Tissue Growth Factor Gene Expression Research Articles

Potential treatment of keloid pathogenesis with follistatin 288 by blocking the activin molecular pathway.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

Connective tissue growth factor gene expression in goat endometrium during estrous cycle and early pregnancy

Embryo implantation is crucial for a successful pregnancy. Although many essential molecular modulators and pathways have been identified, the precise mechanisms of the process in goat remain largely unknown. CCN2 is a connective tissue growth factor participating in many biological processes; however, its presence or function in goat uterus has not yet been reported. In this study, we determined the expression and regulation of CCN2 in goat uterus. CCN2 was not detected by in situ hybridization at ED0 (Day 0 of the estrous cycle), but at ED6 (metestrus), ED12 (dioestrus), and ED16 (proestrus), with high signals in luminal epithelium, superficial glands, and caruncula matrix. During early pregnancy, CCN2 was also detected in these locations on D0 and D6 (pre-receptive uterus). The signals significantly increased on D16 and D19 (receptive uterus), and remained at high levels on D25 and D30. Similarly, the RT-qPCR assays showed that the mRNA level of CCN2 was relatively low on D0 and D6, increased on D16, peaked on D19, and kept high thereafter. Moreover, CCN2 was up-regulated not only in ovariectomized ewes subcutaneously injected with 17β-estradiol and progesterone (separately or together), but also in cultured goat uterine epithelial cells treated with the two hormones or interferon tau (IFNτ). In conclusion, CCN2 expression may be induced by 17β-estradiol, progesterone, and IFNτ in the luminal epithelium of goat receptive uterus, suggesting that CCN2 is involved in goat embryo adhesion during early pregnancy.

5-Aryl-1,3,4-oxadiazol-2-ylthioalkanoic Acids: A Highly Potent New Class of Inhibitors of Rho/Myocardin-Related Transcription Factor (MRTF)/Serum Response Factor (SRF)-Mediated Gene Transcription as Potential Antifibrotic Agents for Scleroderma.

Through a phenotypic high-throughput screen using a serum response element luciferase promoter, we identified a novel 5-aryl-1,3,4-oxadiazol-2-ylthiopropionic acid lead inhibitor of Rho/myocardin-related transcription factor(MRTF)/serum response factor(SRF)-mediated gene transcription with good potency (IC50 = 180 nM). We were able to rapidly improve the cellular potency by 5 orders of magnitude guided by sharply defined and synergistic SAR. The remarkable potency and depth of the SAR, as well as the relatively low molecular weight of the series, suggests, but does not prove, that binding to the unknown molecular target may be occurring through a covalent mechanism. The series nevertheless has no observable cytotoxicity up to 100 μM. Ensuing pharmacokinetic optimization resulted in the development of two potent and orally bioavailable anti-fibrotic agents that were capable of dose-dependently reducing connective tissue growth factor gene expression in vitro as well as significantly reducing the development of bleomycin-induced dermal fibrosis in mice in vivo.

Effects of aging and resistance training in rat tendon remodeling.

In elderly persons, weak tendons contribute to functional limitations, injuries, and disability, but resistance training can attenuate this age-related decline. We evaluated the effects of resistance training on the extracellular matrix (ECM) of the calcaneal tendon (CT) in young and old rats and its effect on tendon remodeling. Wistar rats aged 3 mo (young, n = 30) and 20 mo (old, n = 30) were divided into 4 groups: young sedentary, young trained, old sedentary (OS), and old trained (OT). The training sessions were conducted over a 12-wk period. Aging in sedentary rats showed down-regulation in key genes that regulated ECM remodeling. Moreover, the OS group showed a calcification focus in the distal region of the CT, with reduced blood vessel volume density. In contrast, resistance training was effective in up-regulating connective tissue growth factor, VEGF, and decorin gene expression in old rats. Resistance training also increased proteoglycan content in young and old rats in special small leucine-rich proteoglycans and blood vessels and prevented calcification in OT rats. These findings confirm that resistance training is a potential mechanism in the prevention of aging-related loss in ECM and that it attenuates the detrimental effects of aging in tendons, such as ruptures and tendinopathies.-Marqueti, R. C., Durigan, J. L. Q., Oliveira, A. J. S., Mekaro, M. S., Guzzoni, V., Aro, A. A., Pimentel, E. R., Selistre-de-Araujo, H. S. Effects of aging and resistance training in rat tendon remodeling.

