Expression and biological function of the cellular communication network factor 5 (CCN5) in primary liver cells.

The cellular (centralized) communication network (CCN) factor protein family contains six small secreted cysteine-rich proteins sharing high structural similarity. These matricellular proteins have vital biological functions in cell adhesion, migration, cell cycle progression, and control of production and degradation of extracellular matrix. However, in liver the biological functions of CCN proteins become most visible during hepatic injury, disease, and remodeling. In particular, most of the hepatic functions of CCN proteins were derived from CCN2/CTGF, which becomes highly expressed in damaged hepatocytes and acts as a profibrogenic molecule. On the contrary, CCN1/CYR61 seems to have opposite effects, while the biological activity during hepatic fibrosis is somewhat controversially discussed for other CCN family members. In the present study, we analyzed the expression of CCN5/WISP2 in cultures of different types of primary liver cells and in an experimental model of hepatic fibrosis. We found that CCN5 is expressed in hepatic stellate cells, myofibroblasts and portal myofibroblasts, while CCN5 expression is virtually absent in hepatocytes. During hepatic fibrogenesis, CCN5 is significantly upregulated. Overexpression of CCN5 in portal myofibroblasts reduced expression of transforming growth factor-ß receptor I (ALK5) and concomitant Smad2 activation, whereas JunB expression is upregulated. Moreover, elevated expression of CCN5 induces endoplasmic reticulum stress, unfolded protein response and apoptosis in portal myofibroblasts. We suggest that upregulated expression of CCN5 might be an intrinsic control mechanism that counteracts overshooting fibrotic responses in profibrogenic liver cells.

Chronic mineral oil administration increases hepatic inflammation in wild type mice compared to lipocalin 2 null mice.

Lipocalin 2 (LCN2), an acute-phase protein produced during acute liver injury, plays an important role in the innate immune response against bacterial infection via iron scavenging. LCN2 further influences neutrophil development and physiology leading to increased inflammatory responses. We investigated the roles of LCN2 in chronic inflammation and fibrosis, using repeated carbon tetrachloride (CCl4) in mineral-oil injection. Surprisingly, mice treated with the mineral oil vehicle alone showed liver inflammation, evidenced by neutrophil and monocyte-macrophage infiltration. Fluorescence-activated cell sorting (FACS) of isolated liver leukocytes showed significantly high CD45+ leukocyte concentrations in CCl4 mice, but no difference of Ly6G+ neutrophils between mineral oil and CCl4 application. Liver CD11b+ F4/80+ cells counted higher in CCl4 mice, but the proportions of Gr1high, an indicator of inflammation, were significantly higher in mineral oil groups. Liver myeloperoxidase (MPO), expressed in neutrophils and monocytes, showed higher levels in wild type mice compared to Lcn2-/- in both mineral-oil and CCl4 treated groups. Hepatic and serum LCN2 levels were remarkably higher in the mineral oil-injected wild type group compared to the CCl4. Wild type animals receiving mineral oil showed significantly higher inflammatory cytokine- and chemokine mRNA levels compared to Lcn2-/- mice, with no differences in the CCl4 treated groups. RNA sequencing (RNA-Seq) confirmed significant downregulation of gene sets involved in myeloid cell activation and immune responses in Lcn2 null mice receiving chronic mineral oil versus wild-type. We observed significant upregulation of gene sets and proteins involved in cell cycle DNA replication, with downregulation of collagen-containing extracellular matrix genes in Lcn2-/- mice receiving CCl4, compared to the wild type. Consequently, the wild type mice developed slightly more liver fibrosis compared to Lcn2-/- mice, evidenced by higher levels of collagen type I in the CCl4 groups and no liver fibrosis in mineral oil-treated mice. Our findings indicate that serum and hepatic LCN2 levels correlate with hepatic inflammation rather than fibrosis.

Chronic Carbon Tetrachloride Applications Induced Hepatocyte Apoptosis in Lipocalin 2 Null Mice Through Endoplasmic Reticulum Stress and Unfolded Protein Response.

