Exposure to sufficiently high doses of ionizing radiation is known to cause fibrosis in many different organs and tissues. Connective tissue growth factor (CTGF/CCN2), a member of the CCN family of matricellular proteins, plays an important role in the development of fibrosis in multiple organs. The aim of the present study was to quantify the gene and protein expression of CTGF in a variety of organs from non-human primates (NHP) that were previously exposed to potentially lethal doses of radiation. Tissues from non-irradiated NHP and NHP exposed to whole thoracic lung irradiation (WTLI) or partial-body irradiation with 5% bone marrow sparing (PBI/BM5) were examined by real-time quantitative reverse transcription PCR, western blot, and immunohistochemistry. Expression of CTGF was elevated in the lung tissues of NHP exposed to WTLI relative to the lung tissues of the non-irradiated NHP. Increased expression of CTGF was also observed in multiple organs from NHP exposed to PBI/BM5 compared to non-irradiated NHP; these included the lung, kidney, spleen, thymus, and liver. These irradiated organs also exhibited histological evidence of increased collagen deposition compared to the control tissues. There was significant correlation of CTGF expression with collagen deposition in the lung and spleen of NHP exposed to PBI/BM5. Significant correlations were observed between spleen and multiple organs on CTGF expression and collagen deposition, respectively, suggesting possible crosstalk between spleen and other organs. These data suggest that CTGF levels are increased in multiple organs after radiation exposure and that inflammatory cell infiltration may contribute to the elevated levels of CTGF in multiple organs.

OBJECTIVE

Platelet-rich plasma (PRP) has been widely used to enhance the regeneration of damaged joint tissues, such as osteoarthritic and rheumatoid arthritic cartilage. The aim of this study is to clarify the involvement of all of the CCN family proteins that are crucially associated with joint tissue regeneration.

METHODS

Cyr61-CTGF-NOV (CCN) family proteins in human platelets and megakaryocytic cells were comprehensively analyzed by Western blotting analysis. Production of CCN family proteins in megakaryocytes in vivo was confirmed by immunofluorescence analysis of mouse bone marrow cells. Effects of CCN family proteins found in platelets on chondrocytes were evaluated by using human chondrocytic HCS-2/8 cells.

RESULTS

Inclusion of CCN2, a mesenchymal tissue regenerator, was confirmed. Of note, CCN3, which counteracts CCN2, was newly found to be encapsulated in platelets. Interestingly, these two family members were not detectable in megakaryocytic cells, but their external origins were suggested. Furthermore, we found for the first time CCN5 and CCN1 that inhibits ADAMTS4 in both platelets and megakaryocytes. Finally, application of a CCN family cocktail mimicking platelets onto HCS-2/8 cells enhanced their chondrocytic phenotype.

CONCLUSIONS

Multiple inclusion of CCN1, 2 and 3 in platelets was clarified, which supports the harmonized regenerative potential of PRP in joint therapeutics.

The expression of the CCN family of matricellular proteins is highly dysregulated in connective tissue pathologies such as fibrosis and highly metastatic cancers. Strategies targeting members of this family, especially CCN2, are under development as novel therapeutic approaches to highly metastatic cancers such as pancreatic cancer. In prior reports, the Kleer laboratory and colleagues have linked reduced expression of CCN6 (WISP3) with aggressive breast cancers. Loss of CCN6 was associated with elevated Akt phosphorylation and TAK1 activation. In a recent report, the same group reports that, by modulating Notch signaling, CCN6 can promote the maintenance of an epithelial phenotype and also reduce cancer cell migration and invasion, tumor initiation, and metastasis (Oncotarget in press DOI: 10.18632/oncotarget.7734 ). These results are consistent with the hypothesis that addition of CCN6 peptides may represent a novel, viable therapeutic approach to blocking aggressive breast cancers.

The Cellular Communication Network (CCN) family of matricellular proteins comprises six proteins that share conserved structural features and play numerous biological roles. These proteins can interact with several receptors or soluble proteins, regulating cell signaling pathways in various tissues under physiological and pathological conditions. In the skeletal muscle of mammals, most of the six CCN family members are expressed during embryonic development or in adulthood. Their roles during the adult stage are related to the regulation of muscle mass and regeneration, maintaining vascularization, and the modulation of skeletal muscle fibrosis. This work reviews the CCNs proteins' role in skeletal muscle physiology and disease, focusing on skeletal muscle fibrosis and its regulation by Connective Tissue Growth factor (CCN2/CTGF). Furthermore, we review evidence on the modulation of fibrosis and CCN2/CTGF by the renin-angiotensin system and the kallikrein-kinin system of vasoactive peptides.

