AKI, due to the fact of altered oxygen supply after kidney transplantation, is characterized by renal ischemia-reperfusion injury (IRI). Recent data suggest that AKI to CKD progression may be driven by cellular senescence evolving from prolonged DNA damage response (DDR) following oxidative stress. Cellular communication factor 2 (CCN2, formerly called CTGF) is a major contributor to CKD development and was found to aggravate DNA damage and the subsequent DDR-cellular senescence-fibrosis sequence following renal IRI. We therefore investigated the impact of CCN2 inhibition on oxidative stress and DDR in vivo and in vitro. Four hours after reperfusion, full transcriptome RNA sequencing of mouse IRI kidneys revealed CCN2-dependent enrichment of several signaling pathways, reflecting a different immediate stress response to IRI. Furthermore, decreased staining for γH2AX and p-p53 indicated reduced DNA damage and DDR in tubular epithelial cells of CCN2 knockout (KO) mice. Three days after IRI, DNA damage and DDR were still reduced in CCN2 KO, and this was associated with reduced oxidative stress, marked by lower lipid peroxidation, protein nitrosylation, and kidney expression levels of Nrf2 target genes (i.e., HMOX1 and NQO1). Finally, silencing of CCN2 alleviated DDR and lipid peroxidation induced by anoxia-reoxygenation injury in cultured PTECs. Together, our observations suggest that CCN2 inhibition might mitigate AKI by reducing oxidative stress-induced DNA damage and the subsequent DDR. Thus, targeting CCN2 might help to limit post-IRI AKI.

Keywords: DNA damage response; acute kidney injury; cell cycle arrest; cellular communication network factor 2; ischemia–reperfusion injury; oxidative stress response.

CCNs are a specific type of matricellular protein, which are essential signaling molecules, and play multiple roles in multicellular eukaryotes. This family of proteins consists of six separate members, which exist only in vertebrates. The architecture of CCN proteins is multi-modular comprising four distinct modules. CCN Proteins achieve their primary functional activities by binding with several integrin7 receptors. The CCN family has been linked to cell adhesion, chemotaxis and migration, mitogenesis, cell survival, angiogenesis, differentiation, tumorigenesis, chondrogenesis, and wound healing, among other biological interactions. Breast cancer is the most commonly diagnosed cancer worldwide and CCN regulated breast cancer stands at the top. A favorable or unfavorable association between various CCNs has been reported in patients with breast carcinomas. The pro-tumorigenic CCN1, CCN2, CCN3, and CCN4 may lead to human breast cancer, although the anti-tumorigenic actions of CCN5 and CCN6 are also present. Several studies have been conducted on CCN proteins and cancer in recent years. CCN1 and CCN3 have been shown to exhibit a dual nature of tumor inhibition and tumor suppression to some extent in quiet recent time. Pharmacological advances in treating breast cancer by targeting CCN proteins are also reported. In our study, we intend to provide an overview of these research works while keeping breast cancer in focus. This information may facilitate early diagnosis, early prognosis and the development of new therapeutic strategies.

Keywords: CCN proteins; breast cancer; prospects; therapeutic advances; tumorigenesis.

Background: Cardiotoxicity is a common complication following anthracycline chemotherapy and represents one of the serious adverse reactions affecting life, which severely limits the effective use of anthracyclines in cancer therapy. Although some genes have been investigated by individual studies, the comprehensive analysis of key genes and molecular regulatory network in anthracyclines-induced cardiotoxicity (AIC) is lacking but urgently needed.

Methods: The present study integrating several transcription profiling datasets aimed to identify key genes associated with AIC by weighted correlation network analysis (WGCNA) and differentially expressed analysis (DEA) and also constructed miRNA-transcription factor-gene regulatory network. A total of three transcription profiling datasets involving 47 samples comprising 41 rat heart tissues and 6 human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) samples were enrolled.

Results: The WGCNA and DEA with E-MTAB-1168 identified 14 common genes affected by doxorubicin administrated by 4 weeks or 6 weeks. Functional and signal enrichment analyses revealed that these genes were mainly enriched in the regulation of heart contraction, muscle contraction, heart process, and oxytocin signaling pathway. Ten (Ryr2, Casq1, Fcgr2b, Postn, Tceal5, Ccn2, Tnfrsf12a, Mybpc2, Ankrd23, Scn3b) of the 14 genes were verified by another gene expression profile GSE154603. Importantly, three key genes (Ryr2, Tnfrsf12a, Scn3b) were further validated in a hiPSCMs-based in-vitro model. Additionally, the miRNA-transcription factor-gene regulatory revealed several top-ranked transcription factors including Tcf12, Ctcf, Spdef, Ebf1, Sp1, Rcor1 and miRNAs including miR-124-3p, miR-195-5p, miR-146a-5p, miR-17-5p, miR-15b-5p, miR-424-5p which may be involved in the regulation of genes associated with AIC.