The lack of Lipocalin (LCN2) provokes overwhelming endoplasmic reticulum (ER) stress responses in vitro and in acute toxic liver injury models, resulting in hepatocyte apoptosis. LCN2 is an acute phase protein produced in hepatocytes in response to acute liver injuries. In line with these findings we investigated ER stress responses of Lcn2-/- mice in chronic ER stress using a long-term repetitive carbon tetrachloride (CCl4) injection model. We found chronic CCl4 application to enhance ER stress and unfolded protein responses (UPR), including phosphorylation of eukaryotic initiation factor 2α (eIF2α), increased expression of binding immunoglobulin protein (BiP) and glucose-regulated protein 94 (GRP94). IRE1α/TRAF2/JNK signaling enhanced mitochondrial apoptotic pathways, and showed slightly higher in Lcn2-/- mice compared to the wild type counterparts, leading to increased hepatocyte apoptosis well evidenced by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Hepatocyte injuries were confirmed by significant high serum alanine transaminase (ALT) levels in CCl4-treated Lcn2-/- mice. Lcn2-/- mice furthermore developed mild hepatic steatosis, supporting our finding that ER stress promotes lipogenesis. In a previous report we demonstrated that the pharmacological agent tunicamycin (TM) induced ER stress through altered protein glycosylation and induced high amounts of C/EBP-homologous protein (CHOP), resulting in hepatocyte apoptosis. We compared TM-induced ER stress in wild type, Lcn2-/-, and Chop null (Chop-/-) primary hepatocytes and found Chop-/- hepatocytes to attenuate ER stress responses and resist ER stress-induced hepatocyte apoptosis through canonical eIF2α/GADD34 signaling, inhibiting protein synthesis. Unexpectedly, in later stages of TM incubation, Chop-/- hepatocytes resumed activation of IRE1α/JNK/c-Jun and p38/ATF2 signaling, leading to late hepatocyte apoptosis. This interesting observation indicates Chop-/- mice to be unable to absolutely prevent all types of liver injury, while LCN2 protects the hepatocytes by maintaining homeostasis under ER stress conditions.

Antifibrotic Effects of Amyloid-Beta and Its Loss in Cirrhotic Liver.

The function and regulation of amyloid-beta (Aß) in healthy and diseased liver remains unexplored. Because Aß reduces the integrity of the blood-brain barrier we have examined its potential role in regulating the sinusoidal permeability of normal and cirrhotic liver. Aß and key proteins that generate (beta-secretase 1 and presenilin-1) and degrade it (neprilysin and myelin basic protein) were decreased in human cirrhotic liver. In culture, activated hepatic stellate cells (HSC) internalized Aß more efficiently than astrocytes and HSC degraded Aß leading to suppressed expression of α-smooth muscle actin (α-SMA), collagen 1 and transforming growth factor ß (TGFß). Aß also upregulated sinusoidal permeability marker endothelial NO synthase (eNOS) and decreased TGFß in cultured human liver sinusoidal endothelial cells (hLSEC). Liver Aß levels also correlate with the expression of eNOS in transgenic Alzheimer's disease mice and in human and rodent cirrhosis/fibrosis. These findings suggest a previously unexplored role of Aß in the maintenance of liver sinusoidal permeability and in protection against cirrhosis/fibrosis via attenuation of HSC activation.

α2-Adrenergic Receptor in Liver Fibrosis: Implications for the Adrenoblocker Mesedin.

