Connective Tissue Growth Factor Gene Expression Research Articles

Impact of umbelliprenin-containing niosome nanoparticles on VEGF-A and CTGF genes expression in retinal pigment epithelium cells.

To investigate the impact of niosome nanoparticles carrying umbelliprenin (UMB), an anti-angiogenic and anti-inflammatory plant compound, on the expression of vascular endothelial growth factor (VEGF-A) and connective tissue growth factor (CTGF) genes in a human retinal pigment epithelium (RPE)-like retina-derived cell line. UMB-containing niosomes were created, optimized, and characterized. RPE-like cells were treated with free UMB and UMB-containing niosomes. The IC50 values of the treatments were determined using an MTT assay. Gene expression of VEGF-A and CTGF was evaluated using real-time polymerase chain reaction after RNA extraction and cDNA synthesis. Niosomes' characteristics, including drug entrapment efficiency, size, dispersion index, and zeta potential were assessed. Free UMB had an IC50 of 96.2 µg/mL, while UMB-containing niosomes had an IC50 of 25 µg/mL. Treatment with UMB-containing niosomes and free UMB resulted in a significant reduction in VEGF-A expression compared to control cells (P=0.001). Additionally, UMB-containing niosomes demonstrated a significant reduction in CTGF expression compared to control cells (P=0.05). However, there was no significant reduction in the expression of both genes in cells treated with free UMB. Both free UMB and niosome-encapsulated UMB inhibits VEGF-A and CTGF genes expression. However, the latter demonstrates significantly greater efficacy, potentially due to the lower UMB dosage and gradual delivery. These findings have implications for anti-angiogenesis therapeutic approaches targeting age-related macular degeneration.

Allium jesdianum hydro alcoholic extract ameliorates diabetic nephropathy by suppressing connective tissue growth factor (CTGF) and receptor for advanced glycation endproducts (RAGE) gene expression in diabetic rats with streptozotocin.

Diabetic nephropathy is one of the major complications of diabetes, the use of medicinal plants is increasing due to fewer side effects. This study was designed to examine antidiabetic effects of Allium jesdianum (A.jesdianum) ethanolic extract and evaluate its effects on oxidative stress markers and the expression of connective tissue growth factor (CTGF) and receptor for advanced glycation endproducts (RAGE) genes in the kidney of type 1 diabetic rats. In this study, we randomly divided 24 rats into four groups with six rats in each group as follows: Cnt group: normal control receiving normal saline, Dibt group: diabetic control receiving normal saline daily, Dibt+A.jesdianum 250 group: diabetic rats receiving A.jesdianum at a dose of 250mg/kg bw daily, Dibt+A.jesdianum 500 group: diabetic rats receiving A.jesdianum at a dose of 500mg/kgbw daily. To induce diabetes, we used 55mg/kgbw dose of streptozotocin intraperitoneally. The concentration of fasting blood glucose (FBG) and serum urea, creatinine and albumin, SOD, MDA (using spectrophotometric methods) and gene expression of CTGF and RAGE in kidney tissue (using real-time PCR methods) were quantified in the diabetic rats that received A.jesdianum for 42days, and were compared to control rats. The results showed that in the diabetic group the FBG and serum urea, creatinine and expression of kidney CTGF and RAGE genes and the levels of SOD and MDA significantly increased and serum albumin significantly decreased compared to the Cnt group (p<0.001). Administration of A.jesdianum significantly improved the FBG and serum urea, creatinine and albumin compared to Dibt group (p<0.05). It was shown the A.jesdianum significantly decrease the kidney expression levels of CTGF and RAGE genes and improve oxidative stress (increased SOD and decreased MDA) in the kidney tissues when compared to Dibt group (p<0.001). Also, it was found that the beneficial effects of the A.jesdianum were dose-dependent. The results of this study showed that administration of A.jesdianum for 42days has beneficial anti-diabetic and anti-nephropathic effects in diabetic rats and can be used as an adjunct therapy in the treatment of diabetes.

Cultured Neonatal Rat Cardiomyocytes Continue Beating Through Upregulation of CTGF Gene Expression.

