Activation of nuclear factor kappa B (NF-κB) by connective tissue growth factor (CCN2) is involved in sustaining the survival of primary rat hepatic stellate cells

BACKGROUND/AIMS: Connective tissue growth factor (CCN2) is a matricellular protein that plays a role in hepatic stellate cell (HSC)-mediated fibrogenesis. The aim of this study was to investigate the regulation by CCN2 of cell survival pathways in primary HSC. METHODS: Primary HSC were obtained by in situ enzymatic perfusion of rat liver. NF-κB activation was assessed by immunoblotting for IκBα phosphorylation and degradation and by NF-κB p50 or p65 nuclear accumulation. NF-κB DNA-binding activity was determined by gel mobility shift assay while NF-κB response gene expression was evaluated using a luciferase reporter. Cell viability was assessed by Trypan blue staining or ATP luminescent assay while apoptosis was evaluated by caspase-3 activity. RESULTS: CCN2 induced IκBα phosphorylation and degradation as well as nuclear accumulation of NF-κB. Activated NF-κB comprised three dimers, p65/p65, p65/p50 and p50/p50, that individually bound to DNA-binding sites and subsequently triggered transcriptional activity. This was confirmed by showing that CCN2 promoted activity of a NF-κB luciferase reporter. CCN2 promoted survival of serum-starved HSC and protected the cells from death induced by blocking the NF-κB signaling pathway using Bay-11-7082, a specific inhibitor of IκBα phosphorylation. CONCLUSION: CCN2 contributes to the survival of primary HSC through the NF-κB pathway

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

Background: The pro-fibrogenic cytokine connective tissue growth factor (CTGF) plays an important role in the development and progression of fibrosis in many organ systems, including liver. However, its role in the pathogenesis of hepatitis C virus (HCV)-induced liver fibrosis remains unclear. Methods: In the present study, we assessed CTGF expression in HCV-infected hepatocytes using replicon cells containing full-length HCV genotype 1 and the infectious HCV clone JFH1 (HCV genotype 2) by real-time PCR, Western blot analysis and confocal microscopy. We evaluated transforming growth factor β1 (TGF-β1) as a key upstream mediator of CTGF production using neutralizing antibodies and shRNAs. We also determined the signaling molecules involved in CTGF production using various immunological techniques. Results: We demonstrated an enhanced expression of CTGF in two independent models of HCV infection. We also demonstrated that HCV induced CTGF expression in a TGF-β1-dependent manner. Further dissection of the molecular mechanisms revealed that CTGF production was mediated through sequential activation of MAPkinase and Smad-dependent pathways. Finally, to determine whether CTGF regulates fibrosis, we showed that shRNA-mediated knock-down of CTGF resulted in reduced expression of fibrotic markers in HCV replicon cells. Conclusion: Our studies demonstrate a central role for CTGF expression in HCV-induced liver fibrosis and highlight the potential value of developing CTGF-based anti-fibrotic therapies to counter HCV-induced liver damage

Production of Heparin-Binding Epidermal Growth Factor–like Growth Factor (HB-EGF) at Sites of Thermal Injury in Pediatric Patients

Fluids that accumulate at wound sites may be an important reservoir of growth factors that promote the normal wound healing response. The presence of heparin-binding growth factors was studied in burn wound fluid (BWF) from 45 pediatric patients who had sustained partial thickness burns. One of the growth factors present was similar to platelet-derived growth factor (PDGF) based on its heparin affinity, inhibition of bioactivity by a PDGF antiserum, and detection in a PDGF-AB enzyme-linked iminunosorbent assay. A second growth factor was identified as heparin-binding epidermal growth factor–like growth factor (HB-EGF) based on its heparin affinity, competition with 125I-labeled epidermal growth factor (EGF) for EGF receptor binding, and recognition in biological assays and Western blots by two HB-EGF antisera. Amino acid sequence analysis of one form of this second growth factor verified its identity as an N-terminally truncated form of HB-EGF. Immunohistochemical analysis of partial thickness burns demonstrated the presence of HB-EGF in the advancing epithelial margin, islands of regenerating epithelium within the burn wound, and in the duct and proximal tubules of eccrine sweat glands. HB-EGF in the surface epithelium of burn wounds was uniformally distributed, whereas it was restricted to the basal epithelium in nonburned skin. These data support a role for PDGF and HB-EGF in burn wound healing and suggest that the response to injury includes deposition of HB-EGF and PDGF into blister fluid and a redistribution of HB-EGF in the surface epithelium near the wound site

Strategies for blocking the fibrogenic actions of connective tissue growth factor (CCN2): From pharmacological inhibition in vitro to targeted siRNA therapy in vivo

Connective tissue growth factor (CCN2) is a major pro-fibrotic factor that frequently acts downstream of transforming growth factor beta (TGF-β)-mediated fibrogenic pathways. Much of our knowledge of CCN2 in fibrosis has come from studies in which its production or activity have been experimentally attenuated. These studies, performed both in vitro and in animal models, have demonstrated the utility of pharmacological inhibitors (e.g. tumor necrosis factor alpha (TNF-α), prostaglandins, peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonists, statins, kinase inhibitors), neutralizing antibodies, antisense oligonucleotides, or small interfering RNA (siRNA) to probe the role of CCN2 in fibrogenic pathways. These investigations have allowed the mechanisms regulating CCN2 production to be more clearly defined, have shown that CCN2 is a rational anti-fibrotic target, and have established a framework for developing effective modalities of therapeutic intervention in vivo

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

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

Connective tissue growth factor hammerhead ribozyme attenuates human hepatic stellate cell function

AIM: To determine the effect of hammerhead ribozyme targeting connective tissue growth factor (CCN2) on human hepatic stellate cell (HSC) function

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