Keywords: CCN; CCN2/CTGF; KKS; cellular communication network; fibrosis; skeletal muscle; vasoactive peptides.

Visual environment plays an important role in the occurrence of myopia. We previously showed that the different flashing lights could result in distinct effects on the ocular growth and development of myopia. CCN2 has been reported to regulate various cellular functions and biological processes. However, whether CCN2 signaling was involved in the red flashing light-induced myopia still remains unknown. In the present study, we investigated the effects of the red flashing lights exposure on the refraction and axial length of the eyes in vivo and then evaluated their effects on the expression of CCN2 and TGF- β in sclera tissues. Our data showed that the eyes exposed to the red flashing light became more myopic with a significant increase of the axial length and decrease of the refraction. Both CCN2 and TGF- β , as well as p38 MAPK and PI3K, were highly expressed in the sclera tissues exposed to the red flashing light. Both CCN2 and TGF- β were found to have the same gene expression profile in vivo. In conclusion, our findings found that CCN2 signaling pathway plays an important role in the red flashing light-induced myopia in vivo. Moreover, our study establishes a useful animal model for experimental myopia research.

Connective tissue growth factor (CTGF, CCN2) is induced in response to TGFbeta in fibroblasts. In this report, we show that C2 ceramide reduced the ability of TGFbeta to induce CCN2 protein, mRNA and promoter activity in fibroblasts. C2 ceramide reduced the ability of TGFbeta to induce the generic Smad responsive promoter/reporter construct SBE-luciferase. These results suggest that C2 ceramide reduces the action of TGFbeta in fibroblasts via Smad antagonism.

Supramolecular networks constructed with the tBu--C[triple bond]C superset Ag(n) (n=4 or 5) metal-ligand synthon and trifluoroacetate have been transformed through the introduction of ancillary terminal nitrile ligands, from acetonitrile through propionitrile to tert-butyronitrile, giving rise to a 2D coordination network in AgC[triple chemical bond]CtBu3 AgCF(3)CO(2)H(2)O (1), a 2D hydrogen-bonded network in AgC[triple chemical bond]CtBu5 AgCF(3)CO(2)4 CH(3)CNH(2)O (2), a 2D hybrid coordination/hydrogen-bonded network in AgC[triple chemical bond]CtBu3 AgCF(3)CO(2)CH(3)CH(2)CN2 H(2)O (3), and another 2D coordination network in AgC[triple chemical bond]CtBu4 AgCF(3)CO(2) (CH(3))(3)CCN2 H(2)O (4). Concomitantly, the linkage modes between adjacent ethynide-bound Ag(n) aggregates in these compounds are also changed. A layer-type hydrogen-bonded host lattice in isostructural AgC[triple chemical bond]CtBu4 AgCF(3)CO(2)(R(4)N)(CF(3)CO(2)) 2 H(2)O (R(4)=BnMe(3), 5; R(4)=Et(4), 6; R(4)=nPr(4), 7) is obtained by introducing quaternary ammonium cations as guest templates, which occupy the interstices and thereby mediate the interlayer separation. Use of the bulky nBu(4)N(+) cation leads to disruption of the host network in AgC[triple bond]CtBu4 AgCF(3)CO(2)3[(nBu(4)N)(CF(3)CO(2))]H(2)O (8) with generation of a discrete dense nido-Ag(5) cluster.

The Chagas' disease parasite Trypanosoma cruzi elicits a potent inflammatory response in acutely infected hearts that keeps parasitism in check and triggers cardiac abnormalities. A most-studied mechanism underlying innate immunity in T. cruzi infection is Toll-like receptor (TLR) activation by lipids and other parasite molecules. However, yet-to-be-identified pathways should exist. Here, we show that T. cruzi strongly upregulates monocyte chemoattractant protein 1 (MCP-1)/CCL2 and fractalkine (FKN)/CX3CL1 in cellular and mouse models of heart infection. Mechanistically, upregulation of MCP-1 and FKN stems from the interaction of parasite-derived neurotrophic factor (PDNF)/trans-sialidase with neurotrophic receptors TrkA and TrkC, as assessed by pharmacological inhibition, neutralizing antibodies, and gene silencing studies. Administration of a single dose of intravenous PDNF to naive mice results in a dose-dependent increase in MCP-1 and FKN in the heart and liver with pulse-like kinetics that peak at 3 h postinjection. Intravenous PDNF also augments MCP-1 and FKN in TLR signaling-deficient MyD88-knockout mice, underscoring the MyD88-independent action of PDNF. Although single PDNF injections do not increase MCP-1 and FKN receptors, multiple PDNF injections at short intervals up the levels of receptor transcripts in the heart and liver, suggesting that sustained PDNF triggers cell recruitment at infection sites. Thus, given that MCP-1 and FKN are chemokines essential to the recruitment of immune cells to combat inflammation triggers and to enhance tissue repair, our findings uncover a new mechanism in innate immunity against T. cruzi infection mediated by Trk signaling akin to an endogenous inflammatory and fibrotic pathway resulting from cardiomyocyte-TrkA recognition by matricellular connective tissue growth factor (CTGF/CCN2).

