OBJECTIVE

Promotion of endogenous β-cell mass expansion could facilitate regeneration in patients with diabetes. We discovered that the secreted protein CTGF (aka CCN2) promotes adult β-cell replication and mass regeneration after injury via increasing β-cell immaturity and shortening the replicative refractory period. However, the mechanism of CTGF-mediated β-cell proliferation is unknown. Here we focused on whether CTGF alters cells of the immune system to enhance β-cell replication.

METHODS

Using mouse models for 50% β-cell ablation and conditional, β-cell-specific CTGF induction, we assessed changes in immune cell populations by performing immunolabeling and gene expression analyses. We tested the requirement for macrophages in CTGF-mediated β-cell proliferation via clodronate-based macrophage depletion.

RESULTS

CTGF induction after 50% β-cell ablation increased both macrophages and T-cells in islets. An upregulation in the expression of several macrophage and T-cell chemoattractant genes was also observed in islets. Gene expression analyses suggest an increase in M1 and a decrease in M2 macrophage markers. Depletion of macrophages (without changes in T cell number) blocked CTGF-mediated β-cell proliferation and prevented the increase in β-cell immaturity.

CONCLUSIONS

Our data show that macrophages are critical for CTGF-mediated adult β-cell proliferation in the setting of partial β-cell ablation. This is the first study to link a specific β-cell proliferative factor with immune-mediated β-cell proliferation in a β-cell injury model.

The matricellular protein connective tissue growth factor (CTGF/CCN2) is recognized as key player in the onset of fibrosis in various tissues, including skeletal muscle. In many circumstances, CTGF has been shown to be induced by transforming growth factor beta (TGFβ) and accounting, at least in part, for its biological action. In this study it was verified that in cultured myoblasts CTGF/CCN2 causes their transdifferentiation into myofibroblasts by up-regulating the expression of fibrosis marker proteins α-smooth muscle actin and transgelin. Interestingly, it was also found that the profibrotic effect exerted by CTGF/CCN2 was mediated by the sphingosine kinase (SK)-1/S1P3 signaling axis specifically induced by the treatment with the profibrotic cue. Following CTGF/CCN2-induced up-regulation, S1P3 became the S1P receptor subtype expressed at the highest degree, at least at mRNA level, and was thus capable of readdressing the sphingosine 1-phosphate signaling towards fibrosis rather than myogenic differentiation. Another interesting finding is that CTGF/CCN2 silencing prevented the TGFβ-dependent up-regulation of SK1/S1P3 signaling axis and strongly reduced the profibrotic effect exerted by TGFβ, pointing at a crucial role of endogenous CTGF/CCN2 generated following TGFβ challenge in the transmission of at least part of its profibrotic effect. These results provide new insights into the molecular mechanism by which CTGF/CCN2 drives its biological action and strengthen the concept that SK1/S1P3 axis plays a critical role in the onset of fibrotic cell phenotype.

Efficient myelin regeneration in the central nervous system (CNS) requires the migration, proliferation and differentiation of oligodendrocyte progenitor cells (OPC) into myelinating oligodendrocytes. In demyelinating diseases such as multiple sclerosis (MS), this regenerative process can fail, and therapies targeting myelin repair are currently completely lacking in the clinic. The immune system is emerging as a key regenerative player in many tissues, such as muscle and heart. We recently reported that regulatory T cells (Treg) are required for efficient CNS remyelination. Furthermore, Treg secrete CCN3, a matricellular protein from the CCN family, implicated in regeneration of other tissues. Treg-derived CCN3 promoted oligodendrocyte differentiation and myelination. In contrast, previous studies showed that CCN2 inhibited myelination. These studies highlight the need for further scrutiny of the roles that CCN proteins play in myelin development and regeneration. Collectively, these findings open up exciting avenues of research to uncover the regenerative potential of the adaptive immune system.