Conclusions: Collectively, the current study suggested the important role of the key genes, oxytocin signaling pathway, and the miRNA-transcription factor-gene regulatory network in elucidating the molecular mechanism of AIC.

Keywords: Anthracyclines-induced cardiotoxicity; Differentially expressed analysis; Regulatory network; Weighted correlation network analysis.

Excessive macroautophagy/autophagy is one of the causes of cardiomyocyte death induced by cardiovascular diseases or cancer therapy, yet the underlying mechanism remains unknown. We and other groups previously reported that autophagy might contribute to cardiomyocyte death caused by sunitinib, a tumor angiogenesis inhibitor that is widely used in clinic, which may help to understand the mechanism of autophagy-induced cardiomyocyte death. Here, we found that sunitinib-induced autophagy leads to apoptosis of cardiomyocyte and cardiac dysfunction as the cardiomyocyte-specific Atg7^-/+ heterozygous mice are resistant to sunitinib. Sunitinib-induced maladaptive autophagy selectively degrades the cardiomyocyte survival mediator CCN2 (cellular communication network factor 2) through the TOLLIP (toll interacting protein)-mediated endosome-related pathway and cardiomyocyte-specific knockdown of Ccn2 through adeno-associated virus serotype 9 (AAV9) mimics sunitinib-induced cardiac dysfunction in vivo, suggesting that the autophagic degradation of CCN2 is one of the causes of sunitinib-induced cardiotoxicity and death of cardiomyocytes. Remarkably, deletion of Hmgb1 (high mobility group box 1) inhibited sunitinib-induced cardiomyocyte autophagy and apoptosis, and the HMGB1-specific inhibitor glycyrrhizic acid (GA) significantly mitigated sunitinib-induced autophagy, cardiomyocyte death and cardiotoxicity. Our study reveals a novel target protein of autophagic degradation in the regulation of cardiomyocyte death and highlights the pharmacological inhibitor of HMGB1 as an attractive approach for improving the safety of sunitinib-based cancer therapy.

Keywords: Autophagy; CCN2 degradation; HMGB1; TOLLIP; cardiac dysfunction; glycyrrhizic acid; sunitinib.

Background: Oral submucous fibrosis (OSMF) is one of the common oral potentially malignant disorders that can result in severe morbidity. Depending upon the stage of disease, multiple management therapies exist which include medicinal and surgical approaches. Although the surgical approach is preferred in severe conditions, numerous studies have reported its post-surgical deteriorating outcomes including increased fibrotic changes. To reduce these post-surgical complications, Light amplification by stimulated emission of radiation (Laser) has been introduced and studied as a non-invasive technique to treat oral submucous fibrosis. However, there exists a lack of knowledge about 'which laser shows a better post-treatment outcome'. Accordingly, this review aims to answer this question.

Materials and methods: A systematic review of the published literature was performed using an electronic search in PubMed/Medline, Science Direct, Web of Science, Embase, J- STAGE, Google Scholar, and Scopus databases, from 1952 till 2019 using keywords like, 'Oral submucous fibrosis', 'Treatment', 'Laser', 'Trismus', ' Fibrosis', 'Surgical', 'Non-invasive', and 'Postoperative results'.

Results: The search strategy revealed 20 relevant published studies in which laser had been used to treat 250 patients of OSMF. Effective results were found without any complications in all the cases after follow up.

Conclusion: Observing the current literature, it can be concluded that laser might be used as a potential non-invasive approach in the management of OSMF, however, large scale studies are required to investigate the efficacy and other effects of this technology.

Keywords: AN, Areca nut; CO2, Carbon-dioxide; CTGF/CCN2, Connective tissue growth factor; Er Cr YS GG, Erbium Chromium: Yttrium – Scandium – Gallium – Garnet; Er, YAG Erbium: Yttrium–Aluminium–Garnet; GA, General anaesthesia; GaAs, Gallium Arsenic; H2O, Water; HA, Hydroxyapatite; IF- ά, Interferon ά; KTP, Potassium titanyl phosphate; LA, Local anaesthesia; LPLI, Low-power laser irradiation; Laser; Laser, Light amplification by stimulated emission of radiation; MMP2, Matrix metalloproteinases 2; ND-YAG, Neodymium – doped: Yttrium- Aluminium Garnet; Non-invasive; OSMF, Oral submucous fibrosis; Oral sub mucus fibrosis; PGs, Prostaglandins; TGF- β, Transforming Growth Factor β; TNF, Tumor necrosis factor; Technique; Treatment; UUO, Unilateral ureteral obstruction; WHO, World Health Organization; cAMP, Cyclic adenosine monophosphate.

