Age-related macular degeneration (AMD) is a leading cause of visual impairment. Anti-VEGF drugs are used to treat AMD, but they may induce subretinal fibrosis. We have focused on adrenomedullin (AM), a vasoactive peptide, and its receptor activity-modifying protein, RAMP2, which regulate vascular homeostasis and suppress fibrosis. Here, the therapeutic potential of the AM-RAMP2 system was evaluated after laser-induced choroidal neovascularization (LI-CNV), a mouse model of AMD. Neovascular formation, subretinal fibrosis and macrophage invasion were all enhanced in both AM and RAMP2 knockout (KO) mice as compared to wild-type (WT) mice. These pathological changes were suppressed by intravitreal injection of AM. Comprehensive gene expression analysis of the choroid after LI-CNV with or without AM administration revealed that fibrosis-related molecules including Tgfb, Cxcr4, Ccn2 and Thbs1, were all downregulated by AM. In retinal pigment epithelial cells, co-administration of TGF-β and TNF-α induced epithelial-mesenchymal transition (EMT), which was also prevented by AM. Finally, the effects of TGF-β and CXCR4 inhibitors were assessed, and they eliminated the difference in subretinal fibrosis between RAMP2 KO and WT mice. These findings suggest the AM-RAMP2 system suppresses subretinal fibrosis in LI-CNV by suppressing EMT.

Keywords: RAMP2; adrenomedullin; age-related macular degeneration; genetically engineered mice; laser-induced choroidal neovascularization; subretinal fibrosis.

Activation of hepatic stellate cells (HSCs) is a critical pathogenic feature of liver fibrosis and cirrhosis. BAY 41-2272 is a canonical non-nitric oxide (NO)-based soluble guanylyl cyclase (sGC) stimulator that triggers cyclic guanosine monophosphate (cGMP) signaling for attenuation of fibrotic disorders; however, the impact of BAY 41-2272 on HSC activation remains ill-defined. Transforming growth factor (TGF)β and its downstream connective tissue growth factor (CTGF or cellular communication network factor 2, CCN2) are critical fibrogenic cytokines for accelerating HSC activation. Here, we identified that BAY 41-2272 significantly inhibited the TGFβ1-induced mRNA and protein expression of CTGF in mouse primary HSCs. Indeed, BAY 41-2272 increased the sGC activity and cGMP levels that were potentiated by two NO donors and inhibited by a specific sGC inhibitor, ODQ. Surprisingly, the inhibitory effects of BAY 41-2272 on CTGF expression were independent of the sGC/cGMP pathway in TGFβ1-activated primary HSCs. BAY 41-2272 selectively restricted the TGFβ1-induced phosphorylation of Akt but not canonical Smad2/3 in primary HSCs. Together, we illustrate a unique framework of BAY 41-2272 for inhibiting TGFβ1-induced CTGF upregulation and HSC activation via a noncanonical Akt-dependent but sGC/cGMP-independent pathway.

Keywords: Akt; BAY 41-2272; CTGF; hepatic stellate cell; sGC.

Background: Aged human skin is primarily attributable to the loss of collagen. Hepatocyte growth factor (HGF) acts as an anti-fibrotic factor by suppression of collagen production. In aged human skin, HGF is elevated in dermal fibroblasts and thus contributes to dermal aging (thin dermis) by suppression of collagen production.

Objective: We aimed to investigate the underlying mechanisms of age-related elevation of HGF expression.

Methods: Collagen fibrils in the aged skin dermis are fragmented and disorganized, which impairs collagen-fibroblast interaction, resulting in reduced fibroblast spreading and size. To explore the connection between reduced dermal fibroblast size and age-related elevation of HGF expression, we manipulate dermal fibroblast size, and cell-size dependent regulation of HGF was investigated by laser capture microdissection, immunostaining, capillary electrophoresis immunoassay, and quantitative RT-PCR.

Results: We found that reduced fibroblast size is responsible for age-related elevation of HGF expression. Further investigation indicated that cell size-dependent upregulation of HGF expression was mediated by impeded YAP/TAZ nuclear translocation and their target gene, CCN2. Conversely, restoration of dermal fibroblast size rapidly reversed cell-size-dependent upregulation of HGF in a YAP/TAZ-dependent manner. Finally, we confirmed that elevated HGF expression is accompanied by the reduced expression of YAP/TAZ and CCN2 in the aged human skin in vivo.