Thrombin is a multifunctional serine protease and an important fibrotic mediator that induces CCN2 expression. We previously showed that thrombin induces CCN2 expression via an ASK1-dependent JNK/AP-1 pathway in human lung fibroblasts. In this study, we further investigated the roles of c-Src, JAK2, and STAT3 in thrombin-induced CCN2 expression. Thrombin-induced CCN2 expression and CCN2-Luc activity were attenuated by a JAK inhibitor (AG490) and JAK2DN, STAT3DN, and the STAT decoy ODN. Moreover, transfection of cells with a CCN2-mtSTAT-Luc construct inhibited thrombin-induced CCN2-Luc activity. Treatment of cells with thrombin caused JAK2 phosphorylation at Tyr1007/1008 and STAT3 phosphorylation at Tyr705 in time-dependent manners. Thrombin-induced STAT3 phosphorylation was inhibited by AG490 and JAK2DN. Thrombin-induced STAT3 binding to the CCN2 promoter was analyzed by a DNA-binding affinity pull-down assay. In addition, thrombin-induced CCN2 expression and CCN2-Luc activity were inhibited by c-SrcDN and PP2 (an Src inhibitor). Transfection of cells with c-SrcDN also inhibited thrombin-induced JAK2 and STAT3 phosphorylation. Taken together, these results indicate that thrombin might activate c-Src to induce JAK2 activation, which in turn, causes STAT3 activation, and finally induces CCN2 expression in human lung fibroblasts.

OBJECTIVE

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

METHODS

CCN2 hammerhead ribozyme cDNA plus two self-cleaving sequences were inserted into pTriEx2 to produce pTriCCN2-Rz. Each vector was individually transfected into cultured LX-2 human HSCs, which were then stimulated by addition of transforming growth factor (TGF)-beta1 to the culture medium. Semi-quantitative RT-PCR was used to determine mRNA levels for CCN2 or collagen I, while protein levels of each molecule in cell lysates and conditioned medium were measured by ELISA. Cell-cycle progression of the transfected cells was assessed by flow cytometry.

RESULTS

In pTriEx2-transfected LX-2 cells, TGF-beta1 treatment caused an increase in the mRNA level for CCN2 or collagen I, and an increase in produced and secreted CCN2 or extracellular collagen I protein levels. pTriCCN2-Rz-transfected LX-2 cells showed decreased basal CCN2 or collagen mRNA levels, as well as produced and secreted CCN2 or collagen I protein. Furthermore, the TGF-beta1-induced increase in mRNA or protein for CCN2 or collagen I was inhibited partially in pTriCCN2-Rz-transfected LX-2 cells. Inhibition of CCN2 using hammerhead ribozyme cDNA resulted in fewer of the cells transitioning into S phase.

CONCLUSIONS

Endogenous CCN2 is a mediator of basal or TGF-beta1-induced collagen I production in human HSCs and regulates entry of the cells into S phase.

Non-healing skin wounds remain a significant clinical burden, and in recent years, the regulatory role of matricellular proteins in skin healing has received significant attention. Periostin and CCN2 are both upregulated at day 3 post-wounding in murine skin, where they regulate aspects of the proliferative phase of repair including mesenchymal cell infiltration and myofibroblast differentiation. In this study, we examined 1) the wound phenotype and expression patterns of periostin and CCN2 in non-healing skin wounds in humans and 2) the regulation of their expression in wound fibroblasts by tumor necrosis factor α (TNFα) and transforming growth factor-β1 (TGF-β1). Chronic skin wounds had a pro-inflammatory phenotype, characterized by macrophage infiltration, TNFα immunoreactivity, and neutrophil infiltration. Periostin, but not CCN2, was significantly suppressed in non-healing wound edge tissue at the mRNA and protein level compared with non-involved skin. In vitro, human wound edge fibroblasts populations were still able to proliferate and contract collagen gels. Compared to cells from non-involved skin, periostin and α-SMA mRNA levels increased significantly in the presence of TGF-β1 in wound cells and were significantly decreased by TNFα, but not those of Col1A2 or CCN2. In the presence of both TGF-β1 and TNFα, periostin and α-SMA mRNA levels were significantly reduced compared to TGF-β1 treated wound cells. Effects of TGF-β1 and TNFα on gene expression were also more pronounced in wound edge cells compared to non-involved fibroblasts. We conclude that variations in the expression of periostin and CCN2, are related to an inflammatory microenvironment and the presence of TNFα in human chronic wounds.