Some cultured neonatal rat cardiomyocytes continue spontaneous beating even in serum-free medium. The present study explored the cause and genes responsible for this phenomenon. Ingenuity Pathway Analysis (IPA) software was used to analyze fold changes in gene expression in beating neonatal rat cardiomyocytes, as compared with non-beating cardiomyocytes, which were obtained from DNA microarray data of total RNA extracts of cardiomyocytes. To confirm the involvement of the 8 genes selected by IPA prediction, cellular protein abundances were determined by Western blot. The gene expression of connective tissue growth factor (CTGF) was substantially higher in beating cardiomyocytes than in non-beating cardiomyocytes; thus, CTGF protein content released from cardiomyocytes into the culture medium was examined. IPA showed that the "Apelin Cardiac Fibroblast Signaling Pathway" was significantly inhibited and that microtubule dynamics and cytoskeleton organization were significantly activated. Each fluctuation in the cellular abundances of the 8 proteins in beating cardiomyocytes, as compared with non-beating cardiomyocytes, was primarily in the same direction as that of gene expression. However, the cellular CTGF protein abundance as well as CTGF content released into the medium did not substantially differ between beating and non-beating cardiomyocytes. The present results suggest that the large increase in CTGF gene expression in beating cardiomyocytes is not a cause but a result of beating, which may provide a putative pathway for controlling beating. Beating is sustained by developed cardiomyofibrils and directly upregulates CTGF gene expression, which is not followed by CTGF protein synthesis.

Intratracheal budesonide/surfactant attenuates hyperoxia-induced lung injury in preterm rabbits.

Recent clinical trials have shown improvements in neonatal outcomes after intratracheal administration of a combination of budesonide/surfactant (ITBS) in infants at risk of bronchopulmonary dysplasia. However, the effect of ITBS on lung function and alveolar structure is not known. We aimed to determine the effect of ITBS on lung function, parenchymal structure, and inflammatory cytokine expression in a relevant preterm animal model for bronchopulmonary dysplasia. Premature neonatal rabbits were administered a single dose of ITBS on the day of delivery and exposed to 95% oxygen. Following 7 days of hyperoxia, in vivo forced oscillation and pressure-volume maneuvers were performed to examine pulmonary function. Histological and molecular analysis was performed to assess alveolar and extracellular matrix (ECM) morphology, along with gene expression of connective tissue growth factor (CTGF), IL-8, and CCL-2. ITBS attenuated the functional effect of hyperoxia-induced lung injury and limited the change to respiratory system impedance, measured using the forced oscillation technique. Treatment effects were most obvious in the small airways, with significant effects on small airway resistance and small airway reactance. In addition, ITBS mitigated the decrease in inspiratory capacity and static compliance. ITBS restricted alveolar septal thickening without altering the mean linear intercept and mitigated hyperoxia-induced remodeling of the ECM. These structural changes were associated with improved inspiratory capacity and lung compliance. Gene expression of CTGF, IL-8, and CCL-2 was significantly downregulated in the lung. Treatment with ITBS shortly after delivery attenuated the functional and structural consequences of hyperoxia-induced lung injury to day 7 of life in the preterm rabbit.

Mitochondrial-Targeted Antioxidants Attenuate TGF-β2 Signaling in Human Trabecular Meshwork Cells.