Conclusion: Age-related elevation of HGF is driven by the reduction of fibroblast size in a YAP/TAZ/CCN2 axis-dependent manner. These data reveal a novel mechanism by which reduction of fibroblast size upregulates HGF expression, which in turn contributes to loss of collagen, a prominent feature of aged human skin.

Keywords: CCN2; Cell size; Dermal aging; HGF; YAP/TAZ.

Elevated expression of the multifunctional cytokine transforming growth factor β1 (TGF-β1) is causatively linked to kidney fibrosis progression initiated by diabetic, hypertensive, obstructive, ischemic and toxin-induced injury. Therapeutically relevant approaches to directly target the TGF-β1 pathway (e.g., neutralizing antibodies against TGF-β1), however, remain elusive in humans. TGF-β1 signaling is subjected to extensive negative control at the level of TGF-β1 receptor, SMAD2/3 activation, complex assembly and promoter engagement due to its critical role in tissue homeostasis and numerous pathologies. Progressive kidney injury is accompanied by the deregulation (loss or gain of expression) of several negative regulators of the TGF-β1 signaling cascade by mechanisms involving protein and mRNA stability or epigenetic silencing, further amplifying TGF-β1/SMAD3 signaling and fibrosis. Expression of bone morphogenetic proteins 6 and 7 (BMP6/7), SMAD7, Sloan-Kettering Institute proto-oncogene (Ski) and Ski-related novel gene (SnoN), phosphate tensin homolog on chromosome 10 (PTEN), protein phosphatase magnesium/manganese dependent 1A (PPM1A) and Klotho are dramatically decreased in various nephropathies in animals and humans albeit with different kinetics while the expression of Smurf1/2 E3 ligases are increased. Such deregulations frequently initiate maladaptive renal repair including renal epithelial cell dedifferentiation and growth arrest, fibrotic factor (connective tissue growth factor (CTGF/CCN2), plasminogen activator inhibitor type-1 (PAI-1), TGF-β1) synthesis/secretion, fibroproliferative responses and inflammation. This review addresses how loss of these negative regulators of TGF-β1 pathway exacerbates renal lesion formation and discusses the therapeutic value in restoring the expression of these molecules in ameliorating fibrosis, thus, presenting novel approaches to suppress TGF-β1 hyperactivation during chronic kidney disease (CKD) progression.

Keywords: chronic kidney disease; renal fibrosis; transforming growth factor-beta.

Importin 8, encoded by IPO8, is a ubiquitously expressed member of the importin-β protein family that translocates cargo molecules such as proteins, RNAs, and ribonucleoprotein complexes into the nucleus in a RanGTP-dependent manner. Current knowledge of the cargoes of importin 8 is limited, but TGF-β signaling components such as SMAD1-4 have been suggested to be among them. Here, we report that bi-allelic loss-of-function variants in IPO8 cause a syndromic form of thoracic aortic aneurysm (TAA) with clinical overlap with Loeys-Dietz and Shprintzen-Goldberg syndromes. Seven individuals from six unrelated families showed a consistent phenotype with early-onset TAA, motor developmental delay, connective tissue findings, and craniofacial dysmorphic features. A C57BL/6N Ipo8 knockout mouse model recapitulates TAA development from 8-12 weeks onward in both sexes but most prominently shows ascending aorta dilatation with a propensity for dissection in males. Compliance assays suggest augmented passive stiffness of the ascending aorta in male Ipo8^-/- mice throughout life. Immunohistological investigation of mutant aortic walls reveals elastic fiber disorganization and fragmentation along with a signature of increased TGF-β signaling, as evidenced by nuclear pSmad2 accumulation. RT-qPCR assays of the aortic wall in male Ipo8^-/- mice demonstrate decreased Smad6/7 and increased Mmp2 and Ccn2 (Ctgf) expression, reinforcing a role for dysregulation of the TGF-β signaling pathway in TAA development. Because importin 8 is the most downstream TGF-β-related effector implicated in TAA pathogenesis so far, it offers opportunities for future mechanistic studies and represents a candidate drug target for TAA.

Keywords: Loeys-Dietz syndrome; Shprintzen-Goldberg syndrome; TGF-beta; importin 8; knockout mouse model; thoracic aortic aneurysm.