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). The development and progression of DN might involve multiple factors. Connective tissue growth factor (CCN2, originally known as CTGF) is the one which plays a pivotal role. Therefore, increasing attention is being paid to CCN2 as a potential therapeutic target for DN. Up to date, there are also many drugs or agents which have been shown for their protective effects against DN via different mechanisms. In this review, we only focus on the potential renoprotective therapeutic agents which can specifically abolish CCN2 expression or nonspecifically inhibit CCN2 expression for retarding the development and progression of DN.

Oral submucous fibrosis (OSF) is a potentially malignant disorder that is characterized by a progressive fibrosis in the oral submucosa. Arecoline, an alkaloid compound of the areca nut, is reported to be a major aetiological factor in the development of OSF. Low-power laser irradiation (LPLI) has been reported to be beneficial in fibrosis prevention in different damaged organs. The aim of this study was to investigate the potential therapeutic effects of LPLI on arecoline-induced fibrosis. Arecoline-stimulated human gingival fibroblasts (HGFs) were treated with or without LPLI. The expression levels of the fibrotic marker genes alpha-smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF/CCN2) were analysed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) and western blots. In addition, the transcriptional activity of CCN2 was further determined by a reporter assay. The results indicated that arecoline increased the messenger RNA and protein expression of CCN2 and α-SMA in HGF. Interestingly, both LPLI and forskolin, an adenylyl cyclase activator, reduced the expression of arecoline-mediated fibrotic marker genes and inhibited the transcriptional activity of CCN2. Moreover, pretreatment with SQ22536, an adenylyl cyclase inhibitor, blocked LPLI's inhibition of the expression of arecoline-mediated fibrotic marker genes. Our data suggest that LPLI may inhibit the expression of arecoline-mediated fibrotic marker genes via the cAMP signalling pathway.

BACKGROUND

In Europe and the United States, verteporfin (Visudyne; VP) is registered and used in treating macular degeneration. Research showed that VP decreased expression of fibrotic genes in fibroblasts collected from nodules of patients suffering from Dupuytren's disease, plausibly by de-activating transcription in the Yes Activated Protein (YAP) pathway.

OBJECTIVE

To analyze the effect of VP on myofibroblasts cultured from Peyronie's disease (PD) plaques.

METHODS

At surgery for PD we took biopsies from the plaques of 5 patients. By immunostaining, the presence of the pathologic myofibroblasts was determined. After culturing cells, VP was dispensed in starvation medium for 24 and 48 hours and messenger(m)RNA levels of COL1A1, ACTA2, COL5A1, EDA-FN, LOXL2, CCN2, SERPINH1, PLOD2, and YAP were quantified and compared with controls with real-time polymerase chain reaction.

METHODS

mRNA-levels of COL1A1, ACTA2, COL5A1, EDA-FN, LOXL2, CCN2, SERPINH1, PLOD2, and YAP.

RESULTS

The pathologic phenotype of cells isolated from PD plaques was confirmed with baseline immunofluorescent stainings that showed considerable levels of α-smooth muscle actin, being a marker for the presence of myofibroblasts. The mRNA ratios of all the genes related to fibrosis (COL1A1, etc.) except YAP decreased significantly after treatment with VP within 24 and 48 hours. These results suggest inhibition of fibrosis in the YAP cascade, downstream of YAP.

CONCLUSIONS

In our opinion, urologists must move the focus to disease before deformity, and the search for new oral or intralesional agents, well-tolerated and effective in both the acute and chronic phase of PD must continue. VP blocked the expression of genes related to fibrosis in the YAP cascade in myofibroblasts derived from PD plaque. Mohede DCJ, de Jong IJ, Bank RA, et al. Verteporfin as a medical treatment in Peyronie's disease. Sex Med 2018;6:302-308.