CCN5 is a divergent member of the cellular communication network factor (CCN) family in that it lacks the carboxyl terminal cystine knot domain common to the other CCN family members. CCN5 has been reported to antagonize the profibrotic actions of CCN2 and to inhibit myocardial collagen deposition and fibrosis in chronic pressure overload of the heart. However, what mechanisms that regulate CCN5 activity in the heart remain unknown. Recombinant, replication defective adenovirus encoding firefly luciferase under control of the human CCN5 promoter was prepared and used to investigate what mechanisms regulate CCN5 transcription in relevant cells. Tissue distribution of CCN5 in hearts from healthy mice and from mice subjected to myocardial infarction was investigated. Contrary to the profibrotic immediate early gene CCN2, we find that CCN5 is induced in the late proliferation and maturation phases of scar healing. CCN5 was identified principally in endothelial cells, fibroblasts, smooth muscle cells, and macrophages. Our data show that CCN5 gene transcription and protein levels are induced by catecholamines via β₂-adrenergic receptors. Myocardial induction of CCN5 was further confirmed in isoproterenol-infused mice. We also find that CCN5 transcription is repressed by TNF-α, an inflammatory mediator highly elevated in early phases of wound healing following myocardial infarction. In conclusion, CCN5 predominates in endothelial cells, fibroblasts, and macrophages of the differentiating scar tissue and its transcription is conversely regulated by β₂-adrenergic agonists and TNF-α.

Keywords: CCN2; CCN5; Catecholamines; Myocardial infarction; Primary cardiac fibroblasts; β2-Adrenergic receptor.

The renin-angiotensin system (RAS) controls not only systemic functions, such as blood pressure, but also local tissue-specific events. Previous studies have shown that angiotensin II receptor type 1 (AT₁R) and type 2 (AT₂R), two RAS components, are expressed in chondrocytes. However, the angiotensin II (ANG II) effects exerted through these receptors on chondrocyte metabolism are not fully understood. In this study, we investigated the effects of ANG II and AT₁R blockade on chondrocyte proliferation and differentiation. Firstly, we observed that ANG II significantly suppressed cell proliferation and glycosaminoglycan content in rat chondrocytic RCS cells. Additionally, ANG II decreased CCN2, which is an anabolic factor for chondrocytes, via increased MMP9. In Agtr1a-deficient RCS cells generated by the CRISPR-Cas9 system, Ccn2 and Aggrecan (Acan) expression increased. Losartan, an AT₁R antagonist, blocked the ANG II-induced decrease in CCN2 production and Acan expression in RCS cells. These findings suggest that AT₁R blockade reduces ANG II-induced chondrocyte degeneration. Interestingly, AT₁R-positive cells, which were localized on the surface of the articular cartilage of 7-month-old mice expanded throughout the articular cartilage with aging. These findings suggest that ANG II regulates age-related cartilage degeneration through the ANG II-AT₁R axis.

Keywords: angiotensin II; angiotensin II type I receptor (AT1R); cellular communication network factor 2 (CCN2); losartan; renin–angiotensin system (RAS).

A diet-induced non-alcoholic fatty liver disease (NAFLD) model causing obesity in rodents was used to examine whether sitagliptin and gliclazide therapies have similar protective effects on pathological liver change.

Methods: Male mice were fed a high-fat diet (HFD) or standard chow (Chow) ad libitum for 25 weeks and randomly allocated to oral sitagliptin or gliclazide treatment for the final 10 weeks. Fasting blood glucose and circulating insulin were measured. Inflammatory and fibrotic liver markers were assessed by qPCR. The second messenger ERK and autophagy markers were examined by Western immunoblot. F4/80, collagens and CCN2 were assessed by immunohistochemistry (IHC).

Results: At termination, HFD mice were obese, hyperinsulinemic and insulin-resistant but non-diabetic. The DPP4 inhibitor sitagliptin prevented intrahepatic induction of pro-fibrotic markers collagen-IV, collagen-VI, CCN2 and TGF-β1 and pro-inflammatory markers TNF-α and IL-1β more effectively than sulfonylurea gliclazide. By IHC, liver collagen-VI and CCN2 induction by HFD were inhibited only by sitagliptin. Sitagliptin had a greater ability than gliclazide to normalise ERK-protein liver dysregulation.