The extracellular matrix (ECM) is critical in all aspects of vascular development and health: supporting cell anchorage, providing structure, organization and mechanical stability, and serving as a sink for growth factors and sustained survival signals. Abnormal changes in ECM protein expression, organization, and/or properties, and the ensuing changes in vascular compliance affect vasodilator responses, microvascular pressure transmission, and collateral perfusion. The changes in microvascular compliance are independent factors initiating, driving, and/or exacerbating a plethora of microvascular diseases of the eye including diabetic retinopathy (DR) and vitreoretinopathy, retinopathy of prematurity (ROP), wet age-related macular degeneration (AMD), and neovascular glaucoma. Congruently, one of the major challenges with most vascular regenerative therapies utilizing localized growth factor, endothelial progenitor, or genetically engineered cell delivery, is the regeneration of blood vessels with physiological compliance properties. Interestingly, vascular cells sense physical forces, including the stiffness of their ECM, through mechanosensitive integrins, their associated proteins and the actomyosin cytoskeleton, which generates biochemical signals that culminate in a rapid expression of matricellular proteins such as cellular communication network 1 (CCN1) and CCN2 (aka connective tissue growth factor or CTGF). Loss or gain of function of these proteins alters genetic programs of cell growth, ECM biosynthesis, and intercellular signaling, that culminate in changes in cell behavior, polarization, and barrier function. In particular, the function of the matricellular protein CCN2/CTGF is critical during retinal vessel development and regeneration wherein new blood vessels form and invest a preformed avascular neural retina following putative gradients of matrix stiffness. These observations underscore the need for further in-depth characterization of the ECM-derived cues that dictate structural and functional properties of the microvasculature, along with the development of new therapeutic strategies addressing the ECM-dependent regulation of pathophysiological stiffening of blood vessels in ischemic retinopathies.

Keywords: CCN2; CTGF; angiogenesis; basement membrane; diabetic retinopathy; extracellular matrix; growth factor; ischemia; ischemic retinopathy; neovascularization; retina; stiffness.

Epidermal-specific deletion of integrin α3β1 almost completely prevents the formation of papillomas during 7,12-Dimethylbenz[ a ]anthracene/12- O -tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage skin carcinogenesis. This dramatic decrease in tumorigenesis was thought to be due to an egress and premature differentiation of α3β1-depleted hair bulge (HB) stem cells (SCs), previously considered to be the cancer cells-of-origin in the DMBA/TPA model. Using a reporter mouse line with inducible deletion of α3β1 in HBs, we show that HB SCs remain confined to their niche regardless of the presence of α3β1 and are largely absent from skin tumors. However, tumor formation was significantly decreased in mice deficient for α3β1 in HB SCs. RNA sequencing of HB SCs isolated from short-term DMBA/TPA-treated skin showed α3β1-dependent expression of the matricellular protein connective tissue growth factor (CCN2), which was confirmed in vitro, where CCN2 promoted colony formation and 3D growth of transformed keratinocytes. Together, these findings show that HBs contribute to skin tumorigenesis in an α3β1-dependent manner and suggest a role of HB SCs in creating a permissive environment for tumor growth through the modulation of CCN2 secretion.

Drug-induced gingival enlargement (DIGE) is a fibrotic condition that can be caused by the antihypertensive drug nifedipine and the anti-seizure drug phenytoin, but the molecular etiology of this type of fibrosis is not well understood and the role of confounding factors such as inflammation remains to be fully investigated. The aim of this study was to develop an ex vivo gingival explant system to allow investigation of the effects of nifedipine and phenytoin alone on human gingival tissue. Comparisons were made to the histology of human DIGE tissue retrieved from individuals with DIGE. Increased collagen, fibronectin, and proliferating fibroblasts were evident, but myofibroblasts were not detected in DIGE samples caused by nifedipine and phenytoin. In healthy gingiva cultured in nifedipine or phenytoin-containing media, the number of cells positive for p-SMAD2/3 increased, concomitant with increased CCN2 and periostin immunoreactivity compared to untreated explants. Collagen content assessed through hydroxyproline assays was significantly higher in tissues cultured with either drug compared to control tissues, which was confirmed histologically. Matrix fibronectin levels were also qualitatively greater in tissues treated with either drug. No significant differences in proliferating cells were observed between any of the conditions. Our study demonstrates that nifedipine and phenytoin activate canonical transforming growth factor-beta signaling, CCN2 and periostin expression, as well as increase collagen density, but do not influence cell proliferation or induce myofibroblast differentiation. We conclude that in the absence of confounding variables, nifedipine and phenytoin alter matrix homeostasis in gingival tissue explants ex vivo, and drug administration is a significant factor influencing ECM accumulation in gingival enlargement.

