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CTGF knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload
Abstract
Background:
One of the main contributors to maladaptive cardiac remodeling is fibrosis. Connective tissue growth factor (CTGF), a matricellular protein that is secreted into the cardiac extracellular matrix by both cardiomyocytes and fibroblasts, is often associated with development of fibrosis. However, recent studies have questioned the role of CTGF as a pro-fibrotic factor. Therefore, we aimed to investigate the effect of CTGF on cardiac fibrosis, and on functional, structural, and electrophysiological parameters in a mouse model of CTGF knockout (KO) and chronic pressure overload.
Methods and results:
A new mouse model of global conditional CTGF KO induced by tamoxifen-driven deletion of CTGF, was subjected to 16weeks of chronic pressure overload via transverse aortic constriction (TAC, control was sham surgery). CTGF KO TAC mice presented with hypertrophic hearts, and echocardiography revealed a decrease in contractility on a similar level as control TAC mice. Ex vivo epicardial mapping showed a low incidence of pacing-induced ventricular arrhythmias (2/12 in control TAC vs. 0/10 in CTGF KO TAC, n.s.) and a tendency towards recovery of the longitudinal conduction velocity of CTGF KO TAC hearts. Picrosirius Red staining on these hearts unveiled increased fibrosis at a similar level as control TAC hearts. Furthermore, genes related to fibrogenesis were also similarly upregulated in both TAC groups. Histological analysis revealed an increase in fibronectin and vimentin protein expression, a significant reduction in connexin43 (Cx43) protein expression, and no difference in NaV1.5 expression of CTGF KO ventricles as compared with sham treated animals.
Conclusion:
Conditional CTGF inhibition failed to prevent TAC-induced cardiac fibrosis and hypertrophy. Additionally, no large differences were found in other parameters between CTGF KO and control TAC mice. With no profound effect of CTGF on fibrosis formation, other factors or pathways are likely responsible for fibrosis development.
... Experimental evidence suggests that CCN2 can regulate redox processes linked to proinflammatory responses, as described in vascular smooth muscle cells [39], but kidney data are scarce. In experimental AKI induced by ischemia-reperfusion injury (IRI), we have previously described that CCN2 regulates the redox/NRF2 pathway associated with the activation of cellular senescence [32,40]. Although the activation of several proinflammatory-related signaling pathways was shown to be involved in CCN2 actions in the kidney [33][34][35][36][37][38][39][40][41], to date, the potential role of the NLRP3/RIPK3 pathway has not been investigated. ...
... In experimental AKI induced by ischemia-reperfusion injury (IRI), we have previously described that CCN2 regulates the redox/NRF2 pathway associated with the activation of cellular senescence [32,40]. Although the activation of several proinflammatory-related signaling pathways was shown to be involved in CCN2 actions in the kidney [33][34][35][36][37][38][39][40][41], to date, the potential role of the NLRP3/RIPK3 pathway has not been investigated. To fill this knowledge gap, we evaluated the response of CCN2 conditionally deficient mice to AKI induced with a nephrotoxic compound (folic acid administration) and also the direct effects of CCN2 administration on murine kidneys. ...
... In this study, we found that CCN2 deficiency reduced the inflammatory response caused by FA-AKI, diminishing T cell and macrophage recruitment and the gene expression of proinflammatory factors. Previously, CCN2 deletion also ameliorated inflammatory-related events in the acute phase of IRI [40]. These data extend previous studies describing the role of CCN2 in the regulation of sterile inflammation to pathological processes associated with nephrotoxic or ischemic insults causing AKI. ...
Inflammation is a key characteristic of both acute and chronic kidney diseases. Preclinical data suggest the involvement of the NLRP3/Inflammasome, receptor-interacting protein kinase-3 (RIPK3), and NRF2/oxidative pathways in the regulation of kidney inflammation. Cellular communication network factor 2 (CCN2, also called CTGF in the past) is an established fibrotic biomarker and a well-known mediator of kidney damage. CCN2 was shown to be involved in kidney damage through the regulation of proinflammatory and profibrotic responses. However, to date, the potential role of the NLRP3/RIPK3/NRF2 pathways in CCN2 actions has not been evaluated. In experimental acute kidney injury induced with folic acid in mice, CCN2 deficiency diminished renal inflammatory cell infiltration (monocytes/macrophages and T lymphocytes) as well as the upregulation of proinflammatory genes and the activation of NLRP3/Inflammasome-related components and specific cytokine products, such as IL-1β. Moreover, the NRF2/oxidative pathway was deregulated. Systemic administration of CCN2 to C57BL/6 mice induced kidney immune cell infiltration and activated the NLRP3 pathway. RIPK3 deficiency diminished the CCN2-induced renal upregulation of proinflammatory mediators and prevented NLRP3 modulation. These data suggest that CCN2 plays a fundamental role in sterile inflammation and acute kidney injury by modulating the RIKP3/NLRP3/NRF2 inflammatory pathways.
... CTGF is also involved in initiating processes underlying fibrosis of several organs and tissues, including the heart [6]. However, data on its regulation and its functional role in the pathogenesis of cardiac remodeling and fibrosis in the failing heart are limited and controversial [7][8][9][10][11][12][13][14][15]. ...
... In contrast to the beneficial effects of CTGF inhibition in TAC [13] and PKCε-induced DCM (this report), targeted deletion of most CTGF in adult cardiomyocytes and nonmuscle cells did not appear to prevent the functional changes or fibrosis induced by TAC [14,15], or overexpression of a constitutively active form of TGF-β [14]. However, in some experiments, the reported data exhibited trends consistent with a beneficial effect of loss of CTGF function on fibrillar collagen gene expression [15], but were not significant at the small group sizes examined [14]. ...
... In contrast to the beneficial effects of CTGF inhibition in TAC [13] and PKCε-induced DCM (this report), targeted deletion of most CTGF in adult cardiomyocytes and nonmuscle cells did not appear to prevent the functional changes or fibrosis induced by TAC [14,15], or overexpression of a constitutively active form of TGF-β [14]. However, in some experiments, the reported data exhibited trends consistent with a beneficial effect of loss of CTGF function on fibrillar collagen gene expression [15], but were not significant at the small group sizes examined [14]. Thus, although further investigation is necessary to understand the differences observed from the various animal models used, the cumulative data suggest that inhibition of CTGF in patients with cardiovascular disease is unlikely to be harmful and instead, could provide a significant benefit to patients with DCM. ...
Cardiac structural changes associated with dilated cardiomyopathy (DCM) include cardiomyocyte hypertrophy and myocardial fibrosis. Connective Tissue Growth Factor (CTGF) has been associated with tissue remodeling and is highly expressed in failing hearts. Our aim was to test if inhibition of CTGF would alter the course of cardiac remodeling and preserve cardiac function in the protein kinase Cε (PKCε) mouse model of DCM. Transgenic mice expressing constitutively active PKCε in cardiomyocytes develop cardiac dysfunction that was evident by 3months of age, and that progressed to cardiac fibrosis, heart failure, and increased mortality. Beginning at 3months of age, PKCε mice were treated with a neutralizing monoclonal antibody to CTGF (FG-3149) for an additional 3months. CTGF inhibition significantly improved left ventricular (LV) systolic and diastolic function in PKCε mice, and slowed the progression of LV dilatation. Using gene arrays and quantitative PCR, the expression of many genes associated with tissue remodeling were elevated in PKCε mice, but significantly decreased by CTGF inhibition. However total collagen deposition was not attenuated. The observation of significantly improved LV function by CTGF inhibition in PKCε mice suggests that CTGF inhibition may benefit patients with DCM. Additional studies to explore this potential are warranted.
... Due CCN2-KO mice complete embryonic development but die shortly after birth because of respiratory failure (Ivkovic et al., 2003), a conditional CCN2-KO mice have been developed. In this mice, CCN2 deletion ameliorates experimental renal fibrosis (Rayego-Mateos et al., 2018), but does not improve cardiac fibrosis and hypertrophy following transverse aortic constriction (Fontes et al., 2015). High circulating CCN2 levels have been proposed as a potential risk biomarker for cardiac dysfunction in patients with chronic heart failure and myocardial fibrosis (Koitabashi et al., 2008). ...
... CCN2 deletion: Mice used were CCN2 CCN2flox/floxROSA26-ERT/Cre, henceforth CCN2-KO mice, which are time-conditional knockout (CCN2-KO) mice. They were developed as previously described (Fontes et al., 2015) and used in all experimental models. 13-14 weeks old mice were randomly divided in 2 groups:1) CCN2-KO group, in which tamoxifen were intraperitoneally (IP) injected (0.1 ml of a 10mg/mL solution; C8267, Sigma-Aldrich) four times in alternate days and 2) wild-type (WT) group, which were IP injected with corn oil (0.1 ml) and used as control group. ...
Background and Purpose: Cellular Communication Network Factor 2 (CCN2) is a matricellular protein normally present in the vascular wall but overexpressed in several cardiovascular diseases. CCN2 has been proposed as a downstream mediator of profibrotic actions of Transforming Growth Factor (TGF)-β and Angiotensin II (Ang II). However, its direct role in cardiovascular diseases is not completely understood. Experimental Approach: To investigate the direct role of CCN2 under vascular pathological conditions, a conditionally deficient CCN2 (CCN2-KO) mouse was evaluated infused or not with Ang II. Key Results: In the absence of CCN2, Ang II infusion induced a rapid (within 48 hours) aortic aneurysm generation and increased aneurysm rupture with 80 % lethality at the endpoint. CCN2 deletion caused elastin layer disruption and increased metalloproteinase activity, which were aggravated by Ang II administration. Aortic RNA-seq studies and the subsequent Gene Ontology enriched analysis pointed out the aldosterone biosynthesis process as one of the most enriched terms in absence of CCN2. Pharmacological aldosterone pathway intervention in Ang II-infused CCN2-KO mice, by treatment with the mineralocorticoid receptor antagonist spironolactone, reduced aneurysm formation and mortality after Ang II infusion. Conclusion and Implications: CCN2 deletion induces a rapid aneurysm formation and rupture after Ang II infusion which is partially prevented by blocking the mineralocorticoid receptor. Our present data highlight, for the first time, the potential role of CCN2 as a vascular homeostatic factor and its relevance in the aldosterone synthesis, opening new avenues to future studies in aortic aneurysm treatment.
... FA nephropathy is a model of kidney tubulointerstitial injury that progresses to kidney fibrosis [39,40]. Conditional CCN2-deficient mice (CTGFfl/fl-R26CreER) were developed as described previously [41]. To induce CCN2 deletion, mice received four injections of 10 mg/ml tamoxifen in corn oil (intraperitoneal, over 7 days), and this was followed by a 2-week washout period starting from the last injection. ...
... In several cell types, CCN2(IV) activates the MAPK cascade [38][39][40][41]. We have shown previously that ERK activation is an important EGFR signalling mechanism activated by CCN2(IV) in the kidney and in MCT cells [30]. ...
Connective tissue growth factor (CCN2/CTGF) is a matricellular protein overexpressed in progressive human renal diseases, mainly in fibrotic areas. In vitro studies have demonstrated that CCN2 regulates production of extracellular matrix (ECM) proteins and epithelial mesenchymal transition (EMT), and therefore could contribute to renal fibrosis. CCN2 blockade ameliorates experimental renal damage, including diminution of ECM accumulation. We have described that CCN2 and its C-terminal degradation product CCN2(IV), bind to epidermal growth factor receptor (EGFR) to modulate renal inflammation. However, the receptor involved in CCN2 profibrotic actions has not been described so far. Using a murine model of systemic administration of CCN2(IV) we have unveiled a fibrotic response in the kidney that was diminished by EGFR blockade. Additionally, in CCN2-conditional knockout mice, renal fibrosis elicited by folic acid-induced renal damage was prevented, linked to inhibition of EGFR pathway activation. Our in vitro studies demonstrated a direct effect of CCN2 via the EGFR pathway on ECM production by fibroblasts and the induction of EMT in tubular epithelial cells. Our studies clearly show that the EGFR regulates CCN2 fibrotic signaling in the kidney and suggest that EGFR pathway blockade could be a potential therapeutic option to block CCN2-mediated profibrotic effects in renal diseases.
... CTGF is therefore considered a promising therapeutic target in fibrotic diseases, and indeed clinical trials of monoclonal antibodies targeting CTGF are underway in pulmonary fibrosis and other diseases 30,49 . In heart failure, lineage-specific knockout experiments have shown that CTGF secreted by fibroblasts but not myocardium promotes cardiac fibrosis 50,51 , and . CC-BY-NC-ND 4.0 International license perpetuity. ...
Background
Tissue fibrosis is a common feature of many organ dysfunctions, such as heart failure and chronic kidney disease. However, no fundamental treatment has been developed. This study aims to identify novel molecular mechanisms for antifibrotic intervention, focusing on fibroblast activation.
Methods
We performed a forward genetic screen using a genome-wide CRISPR library in the context of transforming growth factor β (TGF-β)-mediated connective tissue growth factor (CTGF) expression, and used unbiased techniques such as Cleavage Under Targets and Tagmentation (CUT&Tag) and proximity-dependent biotin labeling by TurboID to reveal the detailed molecular mechanisms.