Conclusion: These data indicate that sitagliptin, compared with gliclazide, exhibits greater inhibition of pro-fibrotic and pro-inflammatory changes in an HFD-induced NAFLD model. Sitagliptin therapy, even in the absence of diabetes, may have specific benefits in diet-induced NAFLD.

Keywords: NAFLD; fibrosis; gliclazide; inflammation; sitagliptin.

Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-β signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.

Keywords: Collagen; Fibrillins; Fucosylation; TGF-β; Thrombospondin type I repeats.

Hepatocellular carcinoma (HCC) is a drastic problem in China. Oxaliplatin, a platinum-based chemotherapy drug, has limited efficacy in treating HCC, characterized by intrinsic and acquired resistance. Inositol hexaphosphate (IP6), a carbohydrate abundant in grains, has contributed to the rising popularity of whole grain products consumption for the potential protection against dozens of diseases. However, the therapeutic potential of IP6 in halting the progression of HCC remains unclear, especially in combination with oxaliplatin. The anti-proliferation and anti-migration effects of IP6 were evaluated in vitro and in vivo. The synergistic and sequential anti-proliferative effect with IP6 and oxaliplatin were also evaluated in HCC. Finally, the role of CCN2-LRP6-β-catenin-ABCG1 signaling in oxaliplatin resistance and IP6 treatment was evaluated. We proved that IP6 treatment exhibited independent anticancer effect and synergistic anti-proliferative effects in combination with oxaliplatin in HCC. Specifically, up-regulation of ABCG1 and CCN2 were associated with oxaliplatin resistance. ABCG1 was acting as a downstream molecule of the CCN2-LRP6-Wnt/β-catenin signaling pathway in HCC cells. The IP6 treatment exhibited inhibition of CCN2-LRP6-Wnt/β-catenin signaling pathway and downregulation of ABCG1 in HCC cells. When combined with ABCG1 knocking down in HCC cells, the anti-proliferative effect of oxaliplatin was partly impaired in combination with IP6. We suggested that IP6 treatment renders HCC sensitive to oxaliplatin and breaking the CCN2-LRP6-β-catenin-ABCG1 signaling pathway is one of the mechanism after IP6 treatment.

Keywords: CCN2; Hepatocellular carcinoma; Wnt signaling; inositol hexaphosphate; oxaliplatin.

The function of oleanolic acid (OA) in various types of cancer has been reported frequently, especially for breast cancer. However, the regulation of breast tumor growth in response to OA treatment has not been studied in depth. Here, we first explored the effect of OA treatment on breast tumors in vitro and in vivo and then used RNA-seq technology to study the effect and molecular mechanism of OA treatment of MCF-7 cells, particularly at the level of functional genomics. The results showed that 40 μM OA treatment could significantly inhibit the proliferation and induce the apoptosis of MCF-7 cells. Through analysis of RNA sequencing data quality and differentially expressed genes (DEGs), 67 significantly downregulated genes and 260 significantly upregulated genes were identified to be involved in OA treatment of MCF-7 cells. Among these genes, 43 unique DEGs were enriched in several signaling pathways and Gene Ontology terms, such as p53 signaling pathway, TNF signaling pathway and mTOR signaling pathway. Six downregulated genes, including THBS1, EDN1, CACNG4, CCN2, AXIN2 and BMP4, as well as six upregulated genes, including ATF4, SERPINE1, SESN2, PPARGC1A, EGR1 and JAG1, were selected as target genes in response to OA treatment. The inhibitory effect of OA on breast cancer was also found in the following mouse experiments. Our study provides evidence and molecular support for the treatment of breast cancer with OA.

Keywords: RNA-seq; breast cancer; oleanolic acid; pathway.

Objectives: To clarify the role of cellular communication network factor 2/connective tissue growth factor (CCN2/CTGF) in periodontal tissue regeneration by investigating, the proliferative and tubulogenic responses of human endothelial cells obtained from the periodontal ligament to CCN2/CTGF.

Design: Endothelial cells were seeded on agar gel medium with or without 50 ng/mL recombinant CCN2/CTGF (rCCN2/CTGF) and cultured for 6 h. Cells were morphologically and phenotypically analyzed by immunofluorescent microscopy. A colorimetric assay was used to evaluate cell proliferation, and transmission electron microscopy (TEM) was used for ultrastructural analysis.