OBJECTIVE

To investigate the in vitro effects of CCN2 on odontoblast-like cells proliferation and differentiation.

METHODS

MDPC-23 cells were cultured in DMEM supplemented with 5% FBS. CCN2 was either added to culture media or coated onto culture polystyrene, addition or coating of dH2O was served as control. In the addition group, CCN2 (100 ng/mL) was added into culture media. In the coating group, CCN2 at the concentration of 1000 ng/mL was employed. Cell proliferation was performed using CCK-8 assay. Cell differentiation and mineralization were analyzed by ALPase activity assay, real time RT-PCR and alizarin red staining. One-way ANOVA with post-hoc tukey HSD test was used for statistical analysis.

RESULTS

MDPC-23 cells exhibited robust proliferative activity upon exposure to either soluble or immobilized CCN2. ALP activity of cells cultured on CCN2-modified surface was continuously strengthened from day six (0.831 ± 0.024 units/μg protein versus 0.563 ± 0.006 units/μg protein of control) till day eight (1.035 ± 0.139 units/μg protein versus 0.704 ± 0.061 units/μg protein of control). Gene expression of BSP, OCN and OPN were promoted by soluble CCN2 after 48 h exposure. Moreover, gene expression of BSP, OCN, OPN, ALP, COL1 A1, Runx-2, DSPP and DMP-1 was significantly enhanced by immobilized CCN2. Finally, mineralization of MDPC-23 cells was accelerated by both soluble and immobilized CCN2 to different extent.

CONCLUSIONS

The findings indicate that CCN2 promoted proliferation, odontogenic gene expression and mineralization of MDPC-23 cells. It is proposed that CCN2 may be a promising adjunctive formula for dentin regeneration.

An abnormal tumor growth induces solid stress in tumors, thus reducing blood perfusion. As a result, the impaired blood perfusion, with dense interstitial matrix in tumors significantly reduces the penetration and efficacy of nanotherapeutics. In this study, we have developed a losartan-loaded polydopamine nanoparticle (PLST) for the enhanced delivery of nanoparticles to tumors and improved photothermal cancer therapy. Losartan, an angiotensin inhibitor, is also able to alleviate the solid stress in tumors. It was laden on polydopamine nanoparticles via π-π stacking and was released upon tumor extracellular acidity. PLST reduced collagen production in vitro along with the lowered expression of profibrotic factors of TGF-β1, CCN2, and TIMP-1. The in vivo studies reveal that PLST reduced solid stress in tumors, and the amount of PLST accumulated in tumors was enhanced. The efficiency of the photothermal ablation of tumors was significantly enhanced by using PLST.

BACKGROUND

The aims of this study were to investigate the gene expression of CCN family members in rat intervertebral disc (IVD) cells and to examine whether Wnt-β-catenin signaling regulates the expression of CCN family 2 (CCN2)/connective tissue growth factor (CTGF) in rat nucleus pulposus (NP) cells.

METHODS

The gene expression of CCN family members were assessed in rat IVD cells using real-time reverse transcription polymerase chain reaction (RT-PCR). The expression pattern of CCN2 was also assessed in rat IVD cells using western blot and immunohistochemical analyses. Gain-of-function and loss-of-function experiments were performed to identify the mechanisms by which Wnt-β-catenin signaling influences the activity of the CCN2 promoter. To further determine if the mitogen-activated protein kinase (MAPK) pathway is required for the Wnt-β-catenin signaling-induced regulation of CCN2 expression in the NP cells, CCN2 expression was analyzed by reporter assay, RT-PCR and western blot analysis.