Fibrotic disorders are characterized by an increase in extracellular matrix protein expression and deposition, Duchene Muscular Dystrophy being one of them. Among the factors that induce fibrosis are Transforming Growth Factor type β (TGF-β) and the matricellular protein Connective Tissue Growth Factor (CTGF/CCN2), the latter being a target of the TGF-β/SMAD signaling pathway and is the responsible for the profibrotic effects of TGF-β. Both CTGF and TGF are increased in tissues affected by fibrosis but little is known about the regulation of the expression of CTGF mediated by TGF-β in muscle cells. By using luciferase reporter assays, site directed mutagenesis and specific inhibitors in C2C12 cells; we described a novel SMAD Binding Element (SBE) located in the 5' UTR region of the CTGF gene important for the TGF-β-mediated expression of CTGF in myoblasts. In addition, our results suggest that additional transcription factor binding sites (TFBS) present in the 5' UTR of the CTGF gene are important for this expression and that SP1/SP3 factors are involved in TGF-β-mediated CTGF expression.

Fibrosis is a common feature of several chronic diseases and is characterized by exacerbated accumulation of ECM. An understanding of the cellular and molecular mechanisms involved in the development of this condition is crucial for designing efficient treatments for those pathologies. Connective tissue growth factor (CTGF/CCN2) is a pleiotropic protein with strong profibrotic activity. In this report, we present experimental evidence showing that ECM stimulates the synthesis of CTGF in response to lysophosphatidic acid (LPA).The integrin/focal adhesion kinase (FAK) signaling pathway mediates this effect, since CTGF expression is abolished by the use of the Arg-Gly-Asp-Ser peptide and also by an inhibitor of FAK autophosphorylation at tyrosine 397. Cilengitide, a specific inhibitor of αv integrins, inhibits the expression of CTGF mediated by LPA or transforming growth factor β1. We show that ECM obtained from decellularized myofibroblast cultures or derived from activated fibroblasts from muscles of the Duchenne muscular dystrophy mouse model ( mdx) induces the expression of CTGF. This effect is dependent on FAK phosphorylation in response to its activation by integrin. We also found that the fibrotic ECM inhibits skeletal muscle differentiation. This novel regulatory mechanism of CTGF expression could be acting as a positive profibrotic feedback between the ECM and CTGF, revealing a novel concept in the control of fibrosis under chronic damage.

Degenerative disc disease (DDD) is associated with intervertebral disc degeneration of spinal instability. Here, we report that the cilia of nucleus pulposus (NP) cells mediate mechanotransduction to maintain anabolic activity in the discs. We found that mechanical stress promotes transport of parathyroid hormone 1 receptor (PTH1R) to the cilia and enhances parathyroid hormone (PTH) signaling in NP cells. PTH induces transcription of integrin αvβ6 to activate the transforming growth factor (TGF)-β-connective tissue growth factor (CCN2)-matrix proteins signaling cascade. Intermittent injection of PTH (iPTH) effectively attenuates disc degeneration of aged mice by direct signaling through NP cells, specifically improving intervertebral disc height and volume by increasing levels of TGF-β activity, CCN2, and aggrecan. PTH1R is expressed in both mouse and human NP cells. Importantly, knockout PTH1R or cilia in the NP cells results in significant disc degeneration and blunts the effect of PTH on attenuation of aged discs. Thus, mechanical stress-induced transport of PTH1R to the cilia enhances PTH signaling, which helps maintain intervertebral disc homeostasis, particularly during aging, indicating therapeutic potential of iPTH for DDD.

UNASSIGNED

Human and mouse cardiac beta3-adrenergic receptors (beta3AR) exert antipathetic effects to those of beta1-2AR stimulation. We examined their role in modulating myocardial remodelling, particularly fibrosis in response to haemodynamic stress.