Results: The proliferation of endothelial cells was best promoted by rCCN2/CTGF at 50 ng/mL. In the control group, tube formation was not observed within 6 h. In contrast, endothelial cells seeded on the agar with 50 ng/mL rCCN2/CTGF clearly showed proliferation with network formation. Under a two-dimensional culture condition, a dense network of endothelial cells was not constructed on the plastic bottom. However, drastic morphological change was observed in the endothelial cells on the agar containing rCCN2/CTGF. The endothelial cells in the dense network were interconnected with each other and showed a tube-like structure. Tight junctions or adherens junctions were observed between the adjoining endothelial cells in the dense network.

Conclusions: CCN2/CTGF was found to promote the proliferation and tubulogenesis of endothelial cells from the periodontal ligament. These results suggest that CCN2/CTGF may contribute to the regeneration of damaged periodontal tissue by activating the remaining endothelial cells.

Keywords: Angiogenesis; CCN2/CTGF; Endothelial cells; Periodontal ligament; Periodontal tissue regeneration; Tube formation.

Introduction: Low back pain (LBP), a leading cause of global disability, is often associated with intervertebral disc degeneration (IDD). Exercise therapy is recommended for chronic LBP management and affects many tissues and organ systems. However, the ability of exercise to repair the extracellular matrix (ECM) in degenerating discs is unclear. The aims of the study were to examine mRNA expression of ECM structural components (collagen I, II, X, aggrecan) and regulators of matrix turnover (matrix metalloproteinases (MMP)-3, - 9, - 13, ADAMTS-4, - 5, TIMP1-4, CCN2) between age-matched (a) wild-type and secreted protein acidic and rich in cysteine (SPARC)-null, (b) sedentary and active, and (c) male and female mice.

Methods: At 8 months of age, male and female SPARC-null and wild-type control mice received a home cage running wheel or a control, fixed wheel for 6 months. Deletion of the SPARC gene results in progressive IDD beginning at 2 to 4 months of age. Increased activity was confirmed, and qPCR was performed on excised lumbar discs.

Results: Male SPARC-null mice expressed less aggrecan mRNA than wild-type controls. After 6 months of running, collagen, MMP3, and MMP13 expression was increased in male and MMP3 was increased in female SPARC-null mice. Sex differences were observed in wild-type mice and in response to IDD and long-term running.

Conclusions: Voluntary running results in changes in mRNA consistent with increased ECM turnover and disc regeneration. Improved disc ECM might contribute to the beneficial effects of exercise on LBP and may create an intradiscal environment hospitable to regenerative therapies. Sex-specific differences should be considered in the development of disc-targeting therapies.

Keywords: degeneration; extracellular matrix; pain; pre‐clinical models.

Cellular communication network 2 (CCN2), also known as connective tissue growth factor (CTGF) regulates diverse cellular processes, some at odds with others, including adhesion, proliferation, apoptosis, and extracellular matrix (ECM) protein synthesis. Although a cause-and-effect relationship between CCN2/CTGF expression and local fibrotic reactions has initially been established, CCN2/CTGF manifests cell-, tissue-, and context-specific functions and differentially affects developmental and pathological processes ranging from progenitor cell fate decisions and angiogenesis to inflammation and tumorigenesis. CCN2/CTGF multimodular structure, binding to and activation or inhibition of multiple cell surface receptors, growth factors and ECM proteins, and susceptibility for proteolytic cleavage highlight the complexity to CCN2/CTGF biochemical attributes. CCN2/CTGF expression and dosage in the local environment affects a defined community of its interacting partners, and this results in sequestration of growth factors, interference with or potentiation of ligand-receptor binding, cellular internalization of CCN2/CTGF, inhibition or activation of proteases, and generation of CCN2/CTGF degradome products that add molecular diversity and expand the repertoire of functional modules in the cells and their microenvironment. Through these interactions, different intracellular signals and cellular responses are elicited culminating into physiological or pathological reactions. Thus, the CCN2/CTGF interactome is a defining factor of its tissue- and context-specific effects. Mapping of new CCN2/CTGF binding partners might shed light on yet unknown roles of CCN2/CTGF and provide a solid basis for tissue-specific targeting this molecule or its interacting partners in a therapeutic context.

Keywords: Angiogenesis; CCN2; CTGF; Extracellular matrix protein; Growth factor receptor; Matricellular protein; Protein–protein interaction.