RESULTS

CCN2 messenger RNA (mRNA) and protein were expressed in rat IVDs. Expression of CCN2 was significantly higher than for mRNA of other CCN family members in both rat NP and annulus fibrosus (AF) cells. The relative activity of the CCN2 promoter decreased 24 h after treatment with 6-bromoindirubin-3'-oxime (1.0 μM) (0.773 (95% 0.735, 0.812) P = 0.0077) in NP cells. In addition, treatment with the WT-β-catenin vector (500 ng) significantly decreased CCN2 promoter activity (0.688 (95% 0.535, 0.842) P = 0.0063), whereas β-catenin small interfering RNA (500 ng) significantly increased CCN2 promoter activity (1.775 (95% 1.435, 2.115) P < 0.001). Activation of Wnt-β-catenin signaling decreased the expression of CCN2 mRNA and protein by NP cells. Regulation of CCN2 by Wnt-β-catenin signaling involved the MAPK pathway in rat NP cells.

CONCLUSIONS

This study shows that Wnt-β-catenin signaling regulates the expression of CCN2 through the MAPK pathway in NP cells. Understanding the balance between Wnt-β-catenin signaling and CCN2 is necessary for developing therapeutic alternatives for the treatment of IVD degeneration.

CCN2 is a matricellular protein involved in several critical biological processes. In particular, CCN2 is involved in cartilage development and in osteoarthritis. CCN2 null mice exhibit a range of skeletal dysmorphisms, highlighting its importance in regulating matrix formation during development, however its role in adult cartilage remains unclear. The aim of this study was to determine the role of CCN2 in postnatal chondrocytes in models of post-traumatic osteoarthritis (PTOA). CCN2 deletion was induced in articular chondrocytes of male transgenic mice at 8 weeks of age. PTOA was induced in knees either surgically or non-invasively by repetitive mechanical loading at 10 weeks of age. Knee joints were harvested, scanned with micro-computed tomography and processed for histology. Sections were stained with toluidine blue and scored using the OARSI grading system. In the non-invasive model cartilage lesions were present in the lateral femur but no significant differences were observed between wildtype (WT) and CCN2 knockout (KO) mice 6 weeks post-loading. In the surgical model, severe cartilage degeneration was observed in the medial compartments but no significant differences were observed between WT and CCN2 KO mice at 2, 4, and 8 weeks post-surgery. We conclude that CCN2 deletion in chondrocytes did not modify the development of PTOA in mice, suggesting that chondrocyte expression of CCN2 in adults is not a critical player in protecting cartilage from the degeneration associated with PTOA.

Keywords: CCN2; Cartilage; Osteoarthritis; Post-traumatic; Transgenic mouse; Trauma-induced.

Connective tissue growth factor or cellular communication network 2 (CCN2/CTGF) is a matricellular protein member of the CCN family involved in several crucial biological processes. In skeletal muscle, CCN2/CTGF abundance is elevated in human muscle biopsies and/or animal models for diverse neuromuscular pathologies, including muscular dystrophies, neurodegenerative disorders, muscle denervation, and muscle overuse. In this context, CCN2/CTGF is deeply involved in extracellular matrix (ECM) modulation, acting as a strong pro-fibrotic factor that promotes excessive ECM accumulation. Reducing CCN2/CTGF levels or biological activity in pathological conditions can decrease fibrosis, improve muscle architecture and function. In this work, we summarize information about the role of CCN2/CTGF in fibrosis associated with neuromuscular pathologies and the mechanisms and signaling pathways that regulate their expression in skeletal muscle.

Keywords: ALS, Amyotrophic Lateral Sclerosis; CCN2/CTGF; CCN2/CTGF, connective tissue growth factor; DMD, Duchenne muscular dystrophy; ECM, extracellular matrix; FG-3019; Fibrosis; LPA, lysophosphatidic acid; Neuromuscular diseases; Skeletal muscle.

Fibrosis is a major cause of end-stage renal disease (ESRD) a progressive loss in renal function that occurs over a period of months or years, is characterized by a decreased capability of the kidneys to excrete waste products. There is no specific treatment unequivocally shown to slow the worsening of chronic kidney disease. Plasma levels of CCN2, a fibrogenic agent, is a predictor of ESRD and mortality in patients with type 1 diabetic nephropathy. CCN3 has been hypothesized to have antagonistic effects to CCN2 both in vitro and in vivo, including in cultured mesangial cells. In a recent study, van Roeyen and colleagues (Am J Pathol in press, 2012) showed that in vivo overexpression of CCN3 in a model of anti-Thy1.1-induced experimental glomerulonephritis resulted in decreased albuminuria, glomerulosclerosis and reduced cortical collagen type I accumulation. CCN3 enhanced angiogenesis yes suppressed mesangial cell proliferation. Thus CCN3 protein may represent a novel therapeutic approach to help repair glomerular endothelial damage and mesangioproliferative changes and hence prevent renal failure, glomerulosclerosis and tubulointerstitial fibrosis.