The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-β signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-β signaling. CCN2 acts as a downstream mediator of TGF-β/Smad signaling and acting synergistically with TGF-β to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-β/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin.

In search of direct targets of insulin-like growth factor (IGF)-1 action, we discovered CCN5 (WNT1 inducible signaling pathway protein 2 [WISP2]) as a novel protein expressed in pancreatic β-cells. As a member of the "CCN" ( C ysteine-rich angiogenic inducer 61 [Cyr61], C onnective tissue growth factor [CTGF in humans], and N ephroblastoma overexpressed [Nov; in chickens]) family, the expression of CCN5/WISP2 is stimulated by IGF-1 together with Wnt signaling. When overexpressed in insulinoma cells, CCN5 promotes cell proliferation and cell survival against streptozotocin-induced cell death. The cell proliferation effect seems to be caused by AKT phosphorylation and increased cyclin D1 levels. These properties resemble those of CCN2/CTGF, another isoform of the CCN family, although CCN5 is the only one within the family of six proteins that lacks the C-terminal repeat. Treatment of primary mouse islets with recombinant CCN5 protein produced similar effects to those of gene transfection, indicating that either as a matricellular protein or a secreted growth factor, CCN5 stimulates β-cell proliferation and regeneration in a paracrine fashion. This review also discusses the regulation of CCN5/WISP2 by estrogen and its involvement in angiogenesis and tumorigenesis.

Growth factors are crucial regulators of cell differentiation towards tissue and organ development. Insulin and transforming growth factor-β (TGF-β) have been used as the major factors for chondrogenesis in vitro, by activating the AKT and Smad signaling pathways. Previous reports demonstrated that AKT and Smad3 have a direct interaction that results in the inhibition of TGF-β-mediated cellular responses. However, the result of this interaction between AKT and Smad3 during the chondrogenesis of human bone marrow-derived stem/progenitor cells (hBMSCs) is unknown. In this study, we performed functional analyses by inducing hBMSCs into chondrogenesis with insulin, TGF-β3 or in combination, and found that TGF-β3, when applied concomitantly with insulin, significantly decreases an insulin-induced increase in mRNA levels of the master regulator of chondrogenesis, SOX9, as well as the regulators of the 2 major chondrocyte markers, ACAN and COL2A1. Similarly, the insulin/TGF-β3-treated group presented a significant decrease in the deposition of cartilage matrix as detected by safranin O staining of histological sections of hBMSC micromass cultures when compared to the group stimulated with insulin alone. Intracellular analysis revealed that insulin-induced activation of AKT suppressed Smad3 activation in a dose-dependent manner. Accordingly, insulin/TGF-β3 significantly decreased the TGF-β3-induced increase in mRNA levels of the direct downstream factor of TGF-β/Smad3, CCN2/CGTF, compared to the group stimulated with TGF-β3 alone. On the other hand, insulin/TGF-β3 stimulation did not suppress insulin-induced expression of the downstream targets TSC2 and DDIT4/REDD1. In summary, insulin and TGF-β3 have antagonistic effects when applied concomitantly, with a minimal number of factors. The application of an insulin/TGF-β3 combination without further supplementation should be used with caution in the chondrogenic differentiation of hBMSCs.

CCN family 2/connective tissue growth factor (CCN2/CTGF) is a secreted protein that regulates diverse cellular functions. In addition to being a growth factor to transmit mitogen-activated protein kinase (MAPK) signalling into cells, through largely unknown mechanism, CCN2 antagonizes bone morphogenetic proteins (BMPs) by direct interaction. CCN2 and BMPs co-localize in cartilage, and both of them promote proliferation and differentiation of chondrocytes in vivo and in vitro. However, it was unclear whether these growth factors act cooperatively, or mutually inhibitory in chondrocyte biology. In addition, an information whether the heterooligomer of CCN2 and BMPs has any physiological roles in skeletogenesis was completely missing. Takigawa and his colleagues have recently reported that CCN2 and BMP-2 interacted directly to mutually interfere with their downstream ERK and Smad signalling, whereas the CCN2-BMP-2 complex promoted chondrocyte differentiation [Maeda et al. (2009) J. Biochem. 145, 207-216]. This contradictory finding shed light on a possibility that the complex of CCN2 and BMP-2 is capable of activating an additional non-canonical signalling pathway to promote chondrocyte differentiation.