The noradrenergic system is proposed to play a prominent role in the pathogenesis of liver fibrosis. While α1- and ß-adrenergic receptors (ARs) are suggested to be involved in a multitude of profibrogenic actions, little is known about α2-AR-mediated effects and their expression pattern during liver fibrosis and cirrhosis. We explored the expression of α2-AR in two models of experimental liver fibrosis. We further evaluated the capacity of the α2-AR blocker mesedin to deactivate hepatic stellate cells (HSCs) and to increase the permeability of human liver sinusoidal endothelial cells (hLSECs). The mRNA of α2a-, α2b-, and α2c-AR subtypes was uniformly upregulated in carbon tetrachloride-treated mice vs the controls, while in bile duct-ligated mice, only α2b-AR increased in response to liver injury. In murine HSCs, mesedin led to a decrease in α-smooth muscle actin, transforming growth factor-ß and α2a-AR expression, which was indicated by RT-qPCR, immunocytochemistry, and Western blot analyses. In a hLSEC line, an increased expression of endothelial nitric oxide synthase was detected along with downregulated transforming growth factor-ß. In conclusion, we suggest that the α2-AR blockade alleviates the activation of HSCs and may increase the permeability of liver sinusoids during liver injury.

Dysregulated mesenchymal PDGFR-ß drives kidney fibrosis.

Kidney fibrosis is characterized by expansion and activation of platelet-derived growth factor receptor-ß (PDGFR-ß)-positive mesenchymal cells. To study the consequences of PDGFR-ß activation, we developed a model of primary renal fibrosis using transgenic mice with PDGFR-ß activation specifically in renal mesenchymal cells, driving their pathological proliferation and phenotypic switch toward myofibroblasts. This resulted in progressive mesangioproliferative glomerulonephritis, mesangial sclerosis, and interstitial fibrosis with progressive anemia due to loss of erythropoietin production by fibroblasts. Fibrosis induced secondary tubular epithelial injury at later stages, coinciding with microinflammation, and aggravated the progression of hypertensive and obstructive nephropathy. Inhibition of PDGFR activation reversed fibrosis more effectively in the tubulointerstitium compared to glomeruli. Gene expression signatures in mice with PDGFR-ß activation resembled those found in patients. In conclusion, PDGFR-ß activation alone is sufficient to induce progressive renal fibrosis and failure, mimicking key aspects of chronic kidney disease in humans. Our data provide direct proof that fibrosis per se can drive chronic organ damage and establish a model of primary fibrosis allowing specific studies targeting fibrosis progression and regression.

Identification of Pparγ-modulated miRNA hubs that target the fibrotic tumor microenvironment.

Liver fibrosis interferes with normal liver function and facilitates hepatocellular carcinoma (HCC) development, representing a major threat to human health. Here, we present a comprehensive perspective of microRNA (miRNA) function on targeting the fibrotic microenvironment. Starting from a murine HCC model, we identify a miRNA network composed of 8 miRNA hubs and 54 target genes. We show that let-7, miR-30, miR-29c, miR-335, and miR-338 (collectively termed antifibrotic microRNAs [AF-miRNAs]) down-regulate key structural, signaling, and remodeling components of the extracellular matrix. During fibrogenic transition, these miRNAs are transcriptionally regulated by the transcription factor Pparγ and thus we identify a role of Pparγ as regulator of a functionally related class of AF-miRNAs. The miRNA network is active in human HCC, breast, and lung carcinomas, as well as in 2 independent mouse liver fibrosis models. Therefore, we identify a miRNA:mRNA network that contributes to formation of fibrosis in tumorous and nontumorous organs of mice and humans.

Liver parenchymal cells lacking Lipocalin 2 (LCN2) are prone to endoplasmic reticulum stress and unfolded protein response.