Ocular surface cytokine profile in chronic Stevens-Johnson syndrome and its response to mucous membrane grafting for lid margin keratinisation

BackgroundTo study the tear cytokine and the conjunctival and oral mucosal marker profile in chronic ocular Stevens-Johnson syndrome (SJS) and their alteration following mucous membrane grafting (MMG) for lid margin...

Integrin α2β1 inhibits MST1 kinase phosphorylation and activates Yes-associated protein oncogenic signaling in hepatocellular carcinoma.

The Hippo pathway regulates the down-stream target Yes-associated protein (YAP) to maintain organ homeostasis, which is commonly inactivated in many types of cancers. However, how cell adhesion dysregulates the Hippo pathway activating YAP oncogene in hepatocellular carcinoma (HCC) remains unclear. Our findings demonstrate that α2β1 integrin (but not other β1 integrins) expressed in HCC cells, after binding to collagen extracellular matrix, could inhibit MST1 kinase phosphorylation and activate YAP pro-oncogenic activities. Knockdown of integrin α2 gene (ITGA2) suppressed YAP targeted gene expression in vitro. α2β1 and collagen binding resulted in suppressing Hippo signaling of mammalian sterile 20-like kinase 1 (MST1) and Large tumor suppressor homolog 1 (LATS1) with concomitant activation of YAP-mediated connective tissue growth factor (CTGF) gene expression. In vitro kinase assay showed that MST1 is an immediate downstream target of integrin α2 with S1180 residue as the critical phosphorylation site. Clinical correlational analysis using a gene expression dataset of 228 HCC tumors revealed that ITGA2 expression was significantly associated with tumor progression, and co-expression with YAP targeted genes (AXL receptor tyrosine kinase, CTGF, cyclin D1, glypican 3, insulin like growth factor 1 receptor, and SRY-box 4) correlated with survivals of HCC patients. In conclusion, α2β1 integrin activation through cellular adhesion impacts the Hippo pathway in solid tumors and modulates MST1-YAP signaling cascade. Targeting integrin α2 holds promises for treating YAP-positive HCC.

Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension.

Prostanoids derived from COX-2 and EP receptors are involved in vascular remodelling in different cardiovascular pathologies. This study evaluates the contribution of COX-2 and EP1 receptors to vascular remodelling and function in hypertension. Spontaneously hypertensive rats (SHR) and angiotensin II (AngII)-infused (1.44 mg · kg(-1) · day(-1), 2 weeks) mice were treated with the COX-2 inhibitor celecoxib (25 mg · kg(-1) · day(-1) i.p) or with the EP1 receptor antagonist SC19220 (10 mg · kg(-1) · day(-1) i.p.). COX-2(-/-) mice with or without AngII infusion were also used. Celecoxib and SC19220 treatment did not modify the altered lumen diameter and wall : lumen ratio in mesenteric resistance arteries from SHR-infused and/or AngII-infused animals. However, both treatments and COX-2 deficiency decreased the augmented vascular stiffness in vessels from hypertensive animals. This was accompanied by diminished vascular collagen deposition, normalization of altered elastin structure and decreased connective tissue growth factor and plasminogen activator inhibitor-1 gene expression. COX-2 deficiency and SC19220 treatment diminished the increased vasoconstrictor responses and endothelial dysfunction induced by AngII infusion. Hypertensive animals showed increased mPGES-1 expression and PGE2 production in vascular tissue, normalized by celecoxib. Celecoxib treatment also decreased AngII-induced macrophage infiltration and TNF-α expression. Macrophage conditioned media (MCM) increased COX-2 and collagen type I expression in vascular smooth muscle cells; the latter was reduced by celecoxib treatment. COX-2 and EP1 receptors participate in the increased extracellular matrix deposition and vascular stiffness, the impaired vascular function and inflammation in hypertension. Targeting PGE2 receptors might have benefits in hypertension-associated vascular damage.