Regulation of the mammalian embryo involves cell-signaling molecules produced by the maternal oviduct and endometrium. Here, datasets on the transcriptome of the gestational Days 5 and 6 bovine morula and Day 5 maternal endometrium were examined to identify receptor genes expressed by the morula and expression of the corresponding ligand-related genes in the endometrium. A total of 175 receptor genes were identified in the morula, including 48 encoding for growth factors or WNT signaling molecules, 25 for cytokines and chemokines, 35 involved in juxtacrine and matricellular signaling and 25 encoding for receptors for small molecules. Some of the highly-expressed pairs of endometrial ligand and embryo receptor genes included MDK and its receptors ITGB1, SDC4 and LRP2, WNT5A (RYK), VEGFA (ITGB1), GPI (AMFR), and the hedgehog proteins IHH and DHH (HHIP). The most highly expressed receptors for small molecules were GPRC5C (retinoic acid receptor), PGRMC1 (progesterone), and CHRNB2 (acetylcholine). There were also 84 genes encoding for cell signaling ligands expressed by the morula, with the most highly expressed being GPI, AIMP1, TIMP1, IK, and CCN2. The atlas of receptor and ligand genes should prove useful for understanding details of the communication between the embryo and mother that underlies optimal embryonic development.

Keywords: cell signaling; embryo; endometrium; morula; receptors.

Type 2 diabetes is an independent risk factor for non-alcoholic steatohepatitis (NASH) progression and its mediators have not been resolved. In this study, a pathogenic role of cellular communication network factor 2 (CCN2) protein in NASH pathology, was investigated in an established preclinical NASH model. Male wild type C57BL/6 mice received either Chow or high fat diet (HFD) for 26 weeks, with some mice in each group randomly selected to receive low dose streptozotocin (STZ: 3 i.p. injections, 65 mg/kg) at 15 weeks to induce type 2 diabetes. In the final 10 of the 26 weeks mice from each group were administered i.p. either rabbit anti-CCN2 neutralizing antibody (CCN2Ab) or as control normal rabbit IgG, at a dose of 150 µg per mouse twice/week. NASH developed in the HFD plus diabetes (HFD+DM) group. Administration of CCN2Ab significantly downregulated collagen I and collagen III mRNA induction and prevented pro-inflammatory MCP-1 mRNA induction in HFD+DM mice. At the protein level, CCN2Ab significantly attenuated collagen accumulation by PSR stain and collagen I protein induction in HFD+DM. Phosphorylation of the pro-fibrotic ERK signalling pathway in liver in HFD+DM was attenuated by CCN2Ab treatment. Intrahepatic CCN1 mRNA was induced, whereas CCN3 was downregulated at both the mRNA and protein levels in HFD+DM. CCN3 down-regulation was prevented by CCN2Ab treatment. This in vivo study indicates that CCN2 is a molecular target in NASH with high fat diet and diabetes, and that regulation of ERK signalling is implicated in this process.

Keywords: CCN2; Fibrosis; Inflammation; NAFLD; Neutralizing antibody; Type 2 diabetes.

Purpose: The aim of this study was to establish an in vitro model of nifedipine-induced gingival overgrowth and characterize the anti-fibrotic effect of hepatocyte growth factor (HGF) using this model.

Methods: Human gingival fibroblasts were cultured-treated with 0.1, 1, or 10 µg/mL nifedipine or 10 ng/mL IL-1β + 0.1, 1, or 10 µg/mL nifedipine (0.1N, 1N, 10N, IL + 0.1N, IL + 1N, IL + 10N). Cell proliferation and levels of type I collagen, TGF-β1, CCN2/CTGF, and α-SMA were measured 48 h after the simultaneous addition of 10 and 50 ng/mL HGF (10 and 50HGF) along with IL-1β and nifedipine. Type I collagen was measured after administration of anti-HGF neutralizing antibody.

Results: Significant increases in type I collagen, TGF-β1, and CCN2/CTGF were observed after treatment in the 1N and IL + 0.1N groups. Levels of type I collagen and CCN2/CTGF differed significantly between the IL + 0.1N group and the IL + 0.1N + 50HGF group. Production of type I collagen increased significantly following addition of anti-HGF antibody.

Conclusion: This study demonstrated the establishment of an in vitro model of nifedipine-induced gingival overgrowth by showing increased collagen levels. Experiments using this model suggested that HGF exerts anti-fibrotic effects.

Keywords: drug-induced gingival overgrowth; hepatocyte growth factor; nifedipine.

Background: This study aimed to explore the possible association of single nucleotide polymorphisms (SNPs) in the upstream (rs9402373) and downstream regions (rs9399005 and rs12526196) of the gene encoding connective tissue growth factor (CTGF/CCN2) with relapsing-remitting multiple sclerosis (RRMS) risk and clinical parameters including disability scores and rate of disability progression.