Unfolded protein response (UPR) is an adaptive mechanism allowing the endoplasmic reticulum (ER) to react to an accumulation of unfolded proteins in its lumen, also known as ER stress. The UPR is interconnected with inflammation through several pathways such as reactive oxygen species (ROS) production resulting from the protein folding or alternatively, activation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) via IRE1, or induction of acute phase response (APR). Lipocalin 2 (LCN2) is one of the APR proteins induced under inflammatory conditions and up-regulated during ER stress. Upon incubation of Lcn2-/- and wild type (wt) primary hepatocytes with tunicamycin (TM) or thapsigargin (TG) we found the Lcn2-/- hepatocytes to react with strong UPR to the ER stress, as evidenced by significantly increased levels of Grp94, Bip and Chop mRNA and protein compared to the wt. TM and TG-treated hepatocytes activated p65 NF-κB and JNK, the pathways that respond to stress stimuli and playing a central role in inflammation and apoptosis, respectively. ER stress further activated and cleaved full-length CREBH/CREB3L3, the hepatocyte specific transcription factor to induce systemic inflammatory responses. Upregulation of the C/EBP homologous protein (CHOP) was very prominent in Lcn2-/- hepatocytes and sustained until 48 h, resulting in hepatocyte apoptosis as evidenced by increased cleaved caspase 3. We also explored the UPR of the Lcn2 null mouse livers in acute intoxication and inflammation stages with a single application of lipopolysaccharide (LPS) or carbon tetrachloride (CCl4). The Lcn2 null mice clearly developed stronger UPR in LPS- and CCl4-induced ER stress compared to the wt. Our findings indicate that the upregulation of LCN2 during ER stress-induced inflammatory responses protects hepatocytes from being overwhelmed by UPR upon liver injury.

Portal myofibroblasts are sensitive to CCN-mediated endoplasmic reticulum stress-related apoptosis with potential to attenuate biliary fibrogenesis.

Portal fibroblasts are mesenchyme-derived fibroblasts surrounding the bile ducts, and activated into portal myofibroblasts (pMF) during cholestatic liver injury. pMF express α-smooth muscle actin (α-SMA) and produce the fibrogenic extracellular matrix (ECM) collagen type I and fibronectin, playing important roles in portal fibrosis. A cholestatic bile duct-ligated (BDL) model is characterized by impaired hepatobiliary excretion of bile, leading to increased bile acid accumulation. Accumulation of bile acids is known to induce endoplasmic reticulum (ER) stress leading to liver damage and cell death. Additionally, a BDL fibrotic model is also associated with upregulation of CCN (CYR61, CTGF and NOV) matricellular proteins and reported to induce ER stress both in vitro and in vivo. To explore the effects of CCN proteins, we used adenovirus-mediated CCN1-4 (Ad-CCN1-4) gene transfers into cultured pMF. Overexpression of CCN proteins leads to protein accumulation in the ER lumen, causing ER stress and unfolded protein response (UPR). We further found ER stress and UPR to mitigate fibrogenesis in pMF by decreased cellular production of fibronectin, collagen type 1 and α-SMA. In this scenario, Tauroursodeoxycholic acid, a pharmaceutical chaperone and ER stress inhibitor, attenuated Ad-CCN1-4 induced pMF apoptosis and restored collagen and fibronectin levels. Since hepatic fibrogenesis is accompanied by ER stress and upregulation of CCN proteins in a BDL, we further evaluated ER stress responses after Ad-CCN1 gene transfer in such a model and found overexpressed CCN1 to enhance the ER stress-associated proteins BiP and CHOP with positive cleaved caspase 3 and 9 staining in periportal nonparenchymal cells. This indicates that these nonparenchymal cells, most likely pMF, have the tendency to undergo apoptosis during later stages of BDL. Ad-CCN1 transduction furthermore sensitized pMF for ER stress and apoptosis. We suggest that CCN proteins are key factors in the fibrotic microenvironment impacting pMF survival during fibrogenesis and pMF apoptosis during fibrosis resolution.

N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting.

Lipocalin 2 (LCN2) is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

Data on Lipocalin 2 and phosphatidylinositol 3-kinase signaling in a methionine- and choline-deficient model of non-alcoholic steatohepatitis.

The data presented in this brief report support the research article "Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis" [1, doi: 10.1016/j.bbadis.2017.04.006]. We tested whether the absence of Lipocalin-2 (LCN2) could dysregulate the phosphatidylinositol 3-kinase/protein kinase B (PI3K-PKB) pathway and hepatic homeostasis in Non-Alcoholic-Steatohepatitis (NASH). The article highlights the role of LCN2 in hepatic homeostasis.

Altered mitochondrial and peroxisomal integrity in lipocalin-2-deficient mice with hepatic steatosis.