Connective tissue growth factor (CTGF) or cellular communication network 2 (CCN2) is a matricellular protein essential for normal embryonic development and tissue repair. CTGF exhibits cell- and context-dependent activities, but CTGF function in vascular development and barrier function is unknown. We show that endothelial cells (ECs) are one of the major cellular sources of CTGF in the developing and adult retinal vasculature. Mice lacking CTGF expression either globally or specifically in ECs exhibit impaired vascular cell growth and morphogenesis and blood barrier breakdown. The global molecular signature of CTGF includes cytoskeletal and extracellular matrix protein, growth factor, and transcriptional co-regulator genes such as yes-associated protein (YAP). YAP, itself a transcriptional activator of CTGF, mediates several CTGF-controlled angiogenic and barriergenic transcriptional programs. Re-expression of YAP rescues, at least partially, angiogenesis and barriergenesis in CTGF mutant mouse retinas. Thus, the CTGF-YAP regulatory loop is integral to retinal vascular development and barrier function.

Keywords: Cell Biology; Molecular Biology; Transcriptomics.

Activated factor X (FXa) and thrombin can up-regulate gene expression of connective tissue growth factor (CTGF/CCN2) on fibroblasts. Since tissue factor (TF) is expressed on these cells, we hypothesized that they may assemble the prothrombinase complex leading to CTGF/CCN2 upregulation. In addition, the effect of thrombospondin-1 (TSP1) on this reaction was evaluated. Human foreskin fibroblasts were incubated with purified factor VII (FVII), factor X (FX), factor V (FV), prothrombin and calcium in the presence and absence of TSP1. Generation of FXa and of thrombin were assessed using chromogenic substrates. SMAD pathway phosphorylation was detected via Western-blot analysis. Pre-incubation of fibroblasts with FVII led to its auto-activation by cell-surface expressed TF, which in turn in the presence of FX, FVa, prothrombin and calcium led to FXa (9.7±0.8nM) and thrombin (7.9±0.04 U/mL×10-3) generation. Addition of TSP1 significantly enhanced thrombin (23.3±0.7 U/mL×10-3) but not FXa (8.5±0.6nM) generation. FXa and thrombin generation leads to upregulation of CTGF/CCN2. TSP1 alone upregulated CTGF/CCN2, an effect mediated via activation of transforming growth factor beta (TGFβ) as shown by phosphorylation of the SMAD pathway, an event blunted by using a TGFβ receptor I inhibitor (TGFβRI). FXa- and thrombin-induced upregulation of CTGF/CCN2 was not blocked by TGFβRI. In summary, assembly of the prothrombinase complex occurs on fibroblast's surface leading to serine proteases generation, an event enhanced by TSP1 and associated with CTGF/CCN2 upregulation. These mechanisms may play an important role in human diseases associated with fibrosis.

The process of fracture healing consists of an inflammatory reaction and cartilage and bone tissue reconstruction. The inflammatory cytokine interleukin-1β (IL-1β) signal is an important major factor in fracture healing, whereas its relevance to retinoid receptor (an RAR inverse agonist, which promotes endochondral bone formation) remains unclear. Herein, we investigated the expressions of IL-1β and retinoic acid receptor gamma (RARγ) in a rat fracture model and the effects of IL-1β in the presence of one of several RAR inverse agonists on chondrocytes. An immunohistochemical analysis revealed that IL-1β and RARγ were expressed in chondrocytes at the fracture site in the rat ribs on day 7 post-fracture. In chondrogenic ATDC5 cells, IL-1β decreases the levels of aggrecan and type II collagen but significantly increased the metalloproteinase-13 (Mmp13) mRNA by real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis. An RAR inverse agonist (AGN194310) inhibited IL-1β-stimulated Mmp13 and Ccn2 mRNA in a dose-dependent manner. Phosphorylated extracellular signal regulated-kinases (pERK1/2) and p-p38 mitogen-activated protein kinase (MAPK) were increased time-dependently by IL-1β treatment, and the IL-1β-induced p-p38 MAPK was inhibited by AGN194310. Experimental p38 inhibition led to a drop in the IL-1β-stimulated expressions of Mmp13 and Ccn2 mRNA. MMP13, CCN2, and p-p38 MAPK were expressed in hypertrophic chondrocytes near the invaded vascular endothelial cells. As a whole, these results point to role of the IL-1β via p38 MAPK as important signaling in the regulation of the endochondral bone formation in fracture healing, and to the actions of RAR inverse agonists as potentially relevant modulators of this process.