UNASSIGNED

Mice with cardiac myocyte-specific expression of beta3AR (ADRB3-tg) or tamoxifen-inducible homozygous deletion (c-Adrb3-ko, with loxP-targeted Adrb3) were submitted to transaortic constriction. A superfusion assay was used for proteomic analysis of paracrine mediators between beta3AR-expressing cardiac myocytes and cardiac fibroblasts cultured separately. We show that cardiac beta3AR attenuate myocardial fibrosis in response to haemodynamic stress. Interstitial fibrosis and collagen content were reduced in ADRB3-tg, but augmented in c-Adrb3-ko. ADRB3 and collagen (COL1A1) expression were also inversely related in ventricular biopsies of patients with valve disease. Incubation of cardiac fibroblasts with media conditioned by hypertrophic myocytes induced fibroblast proliferation, myo-differentiation, and collagen production. These effects were abrogated upon ADRB3 expression in myocytes. Comparative shotgun proteomic analysis of the myocyte secretomes revealed a number of factors differentially regulated by beta3AR, among which connective tissue growth factor [CTGF (CCN2)] was prominently reduced. CTGF was similarly reduced in stressed hearts from ADRB3-tg, but increased in hearts from c-Adrb3-ko mice. CTGF expression was mediated by reactive oxygen species production which was reduced by ADRB3 expression in vitro and in vivo. This antioxidant and anti-fibrotic effect involved beta3AR coupling to the neuronal isoform of nitric oxide synthase (nNOS) in cardiac myocytes, as both were abrogated upon nNOS inhibition or Nos1 homozygous deletion.

UNASSIGNED

Cardiac beta3AR protect from fibrosis in response to haemodynamic stress by modulating nitric oxide and oxidant stress-dependent paracrine signaling to fibroblasts. Specific agonism at beta3AR may offer a new therapeutic modality to prevent cardiac fibrosis.

OBJECTIVE

Osteoarthritis (OA) and rheumatoid arthritis (RA) are widespread disabling joint disorders that are considered to be polygenic in nature. This study investigated the spatial expression patterns of all six known human CCN genes using end-stage OA and RA joint samples.

METHODS

We performed in situ hybridization and histological analysis to investigate the spatial expression patterns of human CCN genes using joint tissues obtained during total knee and hip joint replacement procedures on patients with advanced OA or RA. Normal joint tissues taken while performing bipolar hip replacement surgeries were used as controls.

RESULTS

All CCN genes were expressed at higher levels in OA and RA synovial samples as compared with normal controls. Whereas CCN3 and CCN6 were undetectable in control, OA, and RA cartilage, CCN1, CCN2, CCN4, and CCN5 were expressed to a greater extent in OA and RA knee cartilage.

CONCLUSIONS

Our results indicate an involvement of several CCN genes in the pathophysiology of OA and RA.

Knowledge of the microenvironment of articular cartilage in health and disease is the key to accomplishing fundamental disease-modifying treatments for osteoarthritis. The proteins comprising the CCN Family are matricellular proteins with a remarkable relevance within the context of cartilage metabolism. CCN2 displays a great capability for regenerating articular cartilage, and CCN3 has been shown to activate the expression of genes related to articular chondrocytes and to repress genes related to endochondral ossification in epiphyseal chondrocytes. Moreover, mice lacking CCN3 protein have been shown to display ostearthritic changes in their knee articular cartilage. In this study, we employed a monoiodoacetic acid (MIA)-induced osteoarthritic model to investigate whether osteoarthritic changes in the cartilage are reciprocally accompanied by CCN3 down-regulation and an inducible overexpression system to evaluate the effects of CCN3 on articular chondrocytes in vitro. Finally, we also investigated the effects of exogenous CCN3 in vivo during the early stages of MIA-induced osteoarthritis. We discovered that CCN3 is expressed by articular chondrocytes in normal rat knees, whereas it is rapidly down-regulated in osteoarthritic knees. In vitro, we also discovered that CCN3 increases the proteoglycan accumulation, the gene expression of type II collagen, tenascin-C and lubricin, as well as the protein production of tenascin-C and lubricin in articular chondrocytes. In vivo, it was discovered that exogenous CCN3 increased tidemark integrity and produced an increased production of lubricin protein. The potential utility of CCN3 as a future therapeutic agent and possible strategies to improve its therapeutic functions are also discussed.