During the pathogenesis of glaucoma, optic nerve (ON) axons become continuously damaged at the optic nerve head (ONH). This often is associated with reactive astrocytes and increased transforming growth factor (TGF-β) 2 levels. In this study we tested the hypothesis if the presence or absence of decorin (DCN), a small leucine-rich proteoglycan and a natural inhibitor of several members of the TGF family, would affect the expression of the TGF-βs and connective tissue growth factor (CTGF/CCN2) in human ONH astrocytes and murine ON astrocytes. We found that DCN is present in the mouse ON and is expressed by human ONH and murine ON astrocytes. DCN expression and synthesis was significantly reduced after 24 h treatment with 3 nM CTGF/CCN2, while treatment with 4 pM TGF-β2 only reduced expression of DCN significantly. Conversely, DCN treatment significantly reduced the expression of TGF-β1, TGF-β2 and CTGF/CCN2 vis-a-vis untreated controls. Furthermore, DCN treatment significantly reduced expression of fibronectin (FN) and collagen IV (COL IV). Notably, combined treatment with DCN and triciribine, a small molecule inhibitor of protein kinase B (AKT), attenuated effects of DCN on CTGF/CCN2, TGF-β1, and TGF-β2 mRNA expression. We conclude (1) that DCN is an important regulator of TGF-β and CTGF/CCN2 expression in astrocytes of the ON and ONH, (2) that DCN thereby regulates the expression of extracellular matrix (ECM) components and (3) that DCN executes its negative regulatory effects on TGF-β and CTGF/CCN2 via the pAKT/AKT signaling pathway in ON astrocytes.

Keywords: AKT signaling; CTGF/CCN2; TGF-beta; astrocytes; decorin; glaucoma; optic nerve head; reactive gliosis.

Chronic kidney disease (CKD) is characterized by pathological accumulation of extracellular matrix (ECM) proteins in renal structures. Tubulointerstitial fibrosis is observed in glomerular diseases as well as in the regeneration failure of acute kidney injury (AKI). Therefore, finding antifibrotic therapies comprises an intensive research field in Nephrology. Nowadays, ECM is not only considered as a cellular scaffold, but also exerts important cellular functions. In this review, we describe the cellular and molecular mechanisms involved in kidney fibrosis, paying particular attention to ECM components, profibrotic factors and cell-matrix interactions. In response to kidney damage, activation of glomerular and/or tubular cells may induce aberrant phenotypes characterized by overproduction of proinflammatory and profibrotic factors, and thus contribute to CKD progression. Among ECM components, matricellular proteins can regulate cell-ECM interactions, as well as cellular phenotype changes. Regarding kidney fibrosis, one of the most studied matricellular proteins is cellular communication network-2 (CCN2), also called connective tissue growth factor (CTGF), currently considered as a fibrotic marker and a potential therapeutic target. Integrins connect the ECM proteins to the actin cytoskeleton and several downstream signaling pathways that enable cells to respond to external stimuli in a coordinated manner and maintain optimal tissue stiffness. In kidney fibrosis, there is an increase in ECM deposition, lower ECM degradation and ECM proteins cross-linking, leading to an alteration in the tissue mechanical properties and their responses to injurious stimuli. A better understanding of these complex cellular and molecular events could help us to improve the antifibrotic therapies for CKD.

Keywords: ccn2; chronic kidney disease; extracellular matrix components; fibrosis; integrins; matricellular proteins.