Results
CRISPR library screening identified a number of players in both the canonical Smad pathway and the non-canonical pathway. In addition to the known factors, the Keap1-Nrf2 pathway was identified as a predominant regulator of TGF-β-mediated CTGF expression. Keap1 deletion and consequent Nrf2 activation broadly suppressed profibrotic gene expression, independently of conventional antioxidant effects. CUT&Tag revealed that Nrf2 bound to the proximity of fibrosis-related genes including Ctgf and Fn1. Subsequent individual analysis revealed Smad3 and RNA polymerase II binding to the Nrf2 peak site, which was attenuated by Keap1 deletion. TurboID experiments further discovered that Nrf2 interacts with Ddx54, which acts as a corepressor. Consistently, Keap1 deletion-mediated repression of profibrotic gene expression was reversed by additional Ddx54 deletion. The impact of the Keap1-Nrf2 pathway on pathological fibrosis was examined using tamoxifen-inducible fibroblast-specific Keap1 knockout mice. Pressure overload for 4 weeks robustly induced cardiac hypertrophy, fibrosis and contractile dysfunction. However, deletion of Keap1 in the Postn lineage attenuated these cardiac pathologies. The anti-fibrotic effects of Keap1 deletion were also confirmed in renal fibrosis in the unilateral ureteral obstruction (UUO) model.
Conclusions
Fibroblast Nrf2 transcriptionally represses fibrosis-related genes in cooperation with the corepressor Ddx54. Fibroblast-specific deletion of Keap1 attenuated pathological fibrosis in pressure overload heart failure and renal fibrosis.
... Inhibition of CTGF led to reduction of cardiac fibrosis in diabetic rats. However, in the LV pressure overload mice, CTGF was unrelated to fibrosis development (Fontes et al., 2015;Gravning, Ahmed, von Lueder, Edvardsen, & Attramadal, 2013). Taken together, the available data suggests that the role of CTGF in cardiac fibrosis is subtle or diseasespecific. ...
Right ventricular fibrosis is a stress response, predominantly mediated by cardiac fibroblasts. This cell population is sensitive to increased levels of pro-inflammatory cytokines, pro-fibrotic growth factors and mechanical stimulation. Activation of fibroblasts results in the induction of various molecular signaling pathways, most notably the mitogen-activated protein kinase cassettes, leading to increased synthesis and remodeling of the extracellular matrix. While fibrosis confers structural protection in response to damage induced by ischemia or (pressure and volume) overload, it simultaneously contributes to increased myocardial stiffness and right ventricular dysfunction. Here, we review state-of-the-art knowledge of the development of right ventricular fibrosis in response to pressure overload and provide an overview of all published preclinical and clinical studies in which right ventricular fibrosis was targeted to improve cardiac function.
... Thus, despite the increased levels of CCN2 observed in experimental and human cardiovascular diseases, including heart failure, pulmonary hypertension, restenosis, atherosclerosis or aortic aneurysms [9], the preclinical attempts to regulate CCN2 levels have led into mixed results. In this sense, while CCN2 inhibition using a monoclonal antibody attenuates left ventricular remodeling and dysfunction in pressure overload-induced heart failure [21], CCN2 knockout does not affect cardiac hypertrophy and fibrosis formation upon chronic pressure overload [22], and cardiac-restricted overexpression of CCN2 attenuates left ventricular remodeling after myocardial infarction [23]. Concordantly, the post-ischemic administration of recombinant human CCN2 increased the tolerance of ex vivo-perfused murine hearts to ischemia reperfusion injury [24]. ...
Cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a downstream mediator of other profibrotic factors including transforming growth factor (TGF)-β and angiotensin II. However, recent evidence from our group demonstrated the direct role of CCN2 in maintaining aortic wall homeostasis and acute and lethal aortic aneurysm development induced by angiotensin II in the absence of CCN2 in mice. In order to translate these findings to humans, we evaluated the potential association between three polymorphisms in the CCN2 gene and the presence of a thoracic aortic aneurysm (TAA). Patients with and without TAA retrospectively selected were genotyped for rs6918698, rs9402373 and rs12526196 polymorphisms related to the CCN2 gene. Multivariable logistic regression models were performed. In our population of 366 patients (69 with TAA), no associations were found between rs6918698 and rs9402373 and TAA. However, the presence of one C allele from rs12526196 was associated with TAA comparing with the TT genotype, independently of risk factors such as sex, age, hypertension, type of valvulopathy and the presence of a bicuspid aortic valve (OR = 3.17; 95% CI = 1.30–7.88; p = 0.011). In conclusion, we demonstrated an association between the C allele of rs12526196 in the CCN2 gene and the presence of TAA. This study extrapolates to humans the relevance of CCN2 in aortic aneurysm observed in mice and postulates, for the first time, a potential protective role to CCN2 in aortic aneurysm pathology. Our results encourage future research to explore new variants in the CCN2 gene that could be predisposed to TAA development.
... 24 Generation of tamoxifen-inducible CCN2-full KO mice is described elsewhere. 25 In brief, CCN2 Flox/Flox mice were crossbred with ROSA26CreERT2 mice (Gt(ROSA)26Sor tm(cre/ERT2)Tyj /J, the Jackson Laboratory), both on a C57Bl6/J background. For i.p injection, tamoxifen citrate was dissolved in corn oil (Sigma Q9 Aldrich) in a 10 mg/ml concentration. ...
Chronic allograft dysfunction with progressive fibrosis of unknown cause remains a major issue after kidney transplantation, characterized by ischemia-reperfusion injury (IRI). One hypothesis to account for this is that spontaneous progressive tubulointerstitial fibrosis following IRI is driven by cellular senescence evolving from a prolonged, unresolved DNA damage response (DDR). Since cellular communication network factor 2 ((CCN2), formerly called connective tissue growth factor), an established mediator of kidney fibrosis, is also involved in senescence associated pathways, we investigated the relation between CCN2 and cellular senescence following kidney transplantation. Tubular CCN2 overexpression was found to be associated with DDR, loss of kidney function and tubulointerstitial fibrosis in both the early and the late phase in human kidney allograft biopsies. Consistently, CCN2 deficient mice developed reduced senescence and tubulointerstitial fibrosis in the late phase; six weeks after experimental IRI. Moreover, tubular DDR markers and plasma urea were less elevated in CCN2 knockout than in wild type mice. Finally, CCN2 administration or overexpression in epithelial cells induced upregulation of tubular senescence-associated genes including p21, while silencing of CCN2 alleviated DDR induced by anoxia-reoxygenation injury in cultured proximal tubule epithelial cells. Thus, our observations indicate that inhibition of CCN2 can mitigate IRI-induced acute kidney injury, DNA damage, and the subsequent DDR-senescence-fibrosis sequence. Hence, targeting CCN2 might help to protect the kidney from transplantation associated post-IRI chronic kidney dysfunction.
... Regarding gene expression regulation, CCN2 knockout mice die shortly after birth by respiratory failure due to its essential role in coordinating chondrogenesis and angiogenesis during skeletal development [64]. In adult mice, tamoxifen-dependent CCN2 deletion ameliorated renal fibrosis [65], but it did not improve cardiac fibrosis and hypertrophy [66]. Recently, our group has demonstrated the relevance of CCN2 on maintaining vascular wall homeostasis in a model of Ang II-induced vascular damage. ...
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.
... Experiments were performed according to the European community guidelines for animal experiments and the ARRIVE guidelines and with consent of the Experimental Animal Ethics Committee of the University of Utrecht, The Netherlands [22]. Generation of tamoxifen-inducible CCN2 full-knockout mice is extensively described elsewhere [23]. In brief, CCN2 Flox/Flox mice were crossbred with ROSA26CreERT2 mice (Gt(ROSA)26Sor tm(cre/ERT2)Tyj /J, The Jackson Laboratory, Maine, USA), both on a C57Bl6/J background. ...
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.
... CCN2/CTGF-null mice die shortly after birth from respiratory failure caused by the formation of hypoplastic lungs and failed thoracic expansion (Baguma-Nibasheka and Kablar, 2008). Conditional tissuetype-specific deletion of CCN2/CTGF in mice provided further information about the function of CCN2/CTGF in various cell types (Mohiuddin 2021;Chiou et al. 2006;Brigstock, 2010;Arnott et al. 2011;Gravning et al. 2012;Gibson et al. 2014;Fontes et al. 2015;Petrosino et al. 2019). Even though many of those studies focused overwhelmingly on the biological consequences of cell type-specific overexpression or deletion of CCN2/CTGF on cell density, matrix protein gene expression, and ECM remodeling (Doherty et al. 2010;Nagashima et al. 2011;Gibson et al. 2014;Petrosino et al. 2019;Yu et al. 2019), there still is a paucity of information regarding CCN2/CTGF mode of action. ...
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.
... In theory, intervention on these key signaling systems of atrial fibrosis can delay the progress of atrial fibrosis and prevent/treat atrial fibrillation. However, recent studies have found that interventions targeting some essential factors, such as TGF-β and CTGF that play an important role in atrial fibrosis, failed to achieve desired outcomes [7,8]. It is possible that other endogenous (external) factors that cause atrial fibrosis may play a more important role in the formation of atrial fibrosis and the occurrence/ maintenance of atrial fibrillation. ...
Our previous study found that tryptase activated atrial fibroblasts, increased collagen synthesis in atrial fibroblasts through protease activated receptor-2 (PAR2) receptors. Recent studies showed that cytoskeleton-associated protein 4 (CKAP4) played an important role in ventricular fibroblast activation. The present study aimed to investigate the role of CKAP4 in tryptase-induced atrial fibroblast activation, atrial fibrosis, and molecular regulatory mechanisms. We cultured atrial fibroblasts in vitro, gave cells tryptase stimulation, then overexpressed or silenced PAR2 and CKAP4 genes in the cells. Their effects on atrial fibroblast proliferation, migration, extracellular matrix remodeling (Collagen I and fibronectin) and downstream key molecules (TGF-β1, c-jun and c-fos, JNK, p38) were investigated. The results showed that the expression of CKAP4 was significantly increased by tryptase and further increased by pcDNA3.1-PAR2, but decreased by FALLRY-NH2 and PAR2 siRNA. CKAP4 overexpression significantly increased the cell proliferation, migration and levels of Collagen I and fibronectin, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) levels in atrial fibroblasts, while CKAP4 siRNA significantly reduced them. CKAP4 overexpression significantly increased the expression of TGF-β1, c-jun and c-fos, and activated the JNK/p38 pathway, which were suppressed by CKAP4 siRNA. In conclusion, CKAP4 is involved in tryptase-induced phenotypic conversion in atrial fibroblasts through PAR2/p38/JNK pathway, which may provide novel targets in the prevention of atrial fibrosis.
... CTGF is considered to be a weak promoter of cardiac fibrosis on its own, as cardiac overexpression of CTGF did not induce a fibrotic response in mouse or rat hearts [168]. Additionally, global knockout of CTGF did not impact cardiac hypertrophy nor fibrosis following pressure overload [169]. Rather, the role of CTGF in fibrosis is more appropriately attributed to creating an environment conducive for profibrotic signalling [170][171][172]. ...
Cardiac fibroblasts (cFBs) have emerged as a heterogenous cell population. Fibroblasts are considered the main cell source for synthesis of the extracellular matrix (ECM) and as such a dysregulation in cFB function, activity, or viability can lead to disrupted ECM structure or fibrosis. Fibrosis can be initiated in response to different injuries and stimuli, and can be reparative (beneficial) or reactive (damaging). FBs need to be activated to myofibroblasts (MyoFBs) which have augmented capacity in synthesizing ECM proteins, causing fibrosis. In addition to the resident FBs in the myocardium, a number of other cells (pericytes, fibrocytes, mesenchymal, and hematopoietic cells) can transform into MyoFBs, further driving the fibrotic response. Multiple molecules including hormones, cytokines, and growth factors stimulate this process leading to generation of activated MyoFBs. Contribution of different cell types to cFBs and MyoFBs can result in an exponential increase in the number of MyoFBs and an accelerated pro-fibrotic response. Given the diversity of the cell sources, and the array of interconnected signalling pathways that lead to formation of MyoFBs and subsequently fibrosis, identifying a single target to limit the fibrotic response in the myocardium has been challenging. This review article will delineate the importance and relevance of fibroblast heterogeneity in mediating fibrosis in different models of heart failure and will highlight important signalling pathways implicated in myofibroblast activation.
... MR signalling in cardiomyocytes has been shown to induce CTGF expression, 41 although the decisive role of CTGF in fibrosis formation is still subject of debate. 42 Furthermore, MR signalling plays a role in the type of cardiac fibrosis that is formed. Inhibition of MR signalling in mice (via cardiomyocyte-specific genetic deletion) inhibited reactive fibrosis and also reduced the expression of hypertrophy and fibrosis-associated genes such as β-myosin heavy chain, angiotensin-converting enzyme, CTGF and collagens. ...
In the diseased and remodelled heart, increased activity and expression of Ca2+/calmodulin‐dependent protein kinase II (CaMKII), an excess of fibrosis, and a decreased electrical coupling and cellular excitability leads to disturbed calcium homeostasis and tissue integrity. This subsequently leads to increased arrhythmia vulnerability and contractile dysfunction. Here, we investigated the combination of CaMKII inhibition (using genetically modified mice expressing the autocamtide‐3‐related‐peptide (AC3I)) together with eplerenone treatment (AC3I‐Epler) to prevent electrophysiological remodelling, fibrosis and subsequent functional deterioration in a mouse model of chronic pressure overload. We compared AC3I‐Epler mice with mice only subjected to mineralocorticoid receptor (MR) antagonism (WT‐Epler) and mice with only CaMKII inhibition (AC3I‐No). Our data show that a combined CaMKII inhibition together with MR antagonism mitigates contractile deterioration as was manifested by a preservation of ejection fraction, fractional shortening, global longitudinal strain, peak strain and contractile synchronicity. Furthermore, patchy fibrosis formation was reduced, potentially via inhibition of pro‐fibrotic TGF‐β/SMAD3 signalling, which related to a better global contractile performance and a slightly depressed incidence of arrhythmias. Furthermore, the level of patchy fibrosis appeared significantly correlated to eplerenone dose. The addition of eplerenone to CaMKII inhibition potentiates the effects of CaMKII inhibition on pro‐fibrotic pathways. As a result of the applied strategy, limiting patchy fibrosis adheres to a higher synchronicity of contraction and an overall better contractile performance which fits with a tempered arrhythmogenesis.