Lipocalin-2 (LCN2) is a secreted adipokine that transports small hydrophobic molecules such as fatty acids and steroids. LCN2 limits bacterial growth by sequestering iron-containing siderophores and in mammalian liver protects against inflammation, infection, injury and other stressors. Because LCN2 modulates hepatic fat metabolism and homeostasis, we performed a comparative profiling of proteins and lipids of wild type (WT) and Lcn2-deficient mice fed either standard chow or a methionine- and choline-deficient (MCD) diet. Label-free proteomics and 2D-DIGE protein expression profiling revealed differential expression of BRIT1/MCPH1, FABP5, HMGB1, HBB2, and L-FABP, results confirmed by Western blotting. Gene ontology enrichment analysis identified enrichment for genes associated with mitochondrial membrane permeabilization and metabolic processes involving carboxylic acid. Measurements of mitochondrial membrane potential, mitochondrial chelatable iron pool, intracellular lipid peroxidation, and peroxisome numbers in primary hepatocytes confirmed that LCN2 regulates mitochondrial and peroxisomal integrity. Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry imaging identified significant changes to sphingomyelins, triglycerides, and glycerophospholipids in livers of mice fed an MCD diet regardless of LCN2 status. However, two arachidonic acid-containing glycerophospholipids were increased in Lcn2-deficient livers. Thus, LCN2 influences peroxisomal and mitochondrial biology in the liver to maintain triglyceride balance, handle oxidative stress, and control apoptosis.

Adenoviral CCN gene transfers induce in vitro and in vivo endoplasmic reticulum stress and unfolded protein response.

The endoplasmic reticulum (ER) is primarily recognized as the site of synthesis and folding of secreted membrane-bound and certain organelle-targeted proteins. Optimum protein folding requires several factors, including ATP, Ca2+ and an oxidizing environment to allow disulphide-bond formation. ER is highly sensitive to stress that perturb cellular energy levels, the redox state or the Ca2+ concentration. Such stresses reduce the protein folding capacity of the ER, resulting in the accumulation and aggregation of unfolded proteins, a condition referred to as unfolded protein response (UPR). Matricellular proteins of the CCN (CYR61, CTGF, NOV) family play essential roles in extracellular matrix signaling and turnover. They exhibit a similar type of organization and share a closely related primary structure, including 38 conserved cysteine residues. Since CCN1/CYR61 overexpression in hepatic stellate cells (HSC) induces ER stress-related apoptosis, we endeavored to investigate whether the adenovirus mediated gene transfer of other members of CCN proteins incurs ER stress in primary HSC and hepatocytes. We found Ad5-CMV-CCN2, Ad5-CMV-CCN3 and Ad5-CMV-CCN4 to induce ER stress and UPR comparable to Ad5-CMV-CCN1. UPR is a pro-survival response to reduce accumulation of unfolded proteins and restore normal ER functioning. If, however protein aggregation is persistent and the stress cannot be resolved, signaling switches from pro-survival to pro-apoptosis. The observed CCN-induced UPR is relevant in wound healing responses and essential for hepatic tissue repair following liver injury. Adenoviral gene transfer induced massive amounts of matricellular proteins proving to effectively mitigate liver fibrosis if targeted cell specific in HSC and myofibroblasts.

The role of PDGF-D in healthy and fibrotic kidneys.

Platelet-derived growth factor (PDGF)-D, a specific PDGF receptor ß (PDGFR-ß) ligand, mediates mesangial proliferation in vitro and in vivo. However, its role in renal development, physiology, and fibrosis is relatively unknown. In healthy murine kidneys, PDGF-D was found to be expressed on renal mesenchymal cells (mesangial cells, fibroblasts, and vascular smooth muscle cells). During renal fibrosis, PDGF-D and its receptor PDGFR-ß were markedly and similarly upregulated in both human and murine kidneys on activated mesenchymal cells, but PDGF-D was also expressed de novo in injured renal tubular cells. The functional role of PDGF-D was studied in Pdgfd-/- mice, which showed no obvious spontaneous renal phenotype at a young age or during aging. Compared with wild-type littermates, Pdgfd-/- mice had significantly reduced renal interstitial fibrosis in two models of renal scarring: unilateral ureteral obstruction and unilateral ischemia/reperfusion injury. This was associated with reduced phosphorylation of PDGFR-ß and its downstream mediator p38. Systemic adenoviral overexpression of PDGF-D in healthy mice resulted in increased collagen deposition in the kidney interstitium. Thus, PDGF-D is upregulated in murine and human kidney fibrosis, may mediate renal scarring, and is dispensable for normal kidney development and physiological functions. PDGF-D may be a suitable therapeutic target to combat kidney fibrosis.