Excessive production of connective tissue growth factor (CTGF, CCN2) and increased motor ability of the activated fibroblast phenotype contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, molecules and signal pathways regulating CCN2 expression and migration of lung fibroblasts are still elusive. We hypothesize that rapamycin, via binding and blocking mammalian target of rapamycin (mTOR) complex (mTORC), affects CCN2 expression and migration of lung fibroblasts in vitro. Primary normal and fibrotic human lung fibroblasts were isolated from lung tissues of three patients with primary spontaneous pneumothorax and three with IPF. Cells were incubated with regular medium, or medium containing rapamycin, human recombinant transforming growth factor (TGF)-β1, or both. CCN2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression in cells or supernatant was detected. Wound healing and migration assay was used to measure the migratory potential. TGF-β type I receptor (TβRI)/Smad inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. We demonstrated that rapamycin amplified basal or TGF-β1-induced CCN2 mRNA and protein expression in normal or fibrotic fibroblasts by Smad-independent but PI3K-dependent pathway. Additionally, rapamycin also enhanced TIMP-1 expression as indicated by ELISA. However, wound healing and migrating assay showed rapamycin did not affect the mobility of fibroblasts. Collectively, this study implies a significant fibrogenic induction activity of rapamycin by activating AKT and inducing CCN2 expression in vitro and provides the possible mechanisms for the in vivo findings which previously showed no antifibrotic effect of rapamycin on lung fibrosis.

Malignant mesothelioma is an aggressive neoplasm with no particularly effective treatments. We previously reported that overexpression of connective tissue growth factor (CTGF/CCN2) promotes mesothelioma growth, thus suggesting it as a novel molecular target. A human monoclonal antibody that antagonizes CTGF (FG-3019, pamrevlumab) attenuates malignant properties of different kinds of human cancers and is currently under clinical trial for the treatment of pancreatic cancer. This study reports the effects of FG-3019 on human mesothelioma in vitro and in vivo. We analyzed the effects of FG-3019 on the proliferation, apoptosis, migration/invasion, adhesion and anchorage-independent growth in three human mesothelioma cell lines, among which ACC-MESO-4 was most efficiently blocked with FG-3019 and was chosen for in vivo experiments. We also evaluated the coexistent effects of fibroblasts on mesothelioma in vitro, which are also known to produce CTGF in various pathologic situations. Coexistent fibroblasts in transwell systems remarkably promoted the proliferation and migration/invasion of mesothelioma cells. In orthotopic nude mice model, FG-3019 significantly inhibited mesothelioma growth. Histological analyses revealed that FG-3019 not only inhibited the proliferation but also induced apoptosis in both mesothelioma cells and fibroblasts. Our data suggest that FG-3019 antibody therapy could be a novel additional choice for the treatment of mesothelioma.

We recently reported that transgenic mice with cardiac-restricted overexpression of CCN2/CTGF have substantially increased tolerance towards ischemia/reperfusion injury. The purpose of this study was to investigate to what extent fully differentiated cardiac myocytes are direct targets of CCN2, and to resolve the signaling mechanisms that convey the cardioprotective actions of CCN2. Akt and GSK-3β were identified as putative intermediaries of intracellular signaling stimulated by recombinant human CCN2 (rhCCN2). Concentration-effect experiments revealed CCN2-stimulated phosphorylation of Akt (Ser473) and downstream GSK-3β (Ser9) with EC50 ~250 nmol/L. CCN2-stimulated phosphorylation of Akt and GSK-3β was sensitive to inhibition of PI3-kinase (LY294002). Phosphorylation of GSK-3β was also sensitive to Akt-inhibition (API-2), demonstrating CCN2-engendered activation of a PI3-kinase/Akt/GSK-3β-signaling pathway. A C-terminal peptide fragment of CCN2 (11.2 kD) displayed partial agonist activity, while two short peptides derived from the Thrombospondin- and the IGFBP- homology domains of CCN2, respectively, additively inhibited rhCCN2-stimulated Akt-phosphorylation. The viability of cardiac myocytes subjected to hypoxia/reoxygenation injury or doxorubicin-induced oxidative stress was assessed by assays of adenylate kinase and lactate dehydrogenase released from dying cells. Cardiac myocytes exposed to CCN2 displayed increased tolerance towards hypoxia/reoxygenation and doxorubicin-induced oxidative stress, an effect that was abrogated by inhibition of PI3-kinase. The cytoprotective actions of CCN2 reflected in the transcriptome of CCN2-stimulated cardiac myocytes (anti-apoptosis, stress, and wound-response gene programs). In conclusion, this study discloses the novel findings that cardiac myocytes are CCN2 target cells in which CCN2 increases tolerance towards hypoxia and oxidative stress via PI3-kinase-dependent Akt/GSK-3β signaling.