... Inhibition of CCN2 reversed the process of fibrosis and a humanized CCN2 monoclonal antibody (FG-3019) prevented carotid artery vascular stiffness in a diabetic rat model (Lipson et al. 2012). However, the profibrotic effects of CCN2 appeared to be organ-specific because overexpression of CCN2 in the mouse heart promoted age-dependent development of cardiac hypertrophy, but did not induce cardiac fibrosis (Fontes et al. 2015;Shimo et al. 1999;Szabo et al. 2014). The overexpression of CCN2 by itself is not sufficient to induce cardiac fibrosis and the functions of CCN2 in the heart may be different from the observed CCN2 functions in the skin, lung and other tissues (Ahmed et al. 2007;Hviid et al. 2012;Mukudai et al. 2003). ...
The extracellular matrix (ECM) is a deformable dynamic structure that dictates the behavior, function and integrity of blood vessels. The composition, density, chemistry and architecture of major globular and fibrillar proteins of the matrisome regulate the mechanical properties of the vasculature (i.e., stiffness/compliance). ECM proteins are linked via integrins to a protein adhesome directly connected to the actin cytoskeleton and various downstream signaling pathways that enable the cells to respond to external stimuli in a coordinated manner and maintain optimal tissue stiffness. However, cardiovascular risk factors such as diabetes, dyslipidemia, hypertension, ischemia and aging compromise the mechanical balance of the vascular wall. Stiffening of large blood vessels is associated with well-known qualitative and quantitative changes of fibrillar and fibrous macromolecules of the vascular matrisome. However, the mechanical properties of the thin-walled microvasculature are essentially defined by components of the subendothelial matrix. Cellular communication network (CCN) 1 and 2 proteins (aka Cyr61 and CTGF, respectively) of the CCN protein family localize in and act on the pericellular matrix of microvessels and constitute primary candidate markers and regulators of microvascular compliance. CCN1 and CCN2 bind various integrin and non-integrin receptors and initiate signaling pathways that regulate connective tissue remodeling and response to injury, the associated mechanoresponse of vascular cells, and the subsequent inflammatory response. The CCN1 and CCN2 genes are themselves responsive to mechanical stimuli in vascular cells, wherein mechanotransduction signaling converges into the common Rho GTPase pathway, which promotes actomyosin-based contractility and cellular stiffening. However, CCN1 and CCN2 each exhibit unique functional attributes in these processes. A better understanding of their synergistic or antagonistic effects on the maintenance (or loss) of microvascular compliance in physiological and pathological situations will assist more broadly based studies of their functional properties and translational value.
... TGF-b1, MCP-1, and CTGF are important profibrotic cytokines that increase collagen deposition and decrease protease expression to promote cardiac fibrosis (13)(14)(15). In this study, TGF-b1, MCP-1, and CTGF levels were evaluated by immunohistochemistry. ...
Cardiac aging is characterized by myocardial hypertrophy, fibrosis, and diastolic dysfunction. Human kallikrein (hKLK1) protects against fibrosis in various pathogenic states. However, the effects of hKLK1 overexpression on cardiac aging‐related fibrosis and the underlying mechanisms remain unknown. Moreover, the role of hKLK1 in regulating macrophage function leading to cardiac fibrosis has not been investigated. Thus, in this study, we determined the effects of hKLK1 on cardiac aging and explored the mechanisms through which hKLK1 regulated aging‐related fibrosis. Echocardiographic measurements showed that aging caused significant alternations in cardiac morphology, hypertrophy, and fibrosis in rats, and hKLK1 overexpression protected against aging‐induced cardiac dysfunction. Compared with wild‐type hearts, the hKLK1 transgene decreased the expression of monocyte chemoattractant protein 1 and suppressed mitochondrial dysfunction and excess oxidative stress, leading to decreased recruitment and retention of alternatively activated (M2) macrophages and reduced secretion of profibrotic cytokines mediated by the TGF‐β1–Smad3 signaling pathway in hearts of aging rats. Furthermore, these cardioprotective effects of hKLK1 overexpression were associated with the Janus kinase–signal transducer and activator of transcription 3 signaling pathway. H2O2‐induced senescence promoted the differentiation of RAW264.7 cells into M2‐type cells induced by IL‐4 treatment. Bradykinin treatment relieved the migratory capacity of macrophages induced by H2O2. Thus, hKLK1 overexpression reduced cardiac fibrosis and improved aging‐related cardiac dysfunction through reduced shift of macrophages to M2 macrophages, indicating that hKLK1 may alleviate aging‐related cardiac dysfunction.—Hu, D., Dong, R., Yang, Y., Chen, Z., Tang, Y., Fu, M., Wang, D. W., Xu, X., Tu, L. Human kallikrein overexpression alleviates cardiac aging by alternatively regulating macrophage polarization in aged rats. FASEB J. 33, 8436–8452 (2019). www.fasebj.org
... To visualize cardiac fibrosis in murine hearts, 10 μm thick fourchamber-view cryo sections that were fixated in paraformaldehyde were stained with 0.1% Picrosirius Red solution, as described previously [39]. After staining, sections were digitally scanned using Aperio ScanScope XT (Leica Microsystems BV, Son, The Netherlands) and pictures were taken using NDPview2 (Hamamatsu Photonics KK, Shizuoka, Japan). ...
Background:
The intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID.
Methods:
Location of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2.
Results:
First, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures.
Conclusions:
In this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.
... However, our previous work on cardiac fibrosis led us to find that this protein is highly expressed by cardiomyocytes after injury, but that its expression in these cells is unimportant for overall collagen deposition (36). These results have been confirmed by several independent groups and raised the intriguing possibility that CTGF is involved in different functions based on its source (37,38). In skeletal muscle, the injury-repair-regeneration cascade acutely promotes transient collagen deposition (39)(40)(41)(42). ...
In skeletal muscle, extracellular matrix (ECM) remodeling can either support the complete regeneration of injured muscle or facilitate pathologic fibrosis and muscle degeneration. Muscular dystrophy (MD) is a group of genetic disorders that results in a progressive decline in muscle function and is characterized by the abundant deposition of fibrotic tissue. Unlike acute injury, where ECM remodeling is acute and transient, in MD, remodeling persists until fibrosis obstructs the regenerative efforts of diseased muscles. Thus, understanding how ECM is deposited and organized is critical in the context of muscle repair. Connective tissue growth factor (CTGF or CCN2) is a matricellular protein expressed by multiple cell types in response to tissue injury. Although used as a general marker of fibrosis, the cell type–dependent role of CTGF in dystrophic muscle has not been elucidated. To address this question, a conditional Ctgf myofiber and fibroblast‐knockout mouse lines were generated and crossed to a dystrophic background. Only myofiber‐selective inhibition of CTGF protected δ‐sarcoglycan–null (Sgcd−/−) mice from the dystrophic phenotype, and it did so by affecting collagen organization in a way that allowed for improvements in dystrophic muscle regeneration and function. To confirm that muscle‐specific CTGF functions to mediate collagen organization, we generated mice with transgenic muscle‐specific overexpression of CTGF. Again, genetic modulation of CTGF in muscle was not sufficient to drive fibrosis, but altered collagen content and organization after injury. Our results show that the myofibers are critical mediators of the deleterious effects associated with CTGF in MD and acutely injured skeletal muscle.—Petrosino, J. M., Leask, A., Accornero, F. Genetic manipulation of CCN2/CTGF unveils cell‐specific ECM‐remodeling effects in injured skeletal muscle. FASEB J. 33, 2047–2057 (2019). www.fasebj.org
... Moreover, transgenic mice overexpressing CTGF had similar amount of fibrosis after cardiac injury compared to control mice [82][83][84]. Consistent with these findings, Fontes et al. found that CTGF global knockout does not affect cardiac hypertrophy and fibrosis induced by chronic pressure overload [85]. However, there sounds quite a different voice that CTGF cardiac transgenic mice showed significantly increased fibrosis in response to pressure overload [86]. ...
Cardiac fibrosis is defined as the imbalance of extracellular matrix (ECM) production and degradation, thus contributing to cardiac dysfunction in many cardiac pathophysiologic conditions. This review discusses specific markers and origin of cardiac fibroblasts (CFs), and the underlying mechanism involved in the development of cardiac fibrosis. Currently, there are no CFs-specific molecular markers. Most studies use co-labelling with panels of antibodies that can recognize CFs. Origin of fibroblasts is heterogeneous. After fibrotic stimuli, the levels of myocardial pro-fibrotic growth factors and cytokines are increased. These pro-fibrotic growth factors and cytokines bind to its receptors and then trigger the activation of signaling pathway and transcriptional factors via Smad-dependent or Smad independent-manners. These fibrosis-related transcriptional factors regulate gene expression that are involved in the fibrosis to amplify the fibrotic response. Understanding the mechanisms responsible for initiation, progression, and amplification of cardiac fibrosis are of great clinical significance to find drugs that can prevent the progression of cardiac fibrosis.
... Although CTGF expression is strongly induced during cardiac remodeling, its role in cardiac remodeling remains controversial. Recent studies using transgenic mice suggest that CTGF is not involved in cardiac remodeling, hypertrophy, or fibrosis at baseline, nor with aging, after pressure overload, or with neuroendocrine agonist stimulation (Accornero et al., 2015;Fontes M. S. et al., 2015). However, in vitro data indicate that CTGF induces hypertrophy in cardiomyocytes (Hayata et al., 2008). ...
The heart is a highly structured organ consisting of different cell types, including myocytes, endothelial cells, fibroblasts, stem cells, and inflammatory cells. This pluricellularity provides the opportunity of intercellular communication within the organ, with subsequent optimization of its function. Intercellular cross-talk is indispensable during cardiac development, but also plays a substantial modulatory role in the normal and failing heart of adults. More specifically, factors secreted by cardiac microvascular endothelial cells modulate cardiac performance and either positively or negatively affect cardiac remodeling. The role of endothelium-derived small molecules and peptides—for instance NO or endothelin-1—has been extensively studied and is relatively well defined. However, endothelial cells also secrete numerous larger proteins. Information on the role of these proteins in the heart is scattered throughout the literature. In this review, we will link specific proteins that modulate cardiac contractility or cardiac remodeling to their expression by cardiac microvascular endothelial cells. The following proteins will be discussed: IL-6, periostin, tenascin-C, thrombospondin, follistatin-like 1, frizzled-related protein 3, IGF-1, CTGF, dickkopf-3, BMP-2 and−4, apelin, IL-1β, placental growth factor, LIF, WISP-1, midkine, and adrenomedullin. In the future, it is likely that some of these proteins can serve as markers of cardiac remodeling and that the concept of endothelial function and dysfunction might have to be redefined as we learn more about other factors secreted by ECs besides NO.
... Similar to our study, emerging evidence has demonstrated the negligible role of CTGF in the progression of myocardial fibrosis through the use of transgenic mice with myocardial-specific gain or loss of CTGF. 36,37 In the present manuscript, we did not assess other roles of CTGF that have been previously described, such as its role in cardiomyocyte hypertrophy. 38 Thus, future studies regarding the exact role and importance of CTGF in fibrosis progression is warranted. ...
Introduction:
Transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) are often described as the initial pro-fibrotic mediators upregulated early in fibrosis models dependent on angiotensin II (Ang-II). In the present study, we explore the mechanistic link between TGF-β and CTGF expression by using a novel TGF-β trap.
Materials and methods:
NIH/3T3 fibroblasts were subjected to TGF-β with or without TGF-β trap or 1D11 antibody, CTGF or CTGF plus TGF-β for six or 24 hours, and then used for quantitative real-time polymerase chain reaction (qRT-PCR) or immunocytochemistry. Male C57BL/6 mice were infused with Ang-II and randomly assigned TGF-β trap for six or 24 hours. Hearts were harvested for histological analyses, qRT-PCR and western blotting.
Results:
Exogenous TGF-β-induced fibroblasts resulted in significant upregulation of CTGF, TGF-β and type I collagen transcript levels in vitro. Additionally, TGF-β promoted the differentiation of fibroblasts into α-SMA+myofibroblasts. CTGF expression was reduced by the addition of TGF-β trap or neutralizing antibody, confirming that its expression is dependent on TGF-β signaling. In contrast, exogenous CTGF did not appear to have an effect on fibroblast production of pro-fibrotic transcripts or fibroblast differentiation. Ang-II infusion in vivo led to a significant increase in TGF-β and CTGF mRNA expression at six and 24 hours with corresponding changes in Smad2 phosphorylation (pSmad2), indicative of increased TGF-β signaling. Ang-II animals that received the TGF-β trap demonstrated reduced CTGF mRNA levels and pSmad2 at six hours, suggesting that early CTGF expression is dependent on TGF-β signaling.
Conclusions:
We demonstrated that CTGF expression is dependent on TGF-β signaling both in vitro and in vivo in a model of myocardial fibrosis. This also suggests that early myocardial CTGF mRNA expression (six hours) after Ang-II exposure is likely dependent on latent TGF-β activation via the canonical Smad-dependent pathway in resident cardiac cells.