Materials and methods: In total, 200 patients with RRMS and 305 controls were genotyped using real-time PCR (rs1252696 C/T and rs9402373 G/C) or PCR-RFLP (rs9399005 C/T) methods. Furthermore, the association between these genotypes and clinical parameters including Expanded Disability Status Scale (EDSS) score, Multiple Sclerosis Severity Score (MSSS), age at onset, duration of disease, duration of treatment, and presence of contrast-enhancing lesions was analyzed.

Results: rs9399005 genotypes TT and CT in the dominant model were significant predictors of RRMS vs. control status by logistic regression analysis (OR = 1.45, 95% CI = 1.01-2.08, P = .04). Moreover, these genotypes for rs9399005 were associated with a MSSS ≥ 2.4 (OR = 3.54, 95% CI = 1.56-8.05, P = .003). In addition, MSSS was lower in patients who had at least one rs12526196C allele than in the corresponding patients with the TT genotype (P = .02).

Conclusion: To our knowledge, this is the first evidence of the involvement of variants around the CTGF gene in MS risk and disability progression.

Keywords: EDSS; MSSS; Neurodegeneration; Oligodendrocyte; Prognosis; Progression.

The regulation of skeletal muscle growth following pro-hypertrophic stimuli requires a coordinated response by different cell types that leads to extracellular matrix (ECM) remodeling and increases in muscle cross-sectional area. Indeed, matricellular proteins serve a key role as communication vehicles that facilitate the propagation of signaling stimuli required for muscle adaptation to environmental challenges. We found that the matricellular protein cellular communication network factor 2 (CCN2), also known as connective tissue growth factor (CTGF), is induced during a time course of overload-driven skeletal muscle hypertrophy in mice. To elucidate the role of CCN2 in mediating the hypertrophic response, we utilized genetically engineered mouse models for myofiber-specific CCN2 gain- and loss-of-function and then examined their response to mechanical stimuli through muscle overload. Interestingly, myofiber-specific deletion of CCN2 blunted muscle's hypertrophic response to overload without interfering with ECM deposition. On the other hand, when in excess through transgenic CCN2 overexpression, CCN2 was efficient in promoting overload-induced aberrant ECM accumulation without affecting myofiber growth. Altogether, our genetic approaches highlighted independent ECM and myofiber stress adaptation responses, and positioned CCN2 as a central mediator of both. Mechanistically, CCN2 acts by regulating focal adhesion kinase (FAK) mediated transduction of overload-induced extracellular signals, including interleukin 6 (IL6), and their regulatory impact on global protein synthesis in skeletal muscle. Overall, our study highlights the contribution of muscle-derived extracellular matrix factor CCN2 for proper hypertrophic muscle growth.

Keywords: CCN2; CTGF; fibrosis; hypertrophy; muscle.

Lymphangiogenesis is essential for the development of the lymphatic system and is important for physiological processes such as homeostasis, metabolism and immunity. Cellular communication network factor 2 (CCN2, also known as CTGF), is a modular and matricellular protein and a well-known angiogenic factor in physiological and pathological angiogenesis. However, its roles in lymphangiogenesis and intracellular signaling in lymphatic endothelial cells (LECs) remain unclear. Here, we investigated the effects of CCN2 on lymphangiogenesis. In in vivo Matrigel plug assays, exogenous CCN2 increased the number of Podoplanin-positive vessels. Subsequently, we found that CCN2 induced phosphorylation of ERK in primary cultured LECs, which was almost completely inhibited by the blockade of integrin αvβ5 and partially decreased by the blockade of integrin αvβ3. CCN2 promoted direct binding of ERK to dual-specific phosphatase 6 (DUSP6), which regulated the activation of excess ERK by dephosphorylating ERK. In vitro, CCN2 promoted tube formation in LECs, while suppression of Dusp6 further increased tube formation. In vivo, immunohistochemistry also detected ERK phosphorylation and DUSP6 expression in Podoplanin-positive cells on CCN2-supplemented Matrigel. These results indicated that CCN2 promotes lymphangiogenesis by enhancing integrin αvβ5-mediated phosphorylation of ERK and demonstrated that DUSP6 is a negative regulator of excessive lymphangiogenesis by CCN2.

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.

Keywords: CCN2; CTGF; EGFR; SMAD; TGF-β; TGF-β receptors.