SMAD3 and SP1/SP3 Transcription Factors Collaborate to Regulate Connective Tissue Growth Factor Gene Expression in Myoblasts in Response to Transforming Growth Factor β.

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

Noncanonical STAT3 activation regulates excess TGF-β1 and collagen I expression in muscle of stricturing Crohn's disease.

Increased TGF-β1 and TGF-β1-dependent Collagen I production in intestinal mesenchymal cells result in fibrosis in patients with Montreal B2 fibrostenotic Crohn's disease. Numerous cytokines, including IL-6, are produced by activated mesenchymal cells themselves and activate STAT3. The aim of the current study was to determine the mechanisms by which STAT-3 activation might result in intestinal fibrosis. Cytokine levels were measured by ELISA. STAT3 and suppressor of cytokine signaling 3 protein levels were measured by immunoblot, STAT3-TGFB1 DNA-binding activity by chromatin immunoprecipitation, and TGFB1 transcriptional activity by luciferase reporter assay. TGF-β1 (TGFB1), Collagen1α1, and connective tissue growth factor (CTGF) gene expression was measured by quantitative RT-PCR. The role of STAT3 activation was determined using STAT3 inhibitor, Stattic, and by transfection of STAT3 mutants. Autocrine production of cytokines was increased in muscle cells of B2 phenotype patients from strictures and normal intestine in the same patient and compared with other Crohn's phenotypes, ulcerative colitis, and non-Crohn's patients. A unique pattern of STAT3 phosphorylation emerged: high STAT3(S727) and low STAT3(Y705) in strictures and the opposite in unaffected intestine. TGFB1 transcriptional activity was regulated by phospho-STAT3(S727) and was decreased by Stattic or dominant-negative STAT3(S727A). TGF-β1, COL1A1, and CTGF expression was inhibited by Stattic or dominant-negative STAT3(S727A). Treatment of normal muscle cells with IL-6 or expression of constitutively active STAT3(S727E) phenocopied muscle cells from strictured intestine. Neutralization of autocrine IL-6 reversed STAT3 phosphorylation and normalized expression of TGF-β1 in strictured intestinal muscle. The ability of Stattic to improve development of fibrosis was confirmed in mice with 2,4,6-trinitrobenzenesulfonic acid-induced colitis. We observed a unique phospho-STAT3(S727) response in patients with Montreal B2 Crohn's disease, particularly in response to IL-6 leading to increased TGF-β1, collagen, and CTGF production in ileal strictures.

Nonphysiologic blood flow triggers endothelial and arterial remodeling in vivo: Implications for novel left ventricular assist devices with a peripheral anastomosis

Less invasive circulatory support devices have been developed that require anastomosis to a peripheral artery. The Symphony Heart Assist System (Abiomed, Inc, Danvers, Mass) is a volume-displacement pump sewn to the subclavian artery to provide partial circulatory support. The surgical configuration produces nonphysiologic blood pressure and bidirectional flow in the subclavian artery. Our objective was to identify effects of altered hemodynamics on arterial structure and function. In calves (n=23; 80-100 kg), the Symphony pump was sewn end-to-side to the carotid artery. Acutely, carotid blood pressure and flow were recorded to evaluate hemodynamic changes. After medium-term support (1-4 weeks), carotid artery was studied. Histologic and molecular assays evaluated architectural changes. Quantitative real-time polymerase chain reaction evaluated gene expression of matrix metalloproteinase (MMP)-2, MMP-9, and connective tissue growth factor. Invitro carotid arterial-ring studies evaluated physiologic responses. During Symphony support, carotid arterial pressure was 200/15 mm Hg. Antegrade flow increased significantly (P<.05) from 1.40 ± 0.32 to 4.29 ± 0.33 L/min. Flow during native cardiac diastole reversed completely from 0.25 ± 0.05 to -4.15 ± 0.38 L/min in carotid artery proximal to the anastomosis. After medium-term support, the carotid artery was significantly dilated with significantly thinner tunica media and thicker tunica adventitia than in control carotid arteries. MMP-9 gene expression decreased significantly, connective tissue growth factor gene expression increased significantly, and collagen, elastin, and total extracellular matrix increased significantly. Endothelial cells were significantly hypertrophied and produced significantly more von Willebrand factor. Endothelial apoptosis increased significantly. Platelet-endothelial interactions decreased significantly. Endothelial-independent contraction decreased significantly, whereas endothelial-dependent relaxation increased modestly. Assisted circulation with a left ventricular assist device triggered arterial remodeling that allowed a peripheral artery to accommodate the altered hemodynamics of a novel partial-support pump. Further delineation of remodeling pathways may be of significance for the emerging field of partial circulatory support.