BACKGROUND

Connective tissue growth factor (CTGF) has been reported to be upregulated in experimental models of chronic cardiac allograft rejection. We investigated the contribution of CTGF to the development of cardiac allograft vasculopathy (CAV), a surrogate marker for chronic rejection.

METHODS

This prospective study included 72 adult heart allograft recipients. Genotyping of the rs6918698 polymorphism was performed by sequence-specific primer polymerase chain reaction (PCR). CTGF protein levels were measured in serum. CTGF messenger RNA (mRNA) from myocardial biopsy specimens was quantified by quantitative real-time PCR.

RESULTS

Recipient genotype was associated with the development of CAV (p = 0.014) and the carriers of the C allele (CC and CG genotype) were high-risk recipients for the development of CAV (odds ratio, 3.30; 95% confidence interval, 1.12-9.74; p = 0.044). Serum CTGF protein levels could not be associated with the presence of the C allele but were significantly lower in the patients that had developed CAV (p = 0.038). This was attributed to the addition of everolimus to their immunosuppression scheme. Myocardial relative CTGF mRNA expression was estimated to be approximately twice as much in the CAV patients than in the patients without CAV (p = 0.013).

CONCLUSIONS

The important role of CTGF during the development of CAV in heart transplantation was supported by the association of CAV with the recipient CTGF-945 CC/CG genotypes. The CAV patients, who were all receiving everolimus treatment, displayed elevated myocardial CTGF mRNA transcription levels, while everolimus has been observed to reduce serum CTGF protein levels.

Transforming growth factor β (TGFβ) plays a central role in the pathogenesis of gingival overgrowth (GO). Connective tissue growth factor (CTGF; or CCN2) is induced by TGFβ in human gingival fibroblasts (HGFs) and is overexpressed in GO tissues. CCN2 creates an environment favorable for fibrogenesis and is required for the maximal profibrotic effects of TGFβ. We previously showed that Src, JNK, and Smad3 mediate TGFβ1-induced CCN2 protein expression in HGFs. Moreover, Src is an upstream signaling transducer of JNK and Smad3. Recent studies suggested that NADPH oxidase (NOX)-dependent redox mechanisms are involved in mediating the profibrotic effects of TGFβ. In this study, we demonstrated that TGFβ1 upregulated NOX4 protein expression and increased reactive oxygen species (ROS) production in HGFs. Genetic or pharmacologic targeting of NOX4 abrogated TGFβ1-induced ROS production; Src, JNK, and Smad3 activation; and CCN2 and type I collagen protein expression in HGFs. Our results indicated that NOX4-derived ROS play pivotal roles in activating Src kinase activity leading to the activation of canonical (Smad3) and noncanonical (JNK) cascades that cooperate to attain maximum CCN2 expression. Furthermore, we demonstrated that curcumin significantly inhibited the TGFβ1-induced NOX4 protein expression in HGFs. Curcumin potentially qualifies as an agent to control GO by suppressing TGFβ1-induced NOX4 expression in HGFs.

Previous studies have reported the upregulation of CCN proteins early after acute heart injury. The aim of the present work was to evaluate the expression of the CCN1 and CCN2 proteins and their regulation by angiotensin II in the atrial myocardium of a chronically failing heart. Male adult mice were subjected to ligation of the left coronary artery to produce myocardial infarction (the MI group), and 16 of them were treated for 12 weeks with the AT1 receptor antagonist telmisartan (the MI-Tel group). Sham-operated mice served as controls. The expression of proteins was evaluated by immunohistochemistry 12 weeks after the operation. In shamoperated mice, stainings for CCN1 and CCN2 proteins were positive within atrial cardiomyocytes. CCN1-positive reaction revealed diffused cytoplasmic localization, while CCN2 was present mainly within the perinuclear cytoplasm. CCN1 was upregulated in the MI group, while CCN2 remained at basal level. Telmisartan prevented the upregulation of CCN1 and decreased CCN2 level. We compared the experimental data with the expression of CCN1 and CCN2 proteins in human right atrial appendages. We found an inverse, but not significant, relation between the level of either protein and the left ventricular ejection fraction. This suggests a similar atrial regulation of CCN1 and CCN2 expression also in humans. We conclude that in the murine atria, CCN1 and CCN2 proteins are expressed constitutively. In chronic heart failure, CCN proteins tend to be upregulated, which may be related to the action of angiotensin II.