Calcific aortic valve disease (CAVD) is characterized by a fibrocalcific process. The regulatory mechanisms that drive the fibrotic response in the aortic valve (AV) are poorly understood. Long noncoding RNAs derived from super-enhancers (lncRNA-SE) control gene expression and cell fate. Herein, multidimensional profiling including chromatin immunoprecipitation and sequencing, transposase-accessible chromatin sequencing, genome-wide 3D chromatin contacts of enhancer-promoter identified LINC01013 as an overexpressed lncRNA-SE during CAVD. LINC01013 is within a loop anchor, which has contact with the promoter of CCN2 (CTGF) located at ~180 kb upstream. Investigation showed that LINC01013 acts as a decoy factor for the negative transcription elongation factor E (NELF-E), whereby it controls the expression of CCN2. LINC01013-CCN2 is part of a transforming growth factor beta 1 (TGFB1) network and exerts a control over fibrogenesis. These findings illustrate a novel mechanism whereby a dysregulated lncRNA-SE controls, through a looping process, the expression of CCN2 and fibrogenesis of the AV.

Background: CCN2 (cellular communication network factor 2) is a matricellular protein involved in cell communication and microenvironmental signaling responses. CCN2 is known to be overexpressed in several cardiovascular diseases, but its role is not completely understood.

Methods: Here, CCN2 involvement in aortic wall homeostasis and response to vascular injury was investigated in inducible Ccn2-deficient mice, with induction of vascular damage by infusion of Ang II (angiotensin II; 15 days), which is known to upregulate CCN2 expression in the aorta.

Results: Ang II infusion in CCN2-silenced mice lead to 60% mortality within 10 days due to rapid development and rupture of aortic aneurysms, as evidenced by magnetic resonance imaging, echography, and histological examination. Ccn2 deletion decreased systolic blood pressure and caused aortic structural and functional changes, including elastin layer disruption, smooth muscle cell alterations, augmented distensibility, and increased metalloproteinase activity, which were aggravated by Ang II administration. Gene ontology analysis of RNA sequencing data identified aldosterone biosynthesis as one of the most enriched terms in CCN2-deficient aortas. Consistently, treatment with the mineralocorticoid receptor antagonist spironolactone before and during Ang II infusion reduced aneurysm formation and mortality, underscoring the importance of the aldosterone pathway in Ang II-induced aorta pathology.

Conclusions: CCN2 is critically involved in the functional and structural homeostasis of the aorta and in maintenance of its integrity under Ang II-induced stress, at least, in part, by disruption of the aldosterone pathway. Thus, this study opens new avenues to future studies in disorders associated to vascular pathologies.

Keywords: aldosterone; aneurysm; aorta; extracellular matrix; hypertension.

The matricellular glycoprotein thrombospondin1 (TSP1) has complex roles in the extracellular matrix and at cell surfaces, but relatively little is known about its intracellular associations prior to secretion. To search for novel intracellular interactions of TSP1 in situ, we carried out a biotin ligase-based TSP1 interactome screen and identified protein disulphide isomerase A3 (PDIA3/ERp57) as a novel candidate binding protein. In validation, TSP1 and PDIA3 were established to bind in vitro and to colocalise in the endoplasmic reticulum of human dermal fibroblasts (HDF). Loss of PDIA3 function, either by pharmacological inhibition in HDF or in Pdia3^-/- mouse embryo fibroblasts (Pdia3^-/-MEF), led to alterations in the composition of cell-derived ECM, involving changed abundance of fibronectin and TSP1, and was correlated with reduced cell spreading, altered organisation of F-actin and reduced focal adhesions. These cellular phenotypes of Pdia3^-/-MEF were normalised by exposure to conditioned medium (WTCM) or extracellular matrix (WTECM) from wild-type (WT)-MEF. Rescue depended on PDIA3 activity in WT-MEF, and was not prevented by immunodepletion of fibronectin. Heparin-binding proteins in WTCM were found to be necessary for rescue. Comparative quantitative tandem-mass-tag proteomics and functional assays on the heparin-binding secretomes of WT-MEF and Pdia3^-/- MEF identified multiple ECM and growth factor proteins to be down-regulated in the CM of Pdia3^-/- MEF. Of these, CCN2 was identified to be necessary for the adhesion-promoting activity of WTCM on Pdia3^-/- MEF and to bind TSP1. Thus, PDIA3 coordinates fibroblast production of an ECM-rich, pro-adhesive microenvironment, with implications for PDIA3 as a translational target.

Keywords: Cell Adhesion; Extracellular Matrix; Interactome; Secretome; Thrombospondin-1.