Connective tissue growth factor (CTGF, CCN2) is a matricellular protein which plays key roles in normal mammalian development and in tissue homeostasis and repair. In pathological conditions, dysregulated CCN2 has been associated with cancer, cardiovascular disease, and tissue fibrosis. In this study, genetic manipulation of the CCN2 gene was employed to investigate the role of CCN2 expression in vitro and in experimentally-induced models of pulmonary fibrosis and pulmonary arterial hypertension (PAH). Knocking down CCN2 using siRNA, reduced expression of pro-fibrotic markers (fibronectin p < 0.01, collagen type I p < 0.05, α-SMA p < 0.0001, TIMP-1 p < 0.05 and IL-6 p < 0.05) in TGF-β-treated lung fibroblasts derived from systemic sclerosis patients. In vivo studies were performed in mice using a conditional gene deletion strategy targeting CCN2 in a fibroblast-specific and time-dependent manner in two models of lung disease. CCN2 deletion significantly reduced pulmonary interstitial scarring and fibrosis following bleomycin-instillation, as assessed by fibrotic scores (wildtype bleomycin 3.733 ± 0.2667 vs CCN2 knockout (KO) bleomycin 4.917 ± 0.3436, p < 0.05) and micro-CT. In the well-established chronic hypoxia model of pulmonary hypertension, CCN2 gene deletion resulted in a significant decrease in pulmonary vessel remodelling, less right ventricular hypertrophy and a reduction in the haemodynamic measurements characteristic of PAH (RVSP and RV/LV + S were significantly reduced (p < 0.05) in CCN2 KO compared to WT mice in hypoxic/SU5416 conditions). These results support a prominent role for CCN2 in pulmonary fibrosis and in vessel remodelling associated with PAH. Therefore, therapeutics aimed at blocking CCN2 function are likely to benefit several forms of severe lung disease.

Keywords: Bleomycin-induced fibrosis; Connective tissue grown factor (CTGF, CCN2); Interstitial lung disease (ILD); Pulmonary arterial hypertension (PAH); Pulmonary fibrosis; Systemic sclerosis (scleroderma, SSc).

During ischemia/reperfusion (I/R), cardiomyocytes activate pathways that regulate cell survival and death and release factors that modulate fibroblast-to-myofibroblast differentiation. The mechanisms underlying these effects are not fully understood. Polycystin-1 (PC1) is a mechanosensor crucial for cardiac function. This work aims to assess the role of PC1 in cardiomyocyte survival, its role in profibrotic factor expression in cardiomyocytes, and its paracrine effects on I/R-induced cardiac fibroblast function. In vivo and ex vivo I/R and simulated in vitro I/R (sI/R) were induced in wild-type and PC1-knockout (PC1 KO) mice and PC1-knockdown (siPC1) neonatal rat ventricular myocytes (NRVM), respectively. Neonatal rat cardiac fibroblasts (NRCF) were stimulated with conditioned medium (CM) derived from NRVM or siPC1-NRVM supernatant after reperfusion and fibroblast-to-myofibroblast differentiation evaluated. Infarcts were larger in PC1-KO mice subjected to in vivo and ex vivo I/R, and necrosis rates were higher in siPC1-NRVM than control after sI/R. PC1 activated the pro-survival AKT protein during sI/R and induced PC1-AKT-pathway-dependent CTGF expression. Furthermore, conditioned media from sI/R-NRVM induced PC1-dependent fibroblast-to-myofibroblast differentiation in NRCF. This novel evidence shows that PC1 mitigates cardiac damage during I/R, likely through AKT activation, and regulates CTGF expression in cardiomyocytes via AKT. Moreover, PC1-NRVM regulates fibroblast-to-myofibroblast differentiation during sI/R. PC1, therefore, may emerge as a new key regulator of I/R injury-induced cardiac remodeling.

Keywords: AKT; CTGF/CCN2; Cardiac fibroblast differentiation; Cardiac ischemia/reperfusion; Polycystin-1.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin fibrosis. Altered metabolism has recently been described in autoimmune diseases and SSc. Itaconate is a product of the Krebs cycle intermediate cis-aconitate and is an immunomodulator. This work examines the role of the cell-permeable derivative of itaconate, 4-octyl itaconate (4-OI), in SSc. SSc and healthy dermal fibroblasts were exposed to 4-OI. The levels of collagen Nrf2-target genes and pro-inflammatory cytokines interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined. Levels of reactive oxygen species (ROS) as well as the gene expression of collagen and Cellular Communication Network Factor 2 (CCN2) were measured after transforming growth factor beta 1 (TGF-β1) stimulation in the presence or absence of 4-OI. Wild-type or Nrf2-knockout (Nrf2-KO) mouse embryonic fibroblasts (MEFs) were also treated with 4-OI to determine the role of Nrf2 in 4-OI-mediated effects. 4-OI reduced the levels of collagen in SSc dermal fibroblasts. Incubation with 4-OI led to activation of Nrf2 and its target genes heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). 4-OI activated antioxidant response element (ARE)-dependent gene expression, reduced inflammatory cytokine release and reduced TGF-β1-induced collagen and ROS production in dermal fibroblasts. The effects of 4-OI are dependent on Nrf2. The cell-permeable derivative of itaconate 4-OI is anti-fibrotic through upregulation of Nrf2 and could be a potential therapeutic option in an intractable disease.