... Blocking CTGF by RNA interference in primary mouse cardiomyocytes causes attenuation of the expression of several genes such as stretch-induced chemokine, matrix metalloproteases, extracellular and cell-adhesion proteins, suggesting the involvement of CTGF in various pathways [139]. Ubiquitous or heart-specific CTGF knockdown or overexpression in mouse models of cardiac fibrosis did not significantly impact the progression of fibrosis, suggesting other possible drivers of cardiac fibrosis [140,141]. Conversely, CTGF overexpression in the myocardium of mice has also been observed to improve the left ventricular function after myocardial infarction through inhibition of inflammatory responses and apoptosis. Consistently, improved heart function in patients with elevated serum CTGF levels suggested that CTGF might indeed have some protective role in left ventricular remodeling after myocardial infarction [142]. ...
Connective tissue growth factor, also known as CCN2, is a cysteine-rich matricellular protein involved in the control of biological processes, such as cell proliferation, differentiation, adhesion and angiogenesis, as well as multiple pathologies, such as tumor development and tissue fibrosis. Here, we describe the molecular and biological characteristics of CTGF, its regulation and various functions in the spectrum of development and regeneration to fibrosis. We further outline the preclinical and clinical studies concerning compounds targeting CTGF in various pathologies with the focus on heart, lung, liver, kidney and solid organ transplantation. Finally, we address the advances and pitfalls of translational fibrosis research and provide suggestions to move towards a better management of fibrosis.
... Genotype of the animals was confirmed before the experimental procedure (see supplementary materials). Surgeries were performed in the morning and afternoon in a dedicated mouse operation room by an experienced surgeon 21 . Anaesthesia was induced by intraperitoneal (i.p.) injection of medetomidinehydrochloride (1.0 g/kg body weight), midazolam (Dormicum®, Roche, 10.0 mg/kg) and fentanyl (Janssen-Cilag, 0.1 mg/kg). ...
Hypertension is one of the most common risk factors for the development heart failure in the general population. Inflammation plays a central role in this adverse remodeling and eventually to the development of heart failure. Circulating levels of Complement factor 5a (C5a) are increased in hypertensive patients and the C5a receptor is associated with the presence of cardiac fibrosis and inflammation in an experimental hypertension model. To test if C5aR is involved in adverse cardiac remodeling following pressure-overload, we induced transverse aortic constriction (TAC) in wildtype and C5a receptor deficient mice (C5aR−/−). Six weeks after TAC, C5aR-/- animals showed a similar degree of cardiac hypertrophy and decrease in cardiac function as wild type mice (End Systolic Volume; 50.30±5.32 µl vs. 55.81±8.16 µl). In addition, other features of adverse cardiac remodeling like cardiomyocyte cell size (WGA staining), fibrosis (picrosirius red staining) or collagen degradation (matrix metalloproteinase activity assay) did not differ either. In conclusion, full body C5aR deficiency does not affect adverse cardiac remodeling after pressure-overload. However, our finding are in contrast with C5a inhibition studies. Our observations do present the role of C5a-C5aR in adverse cardiac remodeling and heart failure as controversial at the least.
... 1,3 Moreover, in a rabbit model of eccentric hypertrophy, where strains and workload are expected to be high throughout the LV wall, CTGF was also found to be overexpressed. 3 Of note is that recent studies question the importance of CTGF in cardiac structural remodelling, indicating that while it is an important biomarker of myocardial remodelling, it most likely does not play a decisive role in this process 24,25 Cardiac collagen content During the 4-month experimental period, LBBB did not lead to significant ECM remodelling and fibrosis, as collagen content remained similar and collagen gene expression showed a decrease. Possibly, the slightly lower collagen content in the LVfw of LBBB hearts may be explained by 'dilution' of collagen by the increased cardiomyocyte mass, as also shown in previous studies with chronic LV pacing. ...
Aims:
Left bundle branch block (LBBB) creates considerable regional differences in mechanical load within the left ventricle (LV). We investigated expression of selected microRNAs (miRs) in relation to regional hypertrophy and fibrosis in LBBB hearts and their reversibility upon cardiac resynchronization therapy (CRT).
Methods and results:
Eighteen dogs were followed for 4 months after induction of LBBB, 10 of which received CRT after 2 months. Five additional dogs served as control. LV geometric changes were determined by echocardiography and myocardial strain by magnetic resonance imaging tagging. Expression levels of miRs, their target genes: connective tissue growth factor (CTGF), serum response factor (SRF), nuclear factor of activated T cells (NFATc4), and cardiomyocyte diameter and collagen deposition were measured in the septum and LV free wall (LVfw). In LBBB hearts, LVfw and septal systolic circumferential strain were 200% and 50% of control, respectively. This coincided with local hypertrophy in the LVfw. MiR-133a expression was reduced by 33% in the LVfw, which corresponded with a selective increase of CTGF expression in the LVfw (279% of control). By contrast, no change was observed in SRF and NFATc4 expression was decreased in LBBB hearts. CRT normalized strain patterns and reversed miR-133a and CTGF expression towards normal, expression of other miRs, related to remodelling, such as miR-199b and miR-155f, were not affected.
Conclusions:
In the clinically relevant large animal model of LBBB, a close inverse relation exists between local hypertrophy and miR-133a. Reduced miR-133a correlated with increased CTGF levels but not with SRF and NFATc4.
... In the present study, we investigated a role of CTGF in a mouse model of anti-GBM nephritis, using three types of conditional CTGF KO mice. Although a previous study showed that inducible systemic CTGF KO mice lacking exon 4 grow normally 23 , the present study is the first to describe a role of CTGF in kidney diseases using systemic conditional KO mice. Our work revealed that renal injury induced by anti-GBM nephritis is markedly inhibited in inducible systemic CTGF KO mice lacking the whole CTGF coding region. ...
Connective tissue growth factor (CTGF) coordinates the signaling of growth factors and promotes fibrosis. Neonatal death of systemic CTGF knockout (KO) mice has hampered analysis of CTGF in adult renal diseases. We established 3 types of CTGF conditional KO (cKO) mice to investigate a role and source of CTGF in anti-glomerular basement membrane (GBM) glomerulonephritis. Tamoxifen-inducible systemic CTGF (Rosa-CTGF) cKO mice exhibited reduced proteinuria with ameliorated crescent formation and mesangial expansion in anti-GBM nephritis after induction. Although CTGF is expressed by podocytes at basal levels, podocyte-specific CTGF (pod-CTGF) cKO mice showed no improvement in renal injury. In contrast, PDGFRα promoter-driven CTGF (Pdgfra-CTGF) cKO mice, which predominantly lack CTGF expression by mesangial cells, exhibited reduced proteinuria with ameliorated histological changes. Glomerular macrophage accumulation, expression of Adgre1 and Ccl2, and ratio of M1/M2 macrophages were all reduced both in Rosa-CTGF cKO and Pdgfra-CTGF cKO mice, but not in pod-CTGF cKO mice. TGF-β1-stimulated Ccl2 upregulation in mesangial cells and macrophage adhesion to activated mesangial cells were decreased by reduction of CTGF. These results reveal a novel mechanism of macrophage migration into glomeruli with nephritis mediated by CTGF derived from mesangial cells, implicating the therapeutic potential of CTGF inhibition in glomerulonephritis.
... In line with this, dilating cardiomyopathy appears to be CTGF regulated (Koshman et al., 2015), and CTGF reduction attenuates left ventricular remodeling and dysfunction in models of pressure overload (Szabo et al., 2014). However, in other animal models of chronic pressure overload it has been reported that alteration of CTGF levels are of little consequence to the phenotype (Accornero et al., 2015;Fontes et al., 2015). In stark contrast, it has also been reported that CTGF exerts protective effects during experimental cardiac pressure overload (Gravning et al., 2013). ...
Aims:
Connective tissue growth factor (CTGF) plays a key role in tissue fibrogenesis and growing evidence indicates a pathogenic role in cardiovascular disease. Aim of this study is to investigate the association of connective tissue growth factor (CTGF/CCN2) with cardiovascular risk and mortality in patients with manifest vascular disease.
Methods and results:
Plasma CTGF was measured by ELISA in a prospective cohort study of 1227 patients with manifest vascular disease (mean age 59.0 ± 9.9 years). Linear regression analysis was performed to quantify the association between CTGF and cardiovascular risk factors. Results are expressed as beta (β) regression coefficients with 95% confidence intervals (CI). The relation between CTGF and the occurrence of new cardiovascular events and mortality was assessed with Cox proportional hazard analysis. Adjustments were made for potential confounding factors. Plasma CTGF was positively related to total cholesterol (β 0.040;95%CI 0.013-0.067) and LDL cholesterol (β 0.031;95%CI 0.000-0.062) and inversely to glomerular filtration rate (β -0.004;95%CI -0.005 to -0.002). CTGF was significantly lower in patients with cerebrovascular disease. During a median follow-up of 6.5 years (IQR 5.3-7.4) 131 subjects died, 92 experienced an ischemic cardiac complication and 45 an ischemic stroke. CTGF was associated with an increased risk of new vascular events (HR 1.21;95%CI 1.04-1.42), ischemic cardiac events (HR 1.41;95%CI 1.18-1.67) and all-cause mortality (HR 1.18;95%CI 1.00-1.38) for every 1 nmol/L increase in CTGF. No relation was observed between CTGF and the occurrence of ischemic stroke.
Conclusions:
In patients with manifest vascular disease, elevated plasma CTGF confers an increased risk of new cardiovascular events and all-cause mortality.
Hypertrophic cardiomyopathy (HCM) is a common inherited cardiovascular disease, which can cause heart failure and lead to death. In this study, we performed high‐resolution single‐cell RNA‐sequencing of 2115 individual cardiomyocytes obtained from HCM patients and normal controls. Signature up‐ and down‐regulated genes in HCM were identified by integrative analysis across 37 patients and 41 controls from our data and published human single‐cell and single‐nucleus RNA‐seq datasets, which were further classified into gene modules by single‐cell co‐expression analysis. Using our high‐resolution dataset, we also investigated the heterogeneity among individual cardiomyocytes and revealed five distinct clusters within HCM cardiomyocytes. Interestingly, we showed that some extracellular matrix (ECM) genes were up‐regulated in the HCM cardiomyocytes, suggesting that they play a role in cardiac remodelling. Taken together, our study comprehensively profiled the transcriptomic programs of HCM cardiomyocytes and provided insights into molecular mechanisms underlying the pathogenesis of HCM.
Objective:
To investigate the changes in serum levels of endothelin-1 (ET-1) and connective tissue growth factor (CTGF) in patients with atrial fibrillation (AF) and their value for predicting recurrence of AF after radiofrequency ablation (RFCA).
Methods:
Sixty-six patients with paroxysmal AF (PaAF) and 72 with persistent AF (PaAF) admitted in our hospital were recruited as AF group and 80 patients with sinus rhythm as the control group, and in all the participants, serum levels of ET-1 and CTGF were measured using ELISA and Western blotting. From 6 patients with AF and 6 with sinus rhythm undergoing cardiac surgery in our hospital, tissue samples of the right atrial appendage were taken intraoperatively for observation of structural changes of the cardiomyocytes, myocardial fibrosis and expression of ET-1 and CTGF protein. In AF group, the patients receiving RFCA were followed up for 6 months following the procedure for assessment of the outcomes.
Results:
Compared with the control patients, the patients with AF showed obvious damages of the cardiomyocyte structure and myocardial fibrosis. Serum levels of ET-1 and CTGF levels were significantly higher in PaAF and PeAF groups than in the control group, and were higher in PeAF group than in PaAF group. In the patients with AF, serum ET-1 and CTGF levels were positively correlated with left atrial diameter (LAD) (P < 0.05), and ET-1 was positively correlated with CTGF levels (P < 0.05). In patients with postoperative AF recurrence, the serum levels of ET-1 and CTGF were significantly higher than those in patients without recurrence; serum ET-1 and CTGF levels before and after the operation were positively correlated with the recurrence of PeAF, and elevated serum levels of ET- 1 and CTGF were identified by logistic regression analysis as independent risk factors for postoperative recurrence of PeAF.
Conclusion:
Serum levels of ET-1 and CTGF are significantly elevated in AF patients in positive correlation with AF duration. ET-1 and CTGF levels are higher in AF patients with postoperative recurrence, and they both have predictive value for recurrence of PeAF following RFCA.
CCN2 has been shown to be closely involved in the progression of renal fibrosis, indicating the potential of CCN2 inhibition as a therapeutic target. Although the examination of the renal disease phenotypes of adult CCN2 knockout mice has yielded valuable scientific insights, perinatal death has limited studies of CCN2 in vivo. Conditional knockout technology has become widely used to delete genes in the target cell populations or time points using cell-specific Cre recombinase-expressing mice. Therefore, several lines of CCN2-floxed mice have been developed to assess the functional role of CCN2 in adult mice.CCN2 levels are elevated in renal fibrosis and proliferative glomerulonephritis, making them suitable disease models for assessing the effects of CCN2 deletion on the kidney. Renal fibrosis is characterized by glomerulosclerosis and tubulointerstitial fibrosis and transforming growth factor-β. CCN2 is increased in fibrosis and modulates a number of downstream signaling pathways involved in the fibrogenic properties of TGF-β. Unilateral ureteral obstruction is one of the most widely used models of renal tubulointerstitial fibrosis. In addition, anti-glomerular basement membrane antibody glomerulonephritis has become the most widely used model for evaluating the effect of increased renal CCN2 expression. Herein, we describe the construction of CCN2-floxed mice and inducible systemic CCN2 conditional knockout mice and methods for the operation of unilateral ureteral obstruction and the induction of anti-glomerular basement membrane antibody glomerulonephritis.Key wordsCre-loxP systemAnti-glomerular basement membrane (GBM) nephritisPlatelet-derived growth factor receptor (PDGFR)-αRenal fibrosisTransgenic miceDiabetic nephropathyPodocytes
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.
