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.

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.

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 studies showed that EVs purified from normal mouse AML12 hepatocytes ("EV") attenuate the pro-fibrogenic activities of activated hepatic stellate cells (HSCs), a principal fibrosis-producing cell type in the liver. In a 10-day CCl 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 EV.

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.

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 (CCl)- 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).

Pathways of production and delivery of hepatocyte exosomes.

Hepatocyte exosomes (Exo) are proposed to mediate physiological or pathophysiological signaling in a variety of hepatic target cells. Exo 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 Exo 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.

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.

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.

Exosomes mediate intercellular transfer of pro-fibrogenic connective tissue growth factor (CCN2) between hepatic stellate cells, the principal fibrotic cells in the liver.

Background: Fibrogenic pathways in the liver are principally regulated by hepatic stellate cells (HSC), which produce and respond to fibrotic mediators such as connective tissue growth factor (CCN2). The aim of this study was to determine whether CCN2 is shuttled between HSC in membranous nanovesicles, or "exosomes."

Methods: Exosomes were incubated with HSC after isolation from conditioned medium of control or CCN2-green fluorescent protein (GFP)-transfected primary mouse HSC or human LX-2 HSC.

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.

Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived exosomes.

Pancreatitis is an inflammatory condition of the pancreas which, in its chronic form, involves tissue destruction, exocrine and endocrine insufficiency, increased risk of pancreatic cancer, and an extensive fibrotic pathology which is due to unrelenting collagen deposition by pancreatic stellate cells (PSC). In response to noxious agents such as alcohol-excessive consumption of which is a major cause of pancreatitis in the West-normally quiescent PSC undergo a phenotypic and functional transition to activated myofibroblasts which produce and deposit collagen at high levels. This process is regulated by connective tissue growth factor (CCN2), expression of which is highly up-regulated in activated PSC.

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.

Susceptibility to liver fibrosis in mice expressing a connective tissue growth factor transgene in hepatocytes.

Unlabelled: Connective tissue growth factor (CCN2) is a matricellular protein that is up-regulated in many fibrotic disorders and coexpressed with transforming growth factor beta. CCN2 promotes fibrogenesis and survival in activated hepatic stellate cells, and injured or fibrotic liver contains up-regulated levels of CCN2 that are produced by a variety of different cell types, including hepatocytes. To investigate CCN2 action in vivo, transgenic FVB mice were created in which the human CCN2 gene was placed under the control of the albumin enhancer promoter to elevate hepatocyte CCN2 levels.

Generation of neutralizing activity against human immunodeficiency virus type 1 in serum by antibody gene transfer.

Although several human immunodeficiency virus (HIV) vaccine approaches have elicited meaningful antigen-specific T-cell responses in animal models, no single vaccine candidate has engendered antibodies that broadly neutralize primary isolates of HIV type 1 (HIV-1). Thus, there remains a significant gap in the design of HIV vaccines. To address this issue, we exploited the existence of rare human monoclonal antibodies that have been isolated from HIV-infected individuals.