Pro-inflammatory stimulation of meniscus cells increases production of matrix metalloproteinases and additional catabolic factors involved in osteoarthritis pathogenesis

Meniscus injury increases the risk of osteoarthritis; however, the biologic mechanism remains unknown. We hypothesized that pro-inflammatory stimulation of meniscus would increase production of matrix-degrading enzymes, cytokines and chemokines which cause joint tissue destruction and could contribute to osteoarthritis development. Meniscus and cartilage tissue from healthy tissue donors and total knee arthroplasties (TKAs) was cultured. Primary cell cultures were stimulated with pro-inflammatory factors [IL-1β, IL-6, or fibronectin fragments (FnF)] and cellular responses were analyzed by real-time PCR, protein arrays and immunoblots. To determine if NF-κB was required for MMP production, meniscus cultures were treated with inflammatory factors with and without the NF-κB inhibitor, hypoestoxide. Normal and osteoarthritic meniscus cells increased their MMP secretion in response to stimulation, but specific patterns emerged that were unique to each stimulus with the greatest number of MMPs expressed in response to FnF. Meniscus collagen and connective tissue growth factor (CTGF) gene expression was reduced. Expression of cytokines (IL-1α, IL-1β, IL-6), chemokines (IL-8, CXCL1, CXCL2, CSF1) and components of the NF-κB and tumor necrosis factor (TNF) family were significantly increased. Cytokine and chemokine protein production was also increased by stimulation. When primary cell cultures were treated with hypoestoxide in conjunction with pro-inflammatory stimulation, p65 activation was reduced as were MMP-1 and MMP-3 production. Pro-inflammatory stimulation of meniscus cells increased matrix metalloproteinase production and catabolic gene expression. The meniscus could have an active biologic role in osteoarthritis development following joint injury through increased production of cytokines, chemokines, and matrix-degrading enzymes.

Regulation of Connective Tissue Growth Factor Gene Expression and Fibrosis in Human Heart Failure

BackgroundHeart failure (HF) is associated with excessive extracellular matrix (ECM) deposition and abnormal ECM degradation leading to cardiac fibrosis. Connective tissue growth factor (CTGF) modulates ECM production during inflammatory tissue injury, but available data on CTGF gene expression in failing human heart and its response to mechanical unloading are limited. Methods and ResultsLeft ventricle (LV) tissue from patients undergoing cardiac transplantation for ischemic (ICM; n = 20) and dilated (DCM; n = 20) cardiomyopathies and from nonfailing (NF; n = 20) donor hearts were examined. Paired samples (n = 15) from patients undergoing LV assist device (LVAD) implantation as “bridge to transplant” (34–1,145 days) also were analyzed. There was more interstitial fibrosis in both ICM and DCM compared with NF hearts. Hydroxyproline concentration was also significantly increased in DCM compared with NF samples. The expression of CTGF, transforming growth factor (TGF) β1, collagen (COL) 1-α1, COL3-α1, matrix metalloproteinase (MMP) 2, and MMP9 mRNA in ICM and DCM were also significantly elevated compared with NF samples. Although TGF-β1, CTGF, COL1-α1, and COL3-α1 mRNA levels were reduced by unloading, there was only a modest reduction in tissue fibrosis and no difference in protein-bound hydroxyproline concentration between pre- and post-LVAD tissue samples. The persistent fibrosis may be related to a concomitant reduction in MMP9 mRNA and protein levels following unloading. ConclusionsCTGF may be a key regulator of fibrosis during maladaptive remodeling and progression to HF. Although mechanical unloading normalizes most genotypic and functional abnormalities, its effect on ECM remodeling during HF is incomplete.