The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix–cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvβ1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin β1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.
Heart failure is the leading cause of morbidity and mortality worldwide. Several lines of evidence suggest that physical activity and exercise can pre-condition the heart to improve the response to acute cardiac injury such as myocardial infarction or ischemia/reperfusion injury, preventing the progression to heart failure. It is becoming more apparent that cardioprotection is a concerted effort between multiple cell types and converging signaling pathways. However, the molecular mechanisms of cardioprotection are not completely understood. What is clear is that the mechanisms underlying this protection involve acute activation of transcriptional activators and their corresponding gene expression programs. Here, we review the known stress-dependent transcriptional programs that are activated in cardiomyocytes and cardiac fibroblasts to preserve function in the adult heart after injury. Focus is given to prominent transcriptional pathways such as mechanical stress or reactive oxygen species (ROS)-dependent activation of myocardin-related transcription factors (MRTFs)and transforming growth factor beta (TGFβ), and gene expression that positively regulates protective PI3K/Akt signaling. Together, these pathways modulate both beneficial and pathological responses to cardiac injury in a cell-specific manner.
Dilated cardiomyopathy (DCM) is characterized by left ventricular dilation associated with systolic dysfunction. The purpose of the current study is to clarify the effect of connective tissue growth factor (CTGF/CCN2) on myocardial fibrosis and left ventricular hypertrophy (LVH) of rats with DCM through the mitogen-activated protein kinase (MAPK) signaling pathway. First, DCM rat models were established and sh-CTGF/CCN2 lentiviral expression vectors were constructed. Then, by observing the pathological changes and myocardial ultrastructure as well as detecting cardiac functions, myocardial fibrosis, and LVH of rats, the effect of CTGF/CCN2 gene silencing on rats with DCM was investigated. To further explore how CTGF/CCN2 gene silencing affects rats with DCM, the expression of CTGF/CCN2 and the related genes of the MAPK signaling pathway was detected. Sh-CTGF/CCN2-2 and sh-CTGF/CCN2-3 with lower CTGF/CCN2 expression were selected for further experimentation. CTGF/CCN2 gene silencing improved cardiac function and alleviated myocardial fibrosis and LVH of rats with DCM. It was also verified that CTGF/CCN2 gene silencing could relieve the pathology of rats with DCM by inactivation of the MAPK signaling pathway. We conclude that CTGF/CCN2 gene silencing inhibits the activation of the MAPK signaling pathway, thus decreases myocardial fibrosis and LVH, and then improves the pathological symptoms of DCM in rats.
Cardiac fibrosis is a common pathologic consequence of stress insult to the heart and is characterized by abnormal deposition of fibrotic extracellular matrix that compromises cardiac function. Cardiac fibroblasts are key mediators of fibrotic remodeling and are regulated by secreted stress-response proteins. The matricellular protein connective tissue growth factor (CTGF), or CCN2, is strongly produced by injured cardiomyocytes and although it is considered a pro-fibrotic factor in many organ systems, its role in cardiac fibrosis is controversial.
Here we adopted a cell-specific genetic approach to conditionally delete CCN2 in either cardiomyocytes or activated fibroblasts. Fibrosis was induced by angiotensin II-based neurohumoral stimulation, an insult that strongly induces CCN2 expression from cardiomyocytes and to a lesser extent in fibroblasts. Remarkably, only CCN2 deletion from activated fibroblasts inhibited the fibrotic remodeling while deletion from cardiomyocytes (the main source of CCN2 in the heart) had no effects. In vitro experiments revealed that although efficiently secreted by both fibroblasts and cardiomyocytes, only fibroblast-derived CCN2 is proficient in its ability to fully activate fibroblasts. These results overall indicate that although secreted into the extracellular matrix, CCN2 acts in an autocrine fashion. Secretion of CCN2 by cardiomyocytes is not pro-fibrotic, while fibroblast-derived CCN2 can modulate fibrosis in the heart.
In conclusion we found that cardiomyocyte-derived CCN2 is dispensable for cardiac fibrosis, while inhibiting CCN2 induction in activated fibroblasts is sufficient to abrogate the cardiac fibrotic response to angiotensin II. Hence, CCN2 is an autocrine factor in the heart.
The renin-angiotensin-aldosterone system plays crucial roles in cardiovascular physiology and pathophysiology. However, many of the signaling mechanisms have been unclear. The angiotensin II (ANG II) type 1 receptor (AT1R) is believed to mediate most functions of ANG II in the system. AT1R utilizes various signal transduction cascades causing hypertension, cardiovascular remodeling, and end organ damage. Moreover, functional cross-talk between AT1R signaling pathways and other signaling pathways have been recognized. Accumulating evidence reveals the complexity of ANG II signal transduction in pathophysiology of the vasculature, heart, kidney, and brain, as well as several pathophysiological features, including inflammation, metabolic dysfunction, and aging. In this review, we provide a comprehensive update of the ANG II receptor signaling events and their functional significances for potential translation into therapeutic strategies. AT1R remains central to the system in mediating physiological and pathophysiological functions of ANG II, and participation of specific signaling pathways becomes much clearer. There are still certain limitations and many controversies, and several noteworthy new concepts require further support. However, it is expected that rigorous translational research of the ANG II signaling pathways including those in large animals and humans will contribute to establishing effective new therapies against various diseases.
Dilated cardiomyopathy (DCM) leads to disturbed contraction and force transduction, and is associated with substantial mortality in all age groups. Involvement of a disrupted composition of the intercalated disc (ID) has been reported. However, in children, little is established about such subcellular changes during disease, because of the pathological mix-up with the ongoing cardiac maturation. This leaves maladaptive remodeling often undetected. We aimed at illustrating subcellular alterations in children diagnosed with DCM compared to age-matched controls, focusing on ID proteins known to be crucially stable under healthy conditions and destabilized during cardiac injury in adults. Left Ventricular or septal pediatric specimens were collected from 7 individuals diagnosed with DCM (age: 23 weeks in utero - 8 weeks postnatal) and age-matched controls that died of non-cardiovascular cause. We determined the amount of fibrosis and localization of ID proteins by immunohistochemistry. In pediatric DCM, most ID proteins follow similar spatio-temporal changes in localization as in controls. However, although no mutations were found, the signal of the desmosomal protein Desmoglein-2 was reduced in all pediatric DCM specimens, but not in controls or adult DCM patients. Endocardial and transmural fibrosis was increased in all pediatric DCM patients compared to age-matched controls. Composition of the ID in pediatric DCM patients is similar to controls, except for the localization of Desmoglein-2 and presence of severe fibrosis. This suggests that the architecture of desmosomes is already disturbed in the early stages of DCM. These findings contribute to the understanding of pediatric DCM.
Aims:
Atrial fibrillation (AF) is associated with fibrosis that slows electrical conduction and causes perpetuation of the arrhythmia. The molecular characterization of AF pathophysiology may provide novel therapeutic options. This study was designed to elucidate profibrotic signaling and myofibroblast activation in a porcine model of atrial tachypacing-induced AF and reduced left ventricular function.
Materials and methods:
Ten domestic pigs were randomized to sinus rhythm (SR) or AF groups. Prior to AF induction and on day 14 the animals underwent echocardiographic examinations. Profibrotic pathways were analyzed in right atrial tissue obtained from AF animals compared to SR controls using histology, immunofluorescence microscopy, Western blot, and real-time PCR.
Key findings:
AF was associated with atrial dilation, increased atrial fibrosis, and enhanced expression of collagens I and V in right atrial tissue after 14days follow-up. The fraction of α-smooth muscle actin (SMA)-producing activated myofibroblasts was elevated in AF, whereas the abundance of vimentin-expressing inactive fibroblasts was not affected. Profibrotic signaling involved upregulation of TGF-β1, Smad2/3, and CTGF.
Significance:
The transformation of atrial fibroblasts into myofibroblasts through activation of TGF-β1 and CTGF emerged as potential cellular trigger of fibrogenesis. Prevention of fibroblast-to-myofibroblast switching may serve as target for remodeling-based antiarrhythmic AF therapy.
CCN2 has been shown to be closely involved in the progression of renal fibrosis, indicating the potential of CCN2 inhibition as a therapeutic target. Although the examination of the phenotypes of adult CCN2 knockout mice with renal diseases has yielded valuable scientific insights, perinatal death has limited studies of CCN2 in vivo. Conditional knockout technology has become widely used for the deletion of genes in the desired cell populations and time points through the use of cell-specific Cre recombinase-expressing mice. Accordingly, several lines of CCN2 floxed mice have been developed for the assessment of the functional role of CCN2 in adult mice.
CCN2 levels are increased in renal fibrosis and proliferative glomerulonephritis, which represent good disease models for evaluating the effects of CCN2 deletion on the kidney. Of these, anti-glomerular basement membrane antibody glomerulonephritis has become the most widely used model for evaluating the effect of increased renal CCN2 expression. Herein, we describe the construction of CCN2 floxed mice and inducible systemic CCN2 conditional knockout mice and methods for the induction of anti-glomerular basement membrane antibody glomerulonephritis.
The retina is a complex neurovascular structure that conveys light/visual image through the optic nerve to the visual cortex of the brain. Neuronal and vascular activities in the retina are physically and functionally intertwined, and vascular alterations are consequential to the proper function of the entire visual system. In particular, alteration of the structure and barrier function of the retinal vasculature is commonly associated with the development of vasoproliferative ischemic retinopathy, a set of clinically well-defined chronic ocular microvascular complications causing blindness in all age groups. Experimentally, the retinal tissue provides researchers with a convenient, easily accessible, and directly observable model suitable to investigate whether and how newly identified genes regulate vascular development and regeneration. The six mammalian CCN gene-encoded proteins are part of an extracellular network of bioactive molecules that regulate various aspects of organ system development and diseases. Whether and how these molecules regulate the fundamental aspects of blood vessel development and pathology and subsequently the neurovascular link in the retina are open-ended questions. Sophisticated methods have been developed to gain insight into the pathogenesis of retinal vasculopathy. This chapter describes several useful methodologies and animal models to investigate the regulation and potential relevance of the CCN proteins in vasoproliferative diseases of the retina.
The matricellular secreted protein, connective tissue growth factor (CTGF), is upregulated in response to cardiac injury or with transforming growth factor β (TGFβ) stimulation, where it has been suggested to function as a fibrotic effector. Here we generated transgenic mice with inducible cardiac-specific CTGF overexpression, mice with cardiac-specific expression of an activated TGFβ mutant protein, mice with cardiac-specific deletion of Ctgf, and mice in which Ctgf was also deleted from fibroblasts in the heart. Remarkably, neither gain nor loss of CTGF in the heart affected cardiac pathology and propensity toward early lethality due to TGFβ over activation in the heart. Also, neither heart-specific Ctgf deletion nor CTGF overexpression altered cardiac remodeling and function with aging or after multiple acute stress stimuli. Cardiac fibrosis was also unchanged by modulation of CTGF levels in the heart with aging, pressure overload, agonist infusion or TGFβ overexpression. However, CTGF did mildly alter the overall cardiac response to TGFβ when pressure overload stimulation was applied. CTGF has been proposed to function as a critical TGFβ effector in underlying tissue remodeling and fibrosis throughout the body, although our results suggest that CTGF is of minimal importance and is an unlikely therapeutic vantage point for the heart.
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In mice, the calcium-dependent phosphatase calcineurin A (CnA) induces a transcriptional pathway leading to pathological cardiac hypertrophy. Interestingly, induction of CnA has been frequently noticed in human hypertrophic and failing hearts. Independently, the arrhythmia vulnerability of such hearts has been regularly associated with remodeling of parameters determining electrical conduction (expression level of connexin43 (Cx43) and NaV1.5, connective tissue architecture), for which the precise molecular basis and sequence of events is still unknown. Recently, we observed reduced Cx43 and NaV1.5 expression in 4-week old mouse hearts, overexpressing a constitutively active form of CnA (MHC-CnA model), but the order of events is still unknown. Therefore, three key parameters of conduction (Cx43, NaV1.5 and connective tissue expression) were characterized in MHC-CnA ventricles versus wild-type (WT) during postnatal development on a weekly basis. At postnatal week 1, CnA overexpression induced cardiac hypertrophy in MHC-CnA. Moreover, protein and RNA levels of both Cx43 and NaV1.5 were reduced by at least 50% as compared to WT. Cx43 immunoreactive signal was reduced at week 2 in MHC-CnA. At postnatal week 3, Cx43 was less phosphorylated and RNA level of Cx43 normalized to WT values, although the protein level was still reduced. Additionally, MHC-CnA hearts displayed substantial fibrosis relative to WT, which was accompanied by increased RNA levels for genes previously associated with fibrosis such as Col1a1, Col1a2, Col3a1, Tgfb1, Ctgf, Timp1 and microRNA miR-21. In MHC-CnA, reduction in Cx43 and NaV1.5 expression thus coincided with overexpression of CnA and hypertrophy development and preceded significant presence of fibrosis. At postnatal week 4 the alterations in conductional parameters observed in the MHC-CnA model lead to abnormal conduction and arrhythmias, similar to those observed in cardiac remodeling in heart failure patients. The MHC-CnA model, therefore, provides for a unique model to resolve the molecular origin of conductional remodeling in detail.
Myocardial CCN2/CTGF is induced in heart failure of various etiologies. However, its role in the pathophysiology of left ventricular (LV) remodeling after myocardial infarction (MI) remains unresolved. The current study explores the role of CTGF in infarct healing and LV remodeling in an animal model and in patients admitted for acute ST-elevation MI.
Transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) and non-transgenic littermate controls (NLC) were subjected to permanent ligation of the left anterior descending coronary artery. Despite similar infarct size (area of infarction relative to area at risk) 24 hours after ligation of the coronary artery in Tg-CTGF and NLC mice, Tg-CTGF mice disclosed smaller area of scar tissue, smaller increase of cardiac hypertrophy, and less LV dilatation and deterioration of LV function 4 weeks after MI. Tg-CTGF mice also revealed substantially reduced mortality after MI. Remote/peri-infarct tissue of Tg-CTGF mice contained reduced numbers of leucocytes, macrophages, and cells undergoing apoptosis as compared with NLC mice. In a cohort of patients with acute ST-elevation MI (n = 42) admitted to hospital for percutaneous coronary intervention (PCI) serum-CTGF levels (s-CTGF) were monitored and related to infarct size and LV function assessed by cardiac MRI. Increase in s-CTGF levels after MI was associated with reduced infarct size and improved LV ejection fraction one year after MI, as well as attenuated levels of CRP and GDF-15.
Increased myocardial CTGF activities after MI are associated with attenuation of LV remodeling and improved LV function mediated by attenuation of inflammatory responses and inhibition of apoptosis.
Background:
Connective Tissue Growth Factor (CTGF/CCN2) is an important mediator of kidney fibrosis. Previous observations indicated that attenuation of CCN2 expression sufficed to alleviate early kidney damage. However, little is known about the role of CCN2 in fibrosis of severely damaged and more chronically injured kidneys. Therefore, we examined the effects of CCN2 haploinsufficiency on the progression of renal scarring in long-term STZ-induced diabetic nephropathy, in a more advanced stage of obstructive nephropathy following unilateral ureteric obstruction (UUO), and in severe aristolochic acid (AA)-induced tubulotoxic nephritis.
Methods:
Wild-type (WT, CCN2(+/+)) and hemizygous CCN2(+/-) C57Bl/6 mice were studied. In the diabetes experiment, streptozotocin-injected and control mice were followed for 6 months, with regular blood pressure, glycaemia and albuminuria recordings. In the UUO experiment, the left ureter was obstructed for 14 days with the contralateral kidney serving as control. For the AA experiment, mice were followed for 25 days after 5 intraperitoneal injections with AA and compared to control mice injected with buffer alone. Organs were harvested for histology, mRNA and protein measurements. Collagen content was determined by HPLC and expressed as hydroxyproline/proline ratio.
Results:
CCN2 expression was significantly increased in the damaged as compared to control kidneys. In all three models, CCN2 levels in the damaged kidneys of CCN2(+/-) mice averaged about 50% of those in damaged WT kidneys. After 6 months of diabetes, albuminuria was increased 2.5-fold in WT mice, compared to 1.5-fold in CCN2(+/-) mice, mesangial matrix was expanded 5-fold in WT and 4.4-fold in CCN2(+/-) mice and the glomerular basement membrane was thickened 1.3-fold in WT and 1.5-fold in CCN2(+/-) mice (all differences between WT and CCN2(+/-) mice are NS). Tubular damage and interstitial fibrosis scores were also not different between Wt and CCN2(+/-) mice in the diabetes (1.8 vs. 1.7), UUO (2.8 vs. 2.6), and AA (1.4 vs. 1.2) models, as was the case for macrophage influx and collagen content in these three models.
Conclusion:
Unlike in mild and relatively early STZ-induced diabetic nephropathy, scarring of severely and chronically damaged kidneys is not attenuated by a 50% reduction of CCN2 to (near) normal levels. This suggests that CCN2 is either redundant in severe and chronic kidney disease, or that it is a limiting factor only at subnormal concentrations requiring further reduction by available or emerging therapies to prevent fibrosis of the severely injured kidney.
For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the
analysis of scientific images. We discuss the origins, challenges and solutions of these two programs,
and how their history can serve to advise and inform other software projects.
Increased cardiac collagen deposition is observed in almost every cardiac disease and plays an important role in the deteriorating function of the diseased heart. Propeptides of procollagen types I and III, the 2 major collagen types in the heart, can be detected in circulation. Although these propeptides reflect collagen synthesis, also breakdown products of collagen and the matrix metalloproteinases, responsible for the breakdown of the extracellular matrix, can be detected in blood and are used for investigating the turnover of collagen. Clinical trials are performed in recent years to examine the usage of these biomarkers in a diagnostic or prognostic way in heart failure patients. This review aims to discuss the formation of fibrosis, and studies investigating these biomarkers in heart failure are reviewed in this article. In addition, it is conferred what the flaws are of translating these biomarker levels to cardiac fibrosis formation and where we stand in using these biomarkers in clinics.
Beta-MHC-hRARalpha transgenic mice express a constitutively active (truncated) form of the human retinoic acid receptor which triggers development of dilated cardiomyopathy. In those hearts, we studied expression of gap junction proteins in relation to electrical impulse propagation.
As compared to wildtype mice, hearts of 4-6 month old mice with 7-12 inserted hRARalpha copies are marked by an increased heart weight/body weight- and heart weight/tibia length ratio. 3-extremity lead ECGs revealed prolongation of the Q-j interval suggesting delayed ventricular activation. Mapping of electrical activity of epi- and endocardial left ventricular free wall revealed activation delay, increased heterogeneity in conduction and regional conduction block. Ventricular tachycardias did not occur spontaneously nor could be induced by ventricular pacing. Immunohistochemical analysis showed profound and heterogeneous redistribution and down-regulation of the gap junction protein connexin43 (Cx43) in the left ventricular free wall. Here, hRARalpha expression induced re-expression of the hypertrophic markers alpha-skeletal actin and beta-MHC, and in 3 out of 10 severely affected mice, re-expression of Cx40. Concomitant with changes in expression/distribution of Cx43, changes in expression and distribution of beta-catenin and N-cadherin (two other intercalated disk associated proteins) were observed.
Beta-MHC-hRARalpha transgenic hearts show heterogeneous re-expression of (early) sarcomeric genes while expression of connexin43, N-cadherin and beta-catenin is down-regulated. We postulate that the resulting aberrant ventricular activation does not trigger development of lethal arrhythmias due to the small size of remaining healthy ventricular tissue where the transgene is not expressed.
Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure (CHF). To investigate determinants of the increased arrhythmogenic susceptibility, we studied cardiac remodelling and arrhythmogenicity in CHF patients and in a mouse model of chronic pressure overload.
Clinical and (immuno)histological data of myocardial biopsies from CHF patients with (VT+) and without (VT-) documented ventricular arrhythmia were compared with controls. In CHF patients, ejection fraction was decreased and QRS duration was increased. Cell size and interstitial fibrosis were increased, but Connexin43 (Cx43) levels, the most abundant gap junction in ventricular myocardium, were unchanged. No differences were found between VT+ and VT- patients, except for the distribution pattern of Cx43, which was significantly more heterogeneous in VT+. Mice were subjected to transverse aortic constriction (TAC) or sham operated. At 16 weeks, cardiac function was determined by echocardiography and epicardial ventricular activation mapping was performed. Transverse aortic constriction mice had decreased fractional shortening and prolonged QRS duration. Right ventricular conduction velocity was reduced, and polymorphic VTs were induced in 44% TAC and 0% sham mice. Interstitial fibrosis was increased and Cx43 quantity was unchanged in TAC mice with and without arrhythmias. Similar to CHF patients, heterogeneous Cx43 distribution was significantly associated with arrhythmias in TAC mice and with spatial heterogeneity of impulse conduction.
Heterogeneous Cx43 expression during CHF is associated with dispersed impulse conduction and may underlie enhanced susceptibility to ventricular tachyarrhythmias.
Myocardial fibrosis increases arrhythmia vulnerability of the diseased heart. The renin-angiotensin-aldosterone system (RAAS) governs myocardial collagen synthesis. We hypothesized that reducing cardiac fibrosis by chronic RAAS inhibition would result in reduced arrhythmia vulnerability of the senescent mouse heart. Wild-type mice (52 wk old) were treated for 36 wk: 1) untreated control (C); 2) eplerenone (E); 3) losartan (L); and 4) cotreatment with eplerenone and losartan (EL). Ventricular epicardial activation mapping was performed on Langendorff-perfused hearts. Arrhythmia inducibility was tested by one to three premature stimuli and burst pacing. Longitudinal and transverse conduction velocity and dispersion of conduction were determined during pacing at a basic cycle length of 150 ms. Sirius red staining (collagen) was performed. As a result, in the RV of mice in the E, L, and EL groups, transverse conduction velocity was significantly increased and anisotropic ratio was significantly decreased compared with those values of mice in the C group. Anisotropic reentrant arrhythmias were induced in 52% of untreated mice and significantly reduced to 22%, 26%, and 16% in the E, L, and EL groups, respectively. Interstitial fibrosis was significantly decreased in both the RV and LV of all treated groups. Scattered patches of replacement fibrosis were found in 90% of untreated hearts, which were significantly reduced in the E, L, and EL groups. A strong correlation between the abundance of patchy fibrosis and arrhythmia inducibility was found. In conclusion, chronic RAAS inhibition limited aging-related interstitial fibrosis. The lower arrhythmogeneity of treated mice was directly correlated to the reduced amount of patchy fibrosis.
Connective tissue growth factor (CTGF) is a secreted protein that is strongly induced in human and experimental heart failure. CTGF is said to be profibrotic; however, the precise function of CTGF is unclear. We generated transgenic mice and rats with cardiomyocyte-specific CTGF overexpression (CTGF-TG). To investigate CTGF as a fibrosis inducer, we performed morphological and gene expression analyses of CTGF-TG mice and rat hearts under basal conditions and after stimulation with angiotensin II (Ang II) or isoproterenol, respectively. Surprisingly, cardiac tissues of both models did not show increased fibrosis or enhanced gene expression of fibrotic markers. In contrast to controls, Ang II treated CTGF-TG mice displayed preserved cardiac function. However, CTGF-TG mice developed age-dependent cardiac dysfunction at the age of 7 months. CTGF related heart failure was associated with Akt and JNK activation, but not with the induction of natriuretic peptides. Furthermore, cardiomyocytes from CTGF-TG mice showed unaffected cellular contractility and an increased Ca2+ reuptake from sarcoplasmatic reticulum. In an ischemia/reperfusion model CTGF-TG hearts did not differ from controls.
Our data suggest that CTGF itself does not induce cardiac fibrosis. Moreover, it is involved in hypertrophy induction and cellular remodeling depending on the cardiac stress stimulus. Our new transgenic animals are valuable models for reconsideration of CTGF's profibrotic function in the heart.
Protein kinase C (PKC) beta isoform activity is increased in myocardium of diabetic rodents and heart failure patients. Transgenic mice overexpressing PKCbeta2 (PKCbeta2Tg) in the myocardium exhibit cardiomyopathy and cardiac fibrosis. In this study, we characterized the expression of connective tissue growth factor (CTGF) and transforming growth factor beta (TGFbeta) with the development of fibrosis in heart from PKCbeta2Tg mice at 4-16 weeks of age. Heart-to-body weight ratios of transgenic mice increased at 8 and 12 weeks, indicating hypertrophy, and ratios did not differ at 16 weeks. Collagen VI and fibronectin mRNA expression increased in PKCbeta2Tg hearts at 4-12 weeks. Histological examination revealed myocyte hypertrophy and fibrosis in 4- to 16-week PKCbeta2Tg hearts. CTGF expression increased in PKCbeta2Tg hearts at all ages, whereas TGFbeta increased only at 8 and 12 weeks. In 8-week diabetic mouse heart, CTGF and TGFbeta expression increased two- and fourfold, respectively. Similarly, CTGF expression increased in rat hearts at 2-8 weeks of diabetes. This is the first report of increased CTGF expression in myocardium of diabetic rodents suggesting that cardiac injury associated with PKCbeta2 activation, diabetes, or heart failure is marked by increased CTGF expression. CTGF could act independently or together with other cytokines to induce cardiac fibrosis and dysfunction.
Coordinated production and remodeling of the extracellular matrix is essential during development. It is of particular importance for skeletogenesis, as the ability of cartilage and bone to provide structural support is determined by the composition and organization of the extracellular matrix. Connective tissue growth factor (CTGF, CCN2) is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. A role for CTGF in extracellular matrix production is suggested by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. To test whether CTGF is required for extracellular matrix remodeling and/or angiogenesis during development, we examined the pattern of Ctgf expression and generated Ctgf-deficient mice. Ctgf is expressed in a variety of tissues in midgestation embryos, with highest levels in vascular tissues and maturing chondrocytes. We confirmed that CTGF is a crucial regulator of cartilage extracellular matrix remodeling by generating Ctgf(-/-) mice. Ctgf deficiency leads to skeletal dysmorphisms as a result of impaired chondrocyte proliferation and extracellular matrix composition within the hypertrophic zone. Decreased expression of specific extracellular matrix components and matrix metalloproteinases suggests that matrix remodeling within the hypertrophic zones in Ctgf mutants is defective. The mutant phenotype also revealed a role for Ctgf in growth plate angiogenesis. Hypertrophic zones of Ctgf mutant growth plates are expanded, and endochondral ossification is impaired. These defects are linked to decreased expression of vascular endothelial growth factor (VEGF) in the hypertrophic zones of Ctgf mutants. These results demonstrate that CTGF is important for cell proliferation and matrix remodeling during chondrogenesis, and is a key regulator coupling extracellular matrix remodeling to angiogenesis at the growth plate.