Systemic sclerosis (SSc) is characterized by fibrosis of the skin and internal organs. The present study was undertaken to examine the effects of ciprofloxacin, a fluoroquinolone antibiotic implicated in matrix remodeling, on dermal and lung fibroblasts obtained from SSc patients. Dermal and lung fibroblasts from SSc patients and healthy subjects were treated with ciprofloxacin. Western blotting was used to analyze protein levels and RT-PCR was used to measure mRNA expression. The pharmacologic inhibitor UO126 was used to block Erk1/2 signaling. SSc dermal fibroblasts demonstrated a significant decrease in collagen type I mRNA and protein levels after antibiotic treatment, while healthy dermal fibroblasts were less sensitive to ciprofloxacin, downregulating collagen only at the protein levels. Connective tissue growth factor (CCN2) gene expression was significantly reduced and matrix metalloproteinase 1 (MMP1) levels were enhanced after ciprofloxacin treatment to a similar extent in healthy and SSc fibroblasts. Ciprofloxacin induced Erk1/2 phosphorylation, and Erk1/2 blockade completely prevented MMP1 upregulation. However, Smad1 and Smad3 activation in response to TGFβ was not affected. The expression of friend leukemia integration factor 1 (Fli1), a transcriptional repressor of collagen, was increased after treatment with ciprofloxacin only in SSc fibroblasts, and this was accompanied by a decrease in the levels of DNA methyltransferase 1 (Dnmt1). Similar effects were observed in SSc-interstitial lung disease (ILD) lung fibroblasts. In summary, our study demonstrates that ciprofloxacin has antifibrotic actions in SSc dermal and lung fibroblasts via the downregulation of Dnmt1, the upregulation of Fli1 and induction of MMP1 gene expression via an Erk1/2-dependent mechanism. Thus, our data suggest that ciprofloxacin may be an attractive therapy for SSc skin and lung fibrosis.

CCN family protein 2/connective tissue growth factor (CCN2/CTGF) promotes cartilage regeneration in experimental osteoarthritis (OA) models. However, CCN2 production is very low in articular cartilage. The aim of this study was to investigate whether or not CCN2 was promoted by cultured chondrocytes treated with low-intensity pulsed ultrasound (LIPUS) and to clarify its mechanism.

Human chondrocytic cell line (HCS)-2/8, rat primary epiphyseal and articular cartilage cells, and Ccn2-deficient chondrocytes that impaired chondrocyte differentiation, were treated with LIPUS for 20 min at 3.0 MHz frequency and 60 mW/cm2 power. Expressions of chondrocyte differentiation marker mRNAs were examined by real-time PCR (RT-PCR) analysis from HCS-2/8 cells and Ccn2-deficient chondrocytes at 30 min and 1 h after LIPUS treatment, respectively. CCN2 production was examined by Western blotting after 5 h of LIPUS treatment. Moreover, Ca2+ influx was measured by using a Fluo-4 probe.

The gene expression of chondrocyte differentiation markers and CCN2 production were increased in cultured chondrocytes treated with LIPUS. In addition, Ca2+ influx and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)1/2 were increased by LIPUS treatment, and the stability of TRPV4 and BKca channel mRNAs was decreased by siRNA against CCN2. Consistent with those findings, the LIPUS-induced the gene expressions of type II collagen (COL2a1) and Aggrecan (ACAN) observed in wild-type cells were not observed in the Ccn2-deficient chondrocytes.

These data indicate that chondrocyte differentiation represented by CCN2 production was mediated via MAPK pathways activated by LIPUS-stimulated Ca2+ influx, which in turn was supported by the induced CCN2 molecules in articular chondrocytes.