Ciprofloxacin has antifibrotic effects in scleroderma fibroblasts via downregulation of Dnmt1 and upregulation of Fli1

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

Connective tissue growth factor gene expression and decline in renal function in lupus nephritis

In lupus nephritis (LN), kidney inflammation may be followed by fibrosis and progressive decline in function. Transforming growth factor (TGF)-β is a notable mediator of fibrosis, but it has other beneficial roles, thus indicating a need for alternate therapeutic targets for inhibition of fibrosis. Connective tissue growth factor (CTGF) acts as a downstream mediator of TGF-β in promoting fibrosis, without mediating the immunosuppressive effects of TGF-β. Animal studies show that CTGF may have important roles in renal fibrosis, but data are limited in human subjects. The present study tested the hypothesis that renal CTGF mRNA expression is related to TGF-β1 and collagen I expression and is predictive of renal function deterioration in patients with LN (n=39). Gene expression was measured using multiplex real-time quantitative RT-PCR and renal function was estimated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) glomerular filtration rate (GFR) equation. Decline in GFR was assessed by regression of GFR at biopsy to 1 year following biopsy. CTGF mRNA expression was significantly correlated with TGF-β1 and collagen I. GFR at biopsy was 89.2±39.2 ml/ min. Renal CTGF mRNA expression correlated inversely with baseline GFR. Renal CTGF mRNA was significantly higher in patients with moderate to severe CKD compared to those in the milder CKD group (low GFR 4.92±4.34 vs. high GFR 1.52±1.94, p<0.005). CTGF mRNA was also higher in patients with subsequent decline in GFR [GFR decline (5.19±4.46) vs. no GFR decline (1.79±1.97); P<0.01]. In conclusion, renal expression of CTGF was positively related to TGF-β1 and collagen I in patients with LN. Furthermore, high CTGF mRNA expression was associated with poor GFR at baseline and subsequent deterioration of kidney function. CTGF expression in the kidney may serve as an early marker for renal disease progression and could be evaluated as a target for therapeutic intervention to prevent renal failure in LN.

HMG-CoA Reductase Inhibitors (Statins) Reduce Hypertrophic Scar Formation in a Rabbit Ear Wounding Model

3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, also known as statins, are the most commonly prescribed cholesterol-lowering medications in the world. Statins have been shown to inhibit connective tissue growth factor (CTGF) gene and protein expression in vitro, and previous studies in the authors' laboratory have demonstrated that CTGF plays a significant role in wound healing and scarring. The authors explore whether administration of various statins to healing wounds has any effect on hypertrophic scar formation using an established rabbit ear wounding model. Punch wounds were made on the ears of 31 rabbits (n = 372 total wounds). Treatment wounds were injected with simvastatin, lovastatin, or pravastatin at low, medium, or high doses on postwounding days 15, 20, and 25, whereas control wounds were injected in a corresponding fashion. Wounds were harvested after 35 days for histomorphometric analysis. Low-dose (40 μM) simvastatin, lovastatin, and pravastatin each demonstrated significant reductions in scar elevation index when compared with control: 21.9 percent (p = 0.03), 25.8 percent (p = 0.02), and 22.8 percent (p = 0.01), respectively. There were no significant differences in scar elevation index in the medium- (120 μM) and high-dose (400 μM) statin groups. Analysis of mRNA in a subset of rabbits treated with low-dose simvastatin demonstrated a significant reduction in CTGF expression (p = 0.009). 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors reduce hypertrophic scar formation by means of CTGF inhibition when administered at low doses, and the novel application of these commonly prescribed medications may lead to innovative and effective antiscarring therapies.

Runx2/Smad3 Complex Negatively Regulates TGF-β-Induced Connective Tissue Growth Factor gene Expression in Vascular Smooth Muscle Cells