Keywords: Nrf2; fibrosis; itaconate; metabolism; systemic sclerosis.

Angiogenesis, the process of generating new blood vessels from an existing vasculature, is essential in normal developmental processes such as endochondral ossification and in numerous kinds of pathogenesis including tumor growth. A part from the action of angiogenic factor or antiangiogenic factor, it is still unknown at which stage of the angiogenic cascade these agents affect angiogenesis. Here, we describe methods for the use of connective tissue growth factor (CTGF/CCN2) and CCN2 neutralizing antibody in the currently used principal angiogenesis assays, including those in vitro ones for the proliferation, migration, adhesion, and tube formation of endothelial cells and in vivo assays such as those utilizing type I collagen implantation and the chick chorioallantoic membrane (CAM).

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by progressive and excessive accumulation of myofibroblasts and extracellular matrix in the lung. Connective-tissue growth factor (CTGF) exacerbates pulmonary fibrosis in radiation-induced lung fibrosis, and in this study, we demonstrate upregulation of CTGF in a rat lung fibrosis model induced by an adenovirus vector encoding active TGF-β1 (AdTGF-β1). We show that CTGF is also upregulated in patients with IPF. Expression of CTGF was upregulated in vascular smooth muscle cells cultured from fibrotic lungs on days 7 and 14 as well as endothelial cells sorted from fibrotic lungs on days 14 and 28. These findings suggest contributions of different cells in maintaining the fibrotic phenotype during fibrogenesis. Treatment of fibroblasts with recombinant CTGF along with TGF-β increases pro-fibrotic markers in fibroblasts, confirming the synergistic effect of recombinant CTGF with TGF-β in inducing pulmonary fibrosis. Also, fibrotic extracellular matrix upregulated CTGF expression, compared with normal extracellular matrix, suggesting that not only profibrotic mediators, but also a profibrotic environment contributes to fibrogenesis. We also showed that pamrevlumab, a CTGF inhibitory antibody, partially attenuates fibrosis in the model. These results suggest that pamrevlumab could be an option for treatment of pulmonary fibrosis.

Keywords: CTGF; animal model; fibrosis.

Objective: Non-alcoholic fatty liver disease (NAFLD) is one of the main risk factors for cardiovascular mortality and morbidity. This study, for the first time, explored the effects of trans-chalcone on cardiac expressions of myocardial fibrosis-related genes, including transforming growth factor -β1 (TGF-β1), connective tissue growth factor (CTGF/CCN2), and collagen type I.Materials and methods: Twenty-eight rats were randomly divided into four groups: control, received 10% tween 80; chalcone, received trans-chalcone; HFD, received high-fat diet (HFD) and 10% tween 80; HFD + chalcone, received HFD and trans-chalcone, by once-daily gavage for 6 weeks. Finally, cardiac expression levels of TGF-β1, CTGF, and collagen type I were determined.Results: HFD feeding increased mRNA levels of collagen type I, TGF-β1, and CTGF in the heart of rats. However, trans-chalcone inhibited HFD-induced changes.Conclusions:trans-Chalcone can act as a cardioprotective compound by inhibiting TGF-β1 and CTGF-dependent stimulation of collagen type I synthesis in the heart of HFD-fed rats.

CCN family is a novel family of proteins consisting of several modulator molecules. The members display a variety of physiological and pathological functions ; hence they are currently attracting the interest of a number of biologists. In terms of the development and regeneration of cartilage tissues, CCN2/connective tissue growth factor (CTGF) is best known among the CCN family members, since it efficiently promotes endochondral ossification and articular cartilage regeneration. Recently, it has been uncovered how CCN2 gene expression is duly regulated along with the differentiation of chondrocytes, which is uncovering the genetic program leading to cartilage tissue development. Moerover, production of other members, such as CCN1 and CCN4, are occasionally observed in chondrocytes, suggesting the contribution of the entire CCN family members to the developmental process of cartilage in vivo.