OBJECTIVE

Connective tissue growth factor (CTGF/CCN2) is a developmental gene upregulated in pathological conditions, including cardiovascular diseases, whose product is a matricellular protein that can be degraded to biologically active fragments. Among them, the C-terminal module IV [CCN2(IV)] regulates many cellular functions, but there are no data about redox process. Therefore, we investigated whether CCN2(IV) through redox signaling regulates vascular responses.

RESULTS

CCN2(IV) increased superoxide anion (O2(•-)) production in murine aorta (ex vivo and in vivo) and in cultured vascular smooth muscle cells (VSMCs). In isolated murine aorta, CCN2(IV), via O2(•-), increased phenylephrine-induced vascular contraction. CCN2(IV) in vivo regulated several redox-related processes in mice aorta, including increased nonphagocytic NAD(P)H oxidases (Nox)1 activity, protein nitrosylation, endothelial dysfunction, and activation of the nuclear factor-κB (NF-κB) pathway and its related proinflammatory factors. The role of Nox1 in CCN2(IV)-mediated vascular responses in vivo was investigated by gene silencing. The administration of a Nox1 morpholino diminished aortic O2(•-) production, endothelial dysfunction, NF-κB activation, and overexpression of proinflammatory genes in CCN2(IV)-injected mice. The link CCN2(IV)/Nox1/NF-κB/inflammation was confirmed in cultured VSMCs. Epidermal growth factor receptor (EGFR) is a known CCN2 receptor. In VSMCs, CCN2(IV) activates EGFR signaling. Moreover, EGFR kinase inhibition blocked vascular responses in CCN2(IV)-injected mice.

CONCLUSIONS

CCN2(IV) is a novel prooxidant factor that in VSMCs induces O2(•-) production via EGFR/Nox1 activation. Our in vivo data demonstrate that CCN2(IV) through EGFR/Nox1 signaling pathway induces endothelial dysfunction and activation of the NF-κB inflammatory pathway. Therefore, CCN2(IV) could be considered a potential therapeutic target for redox-related cardiovascular diseases.

Postpartum psychosis (PP) is a severe psychiatric disorder affecting a small proportion of new mothers shortly after childbirth. The molecular pathophysiology underlying the disorder is currently poorly understood, and there are no amenable animal models for the condition; maternal deficiency for the enzyme steroid sulfatase has been proposed as a potential risk mechanism. Here we show that inhibition of steroid sulfatase with 667-COUMATE (10mg/kg p.o.) in new mouse mothers results in behavioural abnormalities that can be partially alleviated by the administration of the clinically-efficacious antipsychotic ziprasidone (0.3-1.0mg/kg i.p.). The pattern of behavioural abnormalities in 667-COUMATE-treated mice implicated a genetic substrate at 21-23cM on chromosome 15; of the 17 genes within this chromosomal interval, only one (Nov/Ccn3) was significantly differentially expressed in the brains of vehicle and 667-COUMATE-treated mice. Two additional members of the Ccn family (Ccn2/Ctgf and Ccn4/Wisp1) were also significantly differentially expressed between the two groups, as were three further genes co-expressed with Nov/Ccn3 in brain (Arhgdig) or previously implicated in disorder risk by clinical studies (Adcy8 and Ccl2). The expression of Nov/Ccn3, but not of the other differentially-expressed genes, could be normalised by ziprasidone administration (1.0mg/kg). NOV/CCN3 lies directly under a linkage peak for PP risk at 8q24, and the associated protein possesses numerous characteristics that make it an excellent candidate mediator of PP risk. Our data suggest the 667-COUMATE-treated mouse as a model for PP with some degree of face, construct, and predictive validity, and implicate a novel, and biologically-plausible, molecular risk pathway for PP.