Pharmacological Intervention in Hepatic Stellate Cell Activation and Hepatic Fibrosis.

The activation and transdifferentiation of hepatic stellate cells (HSCs) into contractile, matrix-producing myofibroblasts (MFBs) are central events in hepatic fibrogenesis. These processes are driven by autocrine- and paracrine-acting soluble factors (i.e., cytokines and chemokines). Proof-of-concept studies of the last decades have shown that both the deactivation and removal of hepatic MFBs as well as antagonizing profibrogenic factors are in principle suitable to attenuate ongoing hepatic fibrosis. Although several drugs show potent antifibrotic activities in experimental models of hepatic fibrosis, there is presently no effective pharmaceutical intervention specifically approved for the treatment of liver fibrosis. Pharmaceutical interventions are generally hampered by insufficient supply of drugs to the diseased liver tissue and/or by adverse effects as a result of affecting non-target cells. Therefore, targeted delivery systems that bind specifically to receptors solely expressed on activated HSCs or transdifferentiated MFBs and delivery systems that can improve drug distribution to the liver in general are urgently needed. In this review, we summarize current strategies for targeted delivery of drugs to the liver and in particular to pro-fibrogenic liver cells. The applicability and efficacy of sequestering molecules, selective protein carriers, lipid-based drug vehicles, viral vectors, transcriptional targeting approaches, therapeutic liver- and HSC-specific nanoparticles, and miRNA-based strategies are discussed. Some of these delivery systems that had already been successfully tested in experimental animal models of ongoing hepatic fibrogenesis are expected to translate into clinically useful therapeutics specifically targeting HSCs.

The PDGF system and its antagonists in liver fibrosis.

Platelet derived growth factor (PDGF) signaling plays an important role in activated hepatic stellate cells and portal fibroblast proliferation, chemotaxis, migration and cell survival. PDGF receptors and ligands are upregulated in experimental liver fibrotic models as well as in human liver fibrotic diseases. Blocking of PDGF signaling ameliorates experimental liver fibrogenesis. The plurality of molecular and cellular activities of PDGF and its involvement in initiation, progression and resolution of hepatic fibrogenesis offers an infinite number of therapeutic possibilities. These include the application of therapeutic antibodies (e.g. AbyD3263, MOR8457) which specifically sequester individual PDGF isoforms or the inhibition of PDGF isoforms by synthetic aptamers. In particular, the isolation of innovative slow off-rate modified aptamers (e.g., SOMAmer SL1 and SL5) that carry functional groups absent in natural nucleic acids by the Systematic Evolution of Ligands by EXponential (SELEX) enrichment technique offers the possibility to design high affinity aptamers that target PDGF isoforms for clinical purposes. Dominant-negative soluble PDGF receptors are also effective in attenuation of hepatic stellate cell proliferation and hepatic fibrogenesis. Moreover, some multikinase inhibitors targeting PDGF signaling have been intensively tested during the last decade and are on the way into advanced preclinical studies and clinical trials. This narrative review aims to gauge the recent progression of research into PDGF systems and liver fibrosis.

CCN1/CYR61 overexpression in hepatic stellate cells induces ER stress-related apoptosis.

