre-B differentiation in the presence of interleukin-7. This study provides a better understanding of the functions of connective tissue growth factor within the bone marrow, showing the dual regulatory role of the growth factor in skeletogenesis and in stage-specific B lymphopoiesis.

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.

Degenerative grade affects the responses of human nucleus pulposus cells to link-N, CTGF, and TGFß3.

STUDY DESIGN: Cells isolated from moderately and severely degenerated human intervertebral disks (IVDs) cultured in an alginate scaffold. OBJECTIVE: To compare the regenerative potential of moderately versus severely degenerated cells using 3 proanabolic stimulants. SUMMARY OF BACKGROUND DATA: Injection of soluble cell signaling factors has potential to slow the progression of IVD degeneration. Although degenerative grade is thought to be an important factor in targeting therapeutic interventions it remains unknown whether cells in severely degenerated IVDs have impaired metabolic functions compared to lesser degenerative levels or if they are primarily influenced by the altered microenvironment. METHODS: Nucleus pulposus (NP) cells were cultured in alginate for 21 days and treated with 3 different proanabolic stimulants: a growth factor/anti-inflammatory combination of transforming growth factor ß3 (TGFß3)+dexamethasone (Dex), or matricellular proteins connective tissue growth factor (CTGF) or Link-N. They were assayed for metabolic activity, DNA content, glycosaminoglycan, and qRT-PCR gene profiling. RESULTS: Moderately degenerated cells responded to stimulation with increased proliferation, decreased IL-1ß, MMP9, and COL1A1 expression, and upregulated HAS1 as compared with severely degenerated cells. TGFßR1 (ALK5) receptors were expressed at greater levels in moderately than severely degenerated cells. TGFß3+Dex had a notable stimulatory effect on moderately degenerated NP cells with increased anabolic gene expression and decreased COL1A1 and ADAMTS5 gene expression. Link-N and CTGF had similar responses in all assays, and both treatments upregulated IL-1ß expression and had a more catabolic response than TGFß3+Dex, particularly in the more severely degenerated group. All groups, including different degenerative grades, produced similar amounts of glycosaminoglycan. CONCLUSIONS: Proanabolic stimulants alone had limited capacity to overcome the catabolic and proinflammatory cytokine expression of severely degenerated NP cells and likely require additional anti-inflammatory treatments. Moderately degenerated NP cells had greater TGFß receptor 1 expression and better responded to anabolic stimulation.

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.

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.].

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF(+/+) or HB-EGF-null (HB-EGF(-/-)) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF(+/+) or HB-EGF(-/-) mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF(+/+) and HB-EGF(-/-) mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF(+/+) mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF(-/-) mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF(+/+) mice, TAA-stimulated hepatic mRNA levels in HB-EGF(-/-) mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-ß1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF(+/+) mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.