T. A. B. van Veen, H. V. M. van Rijen, R. F. Wiegerinck, T. Opthof, M. C. Colbert, S. Clement, J. M. T. de Bakker and H. J. Jongsma. Remodeling of Gap Junctions in Mouse Hearts Hypertrophied by Forced Retinoic Acid Signaling.Journal of Molecular and Cellular Cardiology (2002) 34, 1411–1423. Background: β-MHC-hRARα transgenic mice express a constitutively active (truncated) form of the human retinoic acid receptor which triggers development of dilated cardiomyopathy. In those hearts, we studied expression of gap junction proteins in relation to electrical impulse propagation. Methods and Results: As compared to wildtype mice, hearts of 4–6 month old mice with 7–12 inserted hRARα copies are marked by an increased heart weight/body weight- and heart weight/tibia length ratio. 3-extremity lead ECGs revealed prolongation of the Q–j interval suggesting delayed ventricular activation. Mapping of electrical activity of epi- and endocardial left ventricular free wall revealed activation delay, increased heterogeneity in conduction and regional conduction block. Ventricular tachycardia's did not occur spontaneously nor could be induced by ventricular pacing. Immunohistochemical analysis showed profound and heterogeneous redistribution and down-regulation of the gap junction protein connexin43 (Cx43) in the left ventricular free wall. Here, hRARα expression induced re-expression of the hypertrophic markers α-skeletal actin and β-MHC, and in 3 out of 10 severely affected mice, re-expression of Cx40. Concomitant with changes in expression/distribution of Cx43, changes in expression and distribution of β-catenin and N-cadherin (two other intercalated disk associated proteins) were observed. Conclusions: β-MHC-hRARα transgenic hearts show heterogeneous re-expression of (early) sarcomeric genes while expression of connexin43, N-cadherin and β-catenin is down-regulated. We postulate that the resulting aberrant ventricular activation does not trigger development of lethal arrhythmias due to the small size of remaining healthy ventricular tissue where the transgene is not expressed.
Connective tissue growth factor (CTGF) is involved in the pathogenesis of various fibrotic disorders. However, its role in the heart is not clear. To investigate the role of CTGF in regulating the development of cardiac fibrosis and heart failure, we subjected mice to thoracic aortic constriction (TAC) or angiotensin II infusion, and antagonized the function of CTGF with CTGF monoclonal antibody (mAb). After 8 weeks of TAC, mice treated with CTGF mAb had significantly better preserved left ventricular (LV) systolic function and reduced LV dilatation compared with mice treated with control immunoglobulin G. CTGF mAb-treated mice exhibited significantly smaller cardiomyocyte cross-sectional area and reduced expression of hypertrophic marker genes. CTGF mAb treatment reduced the TAC-induced production of collagen 1 but did not significantly attenuate TAC-induced accumulation of interstitial fibrosis. Analysis of genes regulating extracellular matrix proteolysis showed decreased expression of plasminogen activator inhibitor-1 and matrix metalloproteinase-2 in mice treated with CTGF mAb. In contrast to TAC, antagonizing the function of CTGF had no effect on LV dysfunction or LV hypertrophy in mice subjected to 4-week angiotensin II infusion. Further analysis showed that angiotensin II-induced expression of hypertrophic marker genes or collagens was not affected by treatment with CTGF mAb. In conclusion, CTGF mAb protects from adverse LV remodeling and LV dysfunction in hearts subjected to pressure overload by TAC. Antagonizing the function of CTGF may offer protection from cardiac end-organ damage in patients with hypertension.
Myocardial dysfunction is a progressive condition. Early after an insult to the myocardium (e.g., myocardial infarction) there
may be little or no immediate reduction in overall pump function, particularly if the damage has been mild. However, with
time there is a relentless deterioration in both the structure and function of the ventricle by a process referred to as “remodeling”
(1). The specific features of the remodeling process depend, to a large extent, on the nature of the underlying stimulus.
Arrhythmogenic ventricular remodeling is hallmarked by both reduced gap junction expression and increased collagen deposition. We hypothesized that reduced connexin43 (Cx43) expression is responsible for enhanced fibrosis in the remodeled heart, resulting in an arrhythmogenic substrate. Therefore, we investigated the effect of normal or reduced Cx43 expression on the formation of fibrosis in a physiological (aging) and pathophysiological (transverse aortic constriction [TAC]) mouse model.
The Cx43(fl/fl) and Cx43(CreER(T)/fl) mice were aged 18 to 21 months or, at the age of 3 months, either TAC or sham operated and euthanized after 16 weeks. Epicardial activation mapping of the right and left ventricles was performed on Langendorff perfused hearts. Sustained ventricular arrhythmias were induced in 0 of 11 aged Cx43(fl/fl) and 10 of 15 Cx43(Cre-ER(T)/fl) mice (P<0.01). Cx43 expression was reduced by half in aged Cx43(CreER(T)/fl) compared with aged Cx43(fl/fl) mice, whereas collagen deposition was significantly increased from 1.1±0.2% to 7.4±1.3%. Aged Cx43(CreER(T)/fl) mice with arrhythmias had significantly higher levels of fibrosis and conduction heterogeneity than aged Cx43(CreER(T)/fl) mice without arrhythmias. The TAC operation significantly increased fibrosis in control compared with sham (4.0±1.2% versus 0.4±0.06%), but this increase was significantly higher in Cx43(CreER(T)/fl) mice (10.8±1.4%). Discoidin domain receptor 2 expression was unchanged, but procollagen peptide I and III expression and collagen type 1α2 mRNA levels were higher in TAC-operated Cx43HZ mice.
Reduced cellular coupling results in more excessive collagen deposition during aging or pressure overload in mice due to enhanced fibroblast activity, leading to increased conduction in homogeneity and proarrhythmia.
Reduced expression of connexin43 (Cx43) and sodium channel (Nav1.5) and increased expression of collagen (fibrosis) are important determinants of impulse conduction in the heart.
To study the importance and interaction of these factors at very low Cx43 expression, inducible Cx43 knockout mice with and without inducible ventricular tachycardia (VT) were compared through electrophysiology and immunohistochemistry.
Cx43(CreER(T)/fl) mice were induced with tamoxifen and killed after 2 weeks. Epicardial activation mapping was performed on Langendorff-perfused hearts, and arrhythmia vulnerability was tested. Mice were divided into arrhythmogenic (VT+; n = 13) and nonarrhythmogenic (VT-; n = 10) animals, and heart tissue was analyzed for Cx43, Nav1.5, and fibrosis.
VT+ mice had decreased Cx43 expression with increased global, but not local, heterogeneity of Cx43 than did VT- mice. Nav1.5-immunoreactive protein expression was lower in VT+ than in VT- mice, specifically at sites devoid of Cx43. Levels of fibrosis were similar between VT- and VT+ mice. QRS duration was increased and epicardial activation was more dispersed in VT+ mice than in VT- mice. The effective refractory period was similar between the 2 groups. Premature stimulation resulted in a more severe conduction slowing in VT+ than in VT- hearts in the right ventricle. Separate patch-clamp experiments in isolated rat ventricular myocytes confirmed that the loss of Cx43 expression correlated with the decreased sodium current amplitude.
Global heterogeneity in Cx43 expression and concomitant heterogeneous downregulation of sodium-channel protein expression and sodium current leads to slowed and dispersed conduction, which sensitizes the heart for ventricular arrhythmias.
The major gap junction protein expressed in the heart, connexin43 (Cx43), is highly remodeled in the diseased heart. Usually, Cx43 is down-regulated and heterogeneously redistributed to the lateral sides of cardiomyocytes. Reverse remodeling of the impaired Cx43 expression could restore normal cardiac function and normalize electrical stability. In this review, the reduced and heterogeneous Cx43 expression in the heart will be addressed in hypertrophic, dilated and ischemic cardiomyopathy together with its functional consequences of conduction velocity slowing, dispersed impulse conduction, its interaction with fibrosis and propensity to generate arrhythmias. Finally, different therapies are discussed. Treatments aimed to improve the Cx43 expression levels show new potentially anti-arrhythmic therapies during heart failure, but those in the context of acute ischemia can be anti-arrhythmogenic at the cost of larger infarct sizes. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.
No therapy for fibrotic disease is available. The proadhesive matricellular protein connective tissue growth factor CCN2 is a marker of fibrotic cells; however, the specific role of CCN2 in connective tissue biology in general and in fibrogenesis in particular is unclear. The aim of this study was to assess whether adult mice bearing a smooth muscle cell/fibroblast-specific deletion of CCN2 are resistant to bleomycin-induced skin scleroderma.
Cutaneous fibrosis was induced in mice by subcutaneous injection of bleomycin. Untreated control groups were injected with phosphate buffered saline. Mice bearing a fibroblast/smooth muscle cell-specific deletion of CCN2 were investigated for changes in dermal thickness, collagen content, and the number of α-smooth muscle actin (α-SMA)-positive cells. Dermal fibroblasts were isolated to assess whether the induction of collagen and α-SMA messenger RNA in response to transforming growth factor β (TGFβ) was impaired.
The loss of CCN2 resulted in resistance to bleomycin-induced skin fibrosis. In response to bleomycin, wild-type mice possessed, but CCN2-deficient mice lacked, abundant α-SMA-expressing myofibroblasts within fibrotic lesions. Fibroblast responses to TGFβ, a potent inducer of myofibroblast differentiation, were not affected. Collectively, these results indicate that CCN2 is essential for bleomycin-induced skin fibrosis, likely due to a defect in myofibroblast recruitment.
These data indicate that therapeutic strategies that involve blocking CCN2 in vivo may be of benefit in combating fibrotic skin disease.
Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with Connexin distribution in normal and Cx40-deficient mice hearts.
Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine Connexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals.
Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction.
CCN family members are matricellular proteins with diverse roles in cell function. The differential expression of CCN2 and CCN5 during cardiac remodeling suggests that these two members of the CCN family play opposing roles during the development of cardiac hypertrophy and fibrosis. We aimed to evaluate the role of CCN2 and CCN5 in the development of cardiac hypertrophy and fibrosis. In isolated cardiomyocytes, overexpression of CCN2 induced hypertrophic growth, whereas the overexpression of CCN5 inhibited both phenylephrine (PE)- and CCN2-induced hypertrophic responses. Deletion of the C-terminal (CT) domain of CCN2 transformed CCN2 into a CCN5-like dominant negative molecule. Fusion of the CT domain to the Carboxy-terminus of CCN5 transformed CCN5 into a CCN2-like pro-hypertrophic molecule. CCN2 transgenic (TG) mice did not develop cardiac hypertrophy at baseline but showed significantly increased fibrosis in response to pressure overload. In contrast, hypertrophy and fibrosis were both significantly inhibited in CCN5 TG mice. CCN2 TG mice showed an accelerated deterioration of cardiac function in response to pressure overload, whereas CCN5 TG mice showed conserved cardiac function. TGF-beta-SMAD signaling was elevated in CCN2 TG mice, but was inhibited in CCN5 TG mice. CCN2 is pro-hypertrophic and -fibrotic, whereas CCN5 is anti-hypertrophic and -fibrotic. CCN5 lacking the CT domain acts as a dominant negative molecule. CCN5 may provide a novel therapeutic target for the treatment of cardiac hypertrophy and heart failure.
We studied the signal transduction of atrial structural remodeling that contributes to the pathogenesis of atrial fibrillation (AF).
Fibrosis is a hallmark of arrhythmogenic structural remodeling, but the underlying molecular mechanisms are incompletely understood.
We performed transcriptional profiling of left atrial myocardium from patients with AF and sinus rhythm and applied cultured primary cardiac cells and transgenic mice with overexpression of constitutively active V12Rac1 (RacET) in which AF develops at old age to characterize mediators of the signal transduction of atrial remodeling.
Left atrial myocardium from patients with AF showed a marked up-regulation of connective tissue growth factor (CTGF) expression compared with sinus rhythm patients. This was associated with increased fibrosis, nicotinamide adenine dinucleotide phosphate oxidase, Rac1 and RhoA activity, up-regulation of N-cadherin and connexin 43 (Cx43) expression, and increased angiotensin II tissue concentration. In neonatal rat cardiomyocytes and fibroblasts, a specific small molecule inhibitor of Rac1 or simvastatin completely prevented the angiotensin II-induced up-regulation of CTGF, Cx43, and N-cadherin expression. Transfection with small-inhibiting CTGF ribonucleic acid blocked Cx43 and N-cadherin expression. RacET mice showed up-regulation of CTGF, Cx43, and N-cadherin protein expression. Inhibition of Rac1 by oral statin treatment prevented these effects, identifying Rac1 as a key regulator of CTGF in vivo.
The data identify CTGF as an important mediator of atrial structural remodeling during AF. Angiotensin II activates CTGF via activation of Rac1 and nicotinamide adenine dinucleotide phosphate oxidase, leading to up-regulation of Cx43, N-cadherin, and interstitial fibrosis and therefore contributing to the signal transduction of atrial structural remodeling.
Connective tissue growth factor (CCN2) is a major pro-fibrotic factor that frequently acts downstream of transforming growth factor beta (TGF-beta)-mediated fibrogenic pathways. Much of our knowledge of CCN2 in fibrosis has come from studies in which its production or activity have been experimentally attenuated. These studies, performed both in vitro and in animal models, have demonstrated the utility of pharmacological inhibitors (e.g. tumor necrosis factor alpha (TNF-alpha), prostaglandins, peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonists, statins, kinase inhibitors), neutralizing antibodies, antisense oligonucleotides, or small interfering RNA (siRNA) to probe the role of CCN2 in fibrogenic pathways. These investigations have allowed the mechanisms regulating CCN2 production to be more clearly defined, have shown that CCN2 is a rational anti-fibrotic target, and have established a framework for developing effective modalities of therapeutic intervention in vivo.