Muscular fibrosis is caused by excessive accumulation of extracellular matrix (ECM) that replaces functional tissue, and it is a feature of several myopathies and neuropathies. Knowledge of the biology and regulation of pro-fibrotic factors is critical for the development of new therapeutic strategies. Upon unilateral sciatic nerve transection, we observed accumulation of ECM proteins such as collagen and fibronectin in the denervated hindlimb, together with increased levels of the profibrotic factors transforming growth factor type β (TGF-β) and connective tissue growth factor (CTGF/CCN2). In mice hemizygous for CTGF/CCN2 or in mice treated with a blocking antibody against CTGF/CCN2, we observed reduced accumulation of ECM proteins after denervation as compared to control mice, with no changes in fibro/adipogenic progenitors (FAPs), suggesting a direct role of CTGF/CCN2 on denervation-induced fibrosis. During time course experiments, we observed that ECM proteins and CTGF/CCN2 levels are increased early after denervation (2-4 days), while TGF-β signaling shows a delayed kinetics of appearance (1-2 weeks). Furthermore, blockade of TGF-β signaling does not decrease fibronectin or CTGF levels after 4 days of denervation. These results suggest that in our model CTGF/CCN2 is not up-regulated by canonical TGF-β signaling early after denervation and that other factors are likely involved in the early fibrotic response following skeletal muscle denervation.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease with unknown etiology and undefined treatment modality. Fibroblasts are regarded as the major cell type that mediates the onset and progression of lung fibrosis by secreting large amounts of extracellular matrix (ECM) proteins, such as connective tissue growth factor (CTGF/CCN2). Current knowledge confers a crucial role of CCN2 in lung fibrosis. CCN5, another member of the CCN family, has been suggested to play an inhibitory role in some fibrotic diseases, such as cardiac fibrosis. However, the role of CCN5 in the process of IPF remains unknown. In the present study, using western blot analysis, we demonstrate that CCN2 is highly expressed in fibroblasts derived from IPF tissue, but is only slightly expressed in normal human lung fibroblasts. However, CCN5 was weakly expressed in all the above cells. qRT-PCR revealed that transforming growth factor (TGF)-β1 stimulation increased CCN2 expression in the IPF-derived cultures of primary human lung fibroblasts (PIFs) in a time- and concentration-dependent manner, but only slightly affected the expression of CCN5. The overexpression of CCN5 induced by the transfection of PIFs with recombinant plasmid did not affect cell viability, proliferation and apoptosis; however, it significantly suppressed the expression of CCN2, α-smooth muscle actin (α-SMA) and collagen type I. The TGF-β1-induced upregulation of the phosphorylation of Akt was reversed by CCN5 overexpression. Our results also demonstrated that adenovirus-mediated CCN5 overexpression in a mouse model of bleomycin-induced IPF significantly decreased the hydroxyproline content in the lungs, as well as TGF-β1 expression in bronchoalveolar lavage fluid. Taken together, our data demonstrate that CCN5 exerts an inhibitory effect on the fibrotic phenotypes of pulmonary fibroblasts in vitro and in vivo, and as such may be a promising target for the treatment of IPF.

Systemic sclerosis (SSc) is a multi-organ fibrotic disease with few treatment options. Activated fibroblasts are the key effector cells in SSc responsible for the excessive production of collagen and the development of fibrosis. Platelet-derived growth factor (PDGF), a potent mitogen for cells of mesenchymal origin, has been implicated in the activation of SSc fibroblasts. Our aim was to examine the therapeutic potential of crenolanib, an inhibitor of PDGF receptor signaling, in cultured fibroblasts and in angiotensin II-induced skin and heart fibrosis. Crenolanib effectively inhibited proliferation and migration of SSc and healthy control fibroblasts and attenuated basal and transforming growth factor-β-induced expression of CCN2/CTGF and periostin. In contrast to healthy control fibroblasts, SSc fibroblasts proliferated in response to PDGFAA, whereas a combination of PDGFAA and CCN2 was required to elicit a similar response in healthy control fibroblasts. PDGF receptor α mRNA correlated with CCN2 and other fibrotic markers in the skin of SSc patients. In mice challenged with angiotensin II, PDGF receptor α-positive cells were increased in the skin and heart. These PDGF receptor α-positive cells co-localized with PDGF receptor β, procollagen, and periostin. Treatment with crenolanib attenuated the skin and heart fibrosis. Our data indicate that inhibition of PDGF signaling presents an attractive therapeutic approach for SSc.

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.