Connective tissue growth factor (CTGF), a direct target gene of transforming growth factor-β (TGF-β) signaling, plays an important role in the development of atherosclerosis. We previously showed that Runx2, a key transcription factor in osteoblast differentiation, regulates osteogenic conversion and dedifferentiation of vascular smooth muscle cells (VSMCs). In this study, we investigated the hypothesis that Runx2 modulates CTGF gene expression via the regulation of TGF-β signaling. Expression of the Runx2 gene was decreased, and CTGF mRNA levels were reciprocally increased by TGF-β in a time-dependent manner in cultured human aortic smooth muscle cells (HASMCs) and C3H10T1/2 cells. Forced expression of Runx2 decreased and the reduction of Runx2 expression by small interfering RNA enhanced both basal and TGF-β-stimulated CTGF gene expression in HASMCs. Site-directed mutation analysis of the CTGF promoter indicated that transcriptional repression by Runx2 was mediated by the Smad-binding element (SBE) under basal and TGF-β-stimulated conditions. Data obtained from immunoblots of Runx2-, Smad3- or Smad4-transfected cells and chromatin immunoprecipitation analysis indicated that Runx2 interacts with Smad3 at the SBE. Immunohistochemistry revealed that the expression of Runx2 and CTGF was distinct and almost mutually exclusive in human atherosclerotic plaque. These results for the first time demonstrate that Runx2/Smad3 complex negatively regulates endogenous and TGF-β-induced CTGF gene expression in VSMCs. Thus, the induction of Runx2 expression contributes to the phenotypic modulation of VSMCs, in which the TGF-β/Smad pathway plays a major role.

Diastolic dysfunction is associated with cardiac fibrosis in the senescence-accelerated mouse

Diastolic heart failure is a major cause of mortality in the elderly population. It is often preceded by diastolic dysfunction, which is characterized by impaired active relaxation and increased stiffness. We tested the hypothesis that senescence-prone (SAMP8) mice would develop diastolic dysfunction compared with senescence-resistant controls (SAMR1). Pulsed-wave Doppler imaging of the ratio of blood flow velocity through the mitral valve during early (E) vs. late (A) diastole was reduced from 1.3 ± 0.03 in SAMR1 mice to 1.2 ± 0.03 in SAMP8 mice (P < 0.05). Tissue Doppler imaging of the early (E') and late (A') diastolic mitral annulus velocities found E' reduced from 25.7 ± 0.9 mm/s in SAMR1 to 21.1 ± 0.8 mm/s in SAMP8 mice and E'/A' similarly reduced from 1.1 ± 0.02 to 0.8 ± 0.03 in SAMR1 vs. SAMP8 mice, respectively (P < 0.05). Invasive hemodynamics revealed an increased slope of the end-diastolic pressure-volume relationship (0.5 ± 0.05 vs. 0.8 ± 0.14; P < 0.05), indicating increased left ventricular chamber stiffness. There were no differences in systolic function or mean arterial pressure; however, diastolic dysfunction was accompanied by increased fibrosis in the hearts of SAMP8 mice. In SAMR1 vs. SAMP8 mice, interstitial collagen area increased from 0.3 ± 0.04 to 0.8 ± 0.09% and perivascular collagen area increased from 1.0 ± 0.11 to 1.6 ± 0.14%. Transforming growth factor-β and connective tissue growth factor gene expression were increased in the hearts of SAMP8 mice (P < 0.05 for all data). In summary, SAMP8 mice show increased fibrosis and diastolic dysfunction similar to those seen in humans with aging and may represent a suitable model for future mechanistic studies.

Sildenafil Promotes Smooth Muscle Preservation and Ameliorates Fibrosis Through Modulation of Extracellular Matrix and Tissue Growth Factor Gene Expression After Bilateral Cavernosal Nerve Resection in the Rat