Cardiac fibrosis is a major pathogenic factor in a variety of cardiovascular diseases and refers to an excessive deposition of extracellular matrix components in the heart, which leads to cardiac dysfunction and eventually overt heart failure. Evidence is accumulating for a crucial role of connective tissue growth factor (CTGF) in fibrotic processes in several tissues including the heart. CTGF orchestrates the actions of important local factors evoking cardiac fibrosis. The central role of CTGF as a matricellular protein modulating the fibrotic process in cardiac remodelling makes it a possible biomarker for cardiac fibrosis and a potential candidate for therapeutic intervention to mitigate fibrosis in the heart.
"Remodeling" implies changes that result in rearrangement of normally existing structures. This review focuses only on permanent modifications in relation to clinical dysfunction in cardiac remodeling (CR) secondary to myocardial infarction (MI) and/or arterial hypertension and includes a special section on the senescent heart, since CR is mainly a disease of the elderly. From a biological point of view, CR is determined by 1 ) the general process of adaptation which allows both the myocyte and the collagen network to adapt to new working conditions; 2) ventricular fibrosis, i.e., increased collagen concentration, which is multifactorial and caused by senescence, ischemia, various hormones, and/or inflammatory processes; 3) cell death, a parameter linked to fibrosis, which is usually due to necrosis and apoptosis and occurs in nearly all models of CR. The process of adaptation is associated with various changes in genetic expression, including a general activation that causes hypertrophy, isogenic shifts which result in the appearance of a slow isomyosin, and a new Na+-K+-ATPase with a low affinity for sodium, reactivation of genes encoding for atrial natriuretic factor and the renin-angiotensin system, and a diminished concentration of sarcoplasmic reticulum Ca2+-ATPase, beta-adrenergic receptors, and the potassium channel responsible for transient outward current. From a clinical point of view, fibrosis is for the moment a major marker for cardiac failure and a crucial determinant of myocardial heterogeneity, increasing diastolic stiffness, and the propensity for reentry arrhythmias. In addition, systolic dysfunction is facilitated by slowing of the calcium transient and the downregulation of the entire adrenergic system. Modifications of intracellular calcium movements are the main determinants of the triggered activity and automaticity that cause arrhythmias and alterations in relaxation.
Connective tissue growth factor (CTGF), a 36- to 38-kDa peptide, is selectively induced by transforming growth factor-beta and has been suggested to contribute to tissue repair. To test the hypothesis that CTGF is expressed in myocardial infarct tissue following acute myocardial infarction (AMI), we examined CTGF expression after AMI was experimentally induced in rats. Myocardial infarction was induced by left coronary artery ligation in male Sprague-Dawley rats. Northern blotting demonstrated that the CTGF mRNA expression on days 2, 7 and 14 was increased by 6-, 23- and 8-fold, respectively, compared to that in the pre-ligation hearts. In situ hybridization revealed CTGF mRNA signals on day 2 in myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) located between surviving myocytes in the infarct peripheral zone. On day 7, the signals were observed in the inner lesion of the infarct around infarct granulation tissue. Western blotting demonstrated that the CTGF protein expression on days 2, 7 and 14 was increased compared to the pre-ligation hearts. Immunopositive staining for CTGF was observed in the inner lesion of the infarct tissue on day 7. In conclusion, the findings demonstrated the increased expression of CTGF in the infarct tissue. Myocytes in the infarct marginal zone and spindle-shaped mesenchymal cells (presumably myofibroblasts and fibroblasts) were the cells responsible for CTGF production.
Connective tissue growth factor (CTGF) is a cysteine-rich protein induced by transforming growth factor beta (TGF- beta) in connective tissue cells. CTGF can trigger many of the cellular processes underlying fibrosis, such as cell proliferation, adhesion, migration and the synthesis of extracellular matrix; however, its role in acute and chronic cardiac injury is not fully understood. Here, we show that TGF- beta is a specific inducer of CTGF expression in both cardiac fibroblasts and cardiac myocytes. The activity of a CTGF promoter-based reporter construct correlated with endogenous CTGF expression, suggesting that TGF- beta induces CTGF expression most likely by activating its promoter. Upregulation of CTGF coincided with an increase in fibronectin, collagen type I and plasminogen activator inhibitor-1 production. Forskolin, a stimulator of cyclic AMP, blocked TGF- beta induced CTGF expression and reduced the basal level of CTGF, whereas an inhibitor that blocks the MAP kinase signaling pathway (PD 98059) significantly enhanced TGF- beta induced CTGF expression. Furthermore, we found that both TGF- beta and CTGF mRNAs were significantly elevated in the left ventricles and septa of rat hearts 2-16 weeks following myocardial infarction. This correlated well with concomitant increases in fibronectin, and type I and type III collagen mRNA levels in these animal hearts. Significant upregulation of CTGF was also detected in human heart samples derived from patients diagnosed with cardiac ischemia. Based on these findings, we propose that CTGF is an important mediator of TGF- beta signaling in the heart and abnormal expression of this gene could be used as a diagnostic marker for cardiac fibrosis.
Cardiac remodeling, CR, is a complex and rather controversial issue and results from the, sometimes opposite, trophic effects of pure mechanical overload, and susceptibility factors, as senescence, aetiologies, as ischemia, and the neurohormonal reaction. The molecular mechanisms of CR are heritable and had, in the past, increased fitness, as such CR belongs to evolutionary medicine. Aldosterone production plays an important role in the remodeling of the heart. (i) There are numerous evidences that aldosterone induces fibrosis in all the cardiovascular system in the presence of high sodium diet. The aldosterone receptor is a transcriptional factor and the pathways that lead to aldosterone-induced fibrosis are multiple. Aldosterone induces the expression of the angiotensin II receptors subtype I and that of the glucocorticoid receptors. The RALES trial have recently evidenced a significant beneficial effect of spironolactone on both mortality and morbidity in heart failure, and a substudy has shown that these effects are linked to a reduction is fibrosis. (ii) An intracardiac production of aldosterone and corticosterone have been evidenced in the rat. Aldosterone production is regulated by low sodium/high potassium diets and by angiotensin II and is predominant in atria, cardiac production is low as compared to the adrenal production, nevertheless it results in high local concentrations, just like angiotensin II. In rats, myocardial infarction activates aldosterone production and this activation is prevented by losartan. Heart failure, in human, activates aldosterone production and is accompanied by a significant increase of the arteriovenous difference in aldosterone by the myocardium. To conclude (i) after a myocardial infarction local production of aldosterone and angiotensin II are likely to play a major role in regulating collagen turnover and fibrous tissue formation; (ii) during heart failure, the activation of adrenal and cardiovascular production of aldosterone belongs to the neurohormonal reaction and would play a detrimental role in producing reactive fibrosis.
Angiotensin II (Ang II) participates in the development of fibrosis during vascular damage. Connective tissue growth factor (CTGF) is a novel fibrotic mediator. However, the potential link between CTGF and Ang II has not been investigated.
In vivo Ang II effects were studied by systemic infusion into normal rats to evaluate CTGF and extracellular matrix protein (ECM) expression by immunohistochemistry. In aorta of Ang II-infused rats, CTGF staining was markedly increased and ECM overexpression was observed. An AT1 antagonist diminished CTGF and ECM. In growth-arrested vascular smooth muscle cells, Ang II induced CTGF mRNA expression after 1 hour, remained elevated up to 24 hours, and increased CTGF protein production, which was increased up to 72 hours. The AT1 antagonist blocked Ang II-induced CTGF gene and protein expression. Early CTGF upregulation is independent of new protein synthesis. Several intracellular signals elicited by Ang II are involved in CTGF synthesis, including protein kinase C activation, reactive oxygen species, and transforming growth factor-beta endogenous production. Incubation with a CTGF antisense oligonucleotide decreased CTGF and fibronectin upregulation caused by Ang II.
Our results show that Ang II, via AT1, increases CTGF in vascular cells both in vivo and in vitro. This novel finding suggests that CTGF may be a mediator of the profibrogenic effects of Ang II in vascular diseases.
Gene expression signals involved in ischemic injury, extracellular matrix composition and fibrosis defined by global mRNA profiling of the human left ventricular myocardium. The mechanism(s) by which acute and chronic myocardial ischemia translate into the characteristic features of ischemic cardiomyopathy is unresolved at present. We hypothesized that such translation relates to modification of specific gene expression programs during acute and chronic ischemic insults to the myocardium. Global mRNA expression profiles by Affymetrix HG_U133A GeneChip analysis on 33 samples was performed on non-failing human left ventricular myocardium during acute and chronic ischemia in 6 patients undergoing coronary artery by-pass grafting. Results were confirmed by real-time quantitative RT-PCR in 14 patients and supported by histology and immunohistochemistry analyses. Acute ischemia elicited an acute inflammatory response including IL-6, IL-8, MCP-1, VCAM-1 and CYR-61 with an attenuated increase of IL-6 and IL-8 in chronic ischemic myocardium compared to normal myocardium. High mRNA expression of connective tissue growth factor (CTGF) was present in chronic ischemic myocardium with a high degree of correlation between CTGF and mRNA expression of specific genes (e.g. thrombospondin 4, collagen type Ialpha2, versican, adlican, latent transforming growth factor beta binding protein 2 and fibronectin) involved in extracellular matrix remodelling. In conclusion, acute inflammatory induction (e.g. IL-8, IL-6, VCAM-1 and MCP-1) and an acute phase CCN family gene with effects on matrix interactions (CYR-61) might play important roles in the coupling between acute ischemic episodes and chronic myocardial remodelling. In addition, the findings support an important role of CTGF signalling in chronic extracellular matrix remodelling in chronic coronary artery disease.
Excessive fibrosis contributes to an increase in left ventricular stiffness. The goal of the present study was to investigate the role of connective tissue growth factor (CCN2/CTGF), a profibrotic cytokine of the CCN (Cyr61, CTGF, and Nov) family, and its functional interactions with brain natriuretic peptide (BNP), an antifibrotic peptide, in the development of myocardial fibrosis and diastolic heart failure. Histological examination on endomyocardial biopsy samples from patients without systolic dysfunction revealed that the abundance of CTGF-immunopositive cardiac myocytes was correlated with the excessive interstitial fibrosis and a clinical history of acute pulmonary congestion. In a rat pressure overload cardiac hypertrophy model, CTGF mRNA levels and BNP mRNA were increased in proportion to one another in the myocardium. Interestingly, relative abundance of mRNA for CTGF compared with BNP was positively correlated with diastolic dysfunction, myocardial fibrosis area, and procollagen type 1 mRNA expression. Investigation with conditioned medium and subsequent neutralization experiments using primary cultured cells demonstrated that CTGF secreted by cardiac myocytes induced collagen production in cardiac fibroblasts. Further, G protein-coupled receptor ligands induced expression of the CTGF and BNP genes in cardiac myocytes, whereas aldosterone and transforming growth factor-beta preferentially induced expression of the CTGF gene. Finally, exogenous BNP prevented the production of CTGF in cardiac myocytes. These data suggest that a disproportionate increase in CTGF relative to BNP in cardiac myocytes plays a central role in the induction of excessive myocardial fibrosis and diastolic heart failure.
Transforming growth factor-beta (TGF-beta) participates in the pathogenesis of multiple cardiovascular diseases, including hypertension, restenosis, atherosclerosis, cardiac hypertrophy and heart failure. TGF-beta exerts pleiotropic effects on cardiovascular cells, regulating cell growth, fibrosis and inflammation. TGF-beta has long been believed to be the most important extracellular matrix regulator. We review the complex mechanisms involved in TGF-beta-mediated vascular fibrosis that includes the Smad signaling pathway, activation of protein kinases and crosstalk between these pathways. TGF-beta blockade diminishes fibrosis in experimental models, however better antifibrotic targets are needed for an effective therapy in human fibrotic diseases. A good candidate is connective tissue growth factor (CTGF), a downstream mediator of TGF-beta-induced fibrosis. Among the different factors involved in vascular fibrosis, Angiotensin II (AngII) has special interest. AngII can activate the Smad pathway independent of TGF-beta and shares with TGF-beta many intracellular signals implicated in fibrosis. Blockers of AngII have demonstrated beneficial effects on many cardiovascular diseases and are now one of the best options to block TGF-beta fibrotic responses. A better knowledge of the intracellular signals of TGF-beta can provide novel therapeutic approaches for fibrotic diseases.
Connective tissue growth factor (CTGF, CCN2) is a secreted protein with major roles in angiogenesis, chondrogenesis, osteogenesis, tissue repair, cancer and fibrosis. It is a member of the CCN family of immediate-early gene products which are characterised by four discrete protein modules in which reside growth factor binding domains, functional motifs for integrin recognition, heparin and proteoglycan binding, and dimerization motifs. A primary function of CTGF is to modulate and coordinate signaling responses involving cell surface proteoglycans, key components of the extracellular matrix, and growth factors. Integration of these molecular cues regulates growth factor and receptor interactions, cell motility and mesenchymal cell activation and differentiation in tissue remodelling. Abnormal amplification of CTGF dependent signals results in a failure to terminate tissue repair, leading pathological scarring in conditions such as fibrosis and cancer.
- S De Jong
- T A Van Veen
- J M De Bakker
- M A Vos
- H V Van Rijen
S. de Jong, T.A. van Veen, J.M. de Bakker, M.A. Vos, H.V. van Rijen, Biomarkers of
myocardial fibrosis, J. Cardiovasc. Pharmacol. 57 (2011) 522-535.
Connective tissue growth factor overexpression in cardiomyocytes promotes cardiac hypertrophy and protection against pressure overload
- Panek