CCN1/CYR61 is a matricellular protein of the CCN family, comprising six secreted proteins specifically associated with the extracellular matrix (ECM). CCN1 acts as an enhancer of the cutaneous wound healing process by preventing hypertrophic scar formation through induction of myofibroblast senescence. In liver fibrosis, the senescent cells are primarily derived from activated hepatic stellate cells (HSC) that initially proliferate in response to liver damage and are the major source of ECM. We investigate here the possible use of CCN1 as a senescence inducer to attenuate liver fibrogenesis by means of adenoviral gene transfer in primary HSC, myofibroblasts (MFB) and immortalized HSC lines (i.e. LX-2, CFSC-2G). Infection with Ad5-CMV-CCN1 induced large amounts of CCN1 protein in all these cells, resulting in an overload of the endoplasmic reticulum (ER) and in a compensatory unfolded protein response (UPR). The UPR resulted in upregulation of ER chaperones including BIP/Grp78, Grp94 and led to an activation of IRE1α as evidenced by spliced XBP1 mRNA with IRE1α-induced JNK phosphorylation. The UPR arm PERK and eIF2a was phosphorylated, combined with significant CHOP upregulation. Ad5-CMV-CCN1 induced HSC apoptosis that was evident by proteolytic cleavage of caspase-12, caspase-9 and the executor caspase-3 and positive TUNEL stain. Remarkably, Ad5-CMV-CCN1 effectively reduced collagen type I mRNA expression and protein. We conclude that the matricellular protein CCN1 gene transfer induces HSC apoptosis through ER stress and UPR.

PDGF-D signaling in portal myofibroblasts and hepatic stellate cells proves identical to PDGF-B via both PDGF receptor type α and ß.

UNLABELLED: Platelet-derived growth factor-D (PDGF-D) is one member of PDGF growth factors and known to signal by binding to and activating its cognate receptor type ß (PDGFR-ß). Beside PDGF-B, PDGF-D is a potent growth factor for stellate cell growth and proliferation and therefore potentiates the extracellular matrix deposition in liver fibrogenesis. We aimed to explore the signaling and molecular mechanisms of PDGF-D in liver fibrogenesis using the primary liver portal myofibroblasts and hepatic stellate cells. Unexpectedly we found PDGF-D to bind to PDGFR-α, thus inducing receptor endocytosis and decreasing the amount of PDGFR-α significantly. PDGF-D activates PDGFR-α specific tyrosine 754 and -1018 phosphorylation and CrkII, the adaptor protein that is specifically recruited by activated PDGFR-α. As a novel finding we could also demonstrate that recombinant PDGFR-α-Fc chimera homodimer is able to bind PDGF-D and thus prevent PDGF-D signaling. PDGF-D does induce individual PDGFR-ß specific tyrosine phosphorylation similar to the PDGF-B. Additionally, PDGF-D enhances extracellular matrix accumulation comparable to the PDGF-B isoform. CONCLUSION: PDGF-D signaling in pMF and HSC is identical to that of PDGF-B by binding to both PDGFR-α and -ß.

Bile duct ligation in mice: induction of inflammatory liver injury and fibrosis by obstructive cholestasis.

In most vertebrates, the liver produces bile that is necessary to emulsify absorbed fats and enable the digestion of lipids in the small intestine as well as to excrete bilirubin and other metabolic products. In the liver, the experimental obstruction of the extrahepatic biliary system initiates a complex cascade of pathological events that leads to cholestasis and inflammation resulting in a strong fibrotic reaction originating from the periportal fields. Therefore, surgical ligation of the common bile duct has become the most commonly used model to induce obstructive cholestatic injury in rodents and to study the molecular and cellular events that underlie these pathophysiological mechanisms induced by inappropriate bile flow. In recent years, different surgical techniques have been described that either allow reconnection or reanastomosis after bile duct ligation (BDL), e.g., partial BDL, or other microsurgical methods for specific research questions. However, the most frequently used model is the complete obstruction of the common bile duct that induces a strong fibrotic response after 21 to 28 days. The mortality rate can be high due to infectious complications or technical inaccuracies. Here we provide a detailed surgical procedure for the BDL model in mice that induce a highly reproducible fibrotic response in accordance to the 3R rule for animal welfare postulated by Russel and Burch in 1959.