It has been shown that phosphodiesterase type 5 (PDE5) inhibitors preserve smooth muscle (SM) content and ameliorate the fibrotic degeneration normally seen in the corpora cavernosa after bilateral cavernosal nerve resection (BCNR). However, the downstream mechanisms by which these drugs protect the corpora cavernosa remain poorly understood. To provide insight into the mechanism, we aimed to determine the gene expression profile of angiogenesis-related pathways within the penile tissue after BCNR with or without continuous sildenafil (SIL) treatment. Five-month-old Fisher rats were subjected to BCNR or sham operation and treated with or without SIL (20 mg/kg/BW drinking water) for 3 days or 45 days (N = 8 rats per group). Total RNAs isolated from the denuded penile shaft and prostate were subjected to reverse transcription and to angiogenesis real-time-polymerase chain reaction arrays (84 genes). Changes in protein expression of selected genes such as epiregulin (EREG) and connective tissue growth factor (CTGF) were corroborated by Western blot and immunohistochemistry. Genes modulated by BCNR and SIL treatment. A decreased expression of genes related to SM growth factors such as EREG, platelet-derived growth factor (PDGF), extracellular matrix regulators such as metalloproteinases 3 and 9, endothelial growth factors, together with an upregulation of pro-fibrotic genes such as CTGF and transforming growth factor beta 2 were found at both time points after BCNR. SIL treatment reversed this process by upregulating endothelial and SM growth factors and downregulating pro-fibrotic factors. SIL did not affect the expression of EREG, VEGF, and PDGF in the ventral prostate of BCNR animals. SIL treatment after BCNR activates genes related to SM preservation and downregulates genes related to fibrosis in the corpora cavernosa. These results provide a mechanistic justification for the use of SIL and other PDE5 inhibitors as protective therapy against corporal SM loss and fibrosis after radical prostatectomy.

A mouse strain where basal connective tissue growth factor gene expression can be switched from low to high.

Connective tissue growth factor (CTGF) is a signaling molecule that primarily functions in extracellular matrix maintenance and repair. Increased Ctgf expression is associated with fibrosis in chronic organ injury. Studying the role of CTGF in fibrotic disease in vivo, however, has been hampered by perinatal lethality of the Ctgf null mice as well as the limited scope of previous mouse models of Ctgf overproduction. Here, we devised a new approach and engineered a single mutant mouse strain where the endogenous Ctgf-3′ untranslated region (3′UTR) was replaced with a cassette containing two 3′UTR sequences arranged in tandem. The modified Ctgf allele uses a 3′UTR from the mouse FBJ osteosarcoma oncogene (c-Fos) and produces an unstable mRNA, resulting in 60% of normal Ctgf expression (Lo allele). Upon Cre-expression, excision of the c-Fos-3′UTR creates a transcript utilizing the more stable bovine growth hormone (bGH) 3′UTR, resulting in increased Ctgf expression (Hi allele). Using the Ctgf Lo and Hi mutants, and crosses to a Ctgf knockout or Cre-expressing mice, we have generated a series of strains with a 30-fold range of Ctgf expression. Mice with the lowest Ctgf expression, 30% of normal, appear healthy, while a global nine-fold overexpression of Ctgf causes abnormalities, including developmental delay and craniofacial defects, and embryonic death at E10-12. Overexpression of Ctgf by tamoxifen-inducible Cre in the postnatal life, on the other hand, is compatible with life. The Ctgf Lo-Hi mutant mice should prove useful in further understanding the function of CTGF in fibrotic diseases. Additionally, this method can be used for the production of mouse lines with quantitative variations in other genes, particularly with genes that are broadly expressed, have distinct functions in different tissues, or where altered gene expression is not compatible with normal development.

Effects of small interfering RNA approach to silence the connective tissue growth factor gene on the expression of the protein p27

To investigate the effects of the small interfering RNA (siRNA) approach to silence the expression of connective tissue growth factor (CTGF) gene expression on the expression of the protein p27. Experimental research. The specific siRNA of CTGF was designed and synthesized, then transfected into cultured bovine corneal endothelial cells by liposomes. The blank control group was only treated with the culture medium and not be transfected with siRNA. Reverse transfection-polymerase chain reaction (RT-PCR) was applied to show the effect of gene-silencing, and western blot was used to observe the protein p27 expression of bovine corneal endothelial cells, which were treated with 1.00 µg/L transforming growth factor β(2) (TGF-β(2)). The results were analyzed by one-factor analysis of variance. CTGF siRNA was successfully transfected into cultured bovine corneal endothelial cells and effectively silence the expression of CTGF mRNA. The CTGF mRNA expression decreased to 17.3%, 24.4% and 41.7% of control values at 24, 48 and 72 h after transfection, there was significant difference between these groups (F = 389.9, P < 0.05). Significant decrease of protein p27 expression was detected at 36 h after transfection (F = 299.3, P < 0.05). CTGF-specific siRNA was able to inhibit the expression of CTGF mRNA effectively and down-regulate the expression of p27.