POAG is a progressive optic neuropathy that is currently the leading cause of irreversible blindness worldwide. While the underlying cause of POAG remains unclear, TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment is considered an early pathologic consequence associated with impaired aqueous humor (AH) outflow and elevated IOP. Early studies have also demonstrated markedly elevated levels of oxidative stress markers in AH from POAG patients along with altered expression of antioxidant defenses. Here, using cultured primary or transformed human TM cells, we investigated the role oxidative stress plays at regulating TGF-β2-mediated remodeling of the ECM. Primary or transformed (GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-β2 and relative changes in intracellular reactive oxygen species (ROS) were measured using oxidation-sensitive fluorogenic dyes CellROX green or 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). TGF-β2-mediated changes in the content of connective tissue growth factor (CTGF) and collagen types 1α1 (COL1A1) and 4α1 (COL4A1) mRNA or collagens I and IV isoform proteins were determined in the absence or presence of mitochondrial-targeted antioxidants (XJB-5-131 or MitoQ) and quantified by quantitative PCR or by immunoblot and immunocytochemistry. Smad-dependent canonic signaling was determined by immunoblot, whereas Smad-dependent transcriptional activity was quantified using a Smad2/3-responsive SBE-luciferase reporter assay. Primary or transformed human TM cells cultured in the presence of TGF-β2 (5 ng/mL; 2 hours) exhibited marked increases in CellROX or fluorescein fluorescence. Consistent with previous reports, challenging cultured human TM cells with TGF-β2 elicited measurable increases in regulated Smad2/3 signaling as well as increases in CTGF, COL1A1, and COL4A1 mRNA and collagen protein content. Pretreating human TM cells with mitochondrial-targeted antioxidants XJB-5-131 (10 μM) or MitoQ (10 nM) attenuated TGF-β2-mediated changes in Smad-dependent transcriptional activity. The multifunctional profibrotic cytokine TGF-β2 elicits a marked increase in oxidative stress in human TM cells. Mitochondrial-targeted antioxidants attenuate TGF-β2-mediated changes in Smad-dependent transcriptional activity, including marked reductions in CTGF and collagen isoform gene and protein expression. These findings suggest that mitochondrial-targeted antioxidants, when delivered directly to the TM, exhibit potential as a novel strategy by which to slow the progression of TGF-β2-mediated remodeling of the ECM within the TM.

Connective tissue growth factor (CTGF) methylation status is associated with prognosis of patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy (RCHT): A multicenter study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).

6050 Background: CTGF plays a central role in tissue remodeling and has emerged as an attractive novel therapeutic target. We sought to investigate the impact of CTGF methylation status (CTGF-M) in predicting outcome of HNSCC patients undergoing RCHT. Methods: CTGF-M was discovered by applying Illumina 450K/850K arrays to DNA extracted from FFPE material of patients homogeneously treated with surgery followed by adjuvant cisplatin-based RCHT in frame of the DKTK-ROG multicenter retrospective trial (n = 194, training cohort). Methylation probes correlating with overall survival (OS) and progression were identified using random forest. Validation was done in 4 cohorts including DKTK-ROG definitive RCHT (n = 110) and 3 RCHT cohorts from Heidelberg, Dresden and Munich (n = 222). CTGF-M and RNAseq data from The Cancer Genome Atlas (TCGA) (n = 206) were analyzed to identify differentially expressed genes (DEG) as a function of CTGF-M. Results: Increased methylation of 2 probes (mapping to CTGF 3’UTR and gene body) was associated with significantly improved OS in the training cohort (HR = 0.51, p = 0.044) and the validation cohort (HR = 0.67, p = 0.016), in multivariate cox regressions adjusting for HPV status, age, T and N stages, location and treatment (adjuvant vs definitive RCHT). In the TCGA, probes’ methylation was significantly inversely correlated with CTGF gene expression (r = -0.18 and -0.51, p &lt; 0.05). 1843 DEGs were found at FDR &lt; 0.05 as a function of CTGF-M. Pathways mapping to tissue remodeling were significantly enriched for among downregulated genes in CTGF hypermethylated tumors. Increased CTGF methylation was inversely correlated with the mesenchymal subtype mRNA gene signature (r = -0.21, p = 0.0026) and correlated with the atypical subtype (r = 0.32, p = 0.000002). Conclusions: Implementation of CTGF-M in routine diagnostic is feasible and correlates well with CTGF gene-expression levels and activation of tissue remodeling pathways. Therefore, CTGF-M might be instructive for stratifying HNSCC patients for CTGF targeting therapies. CTGF emerges as a promising prognostic marker independently of HPV status.

Adipose Tissue CTGF Expression is Associated with Adiposity and Insulin Resistance in Humans.

Connective tissue growth factor (CTGF) is an important regulator of fibrogenesis in many organs. This study evaluated the interrelationship among adipose tissue CTGF expression, fat mass, and insulin resistance in humans. This study examined (1) CTGF gene expression in human subcutaneous preadipocytes before and after inducing adipogenesis; (2) relationships among abdominal subcutaneous adipose tissue CTGF gene expression, body fat mass, and indices of insulin sensitivity, including the hepatic insulin sensitivity index and the hyperinsulinemic-euglycemic clamp procedure in conjunction with stable isotope glucose tracer infusion, in 72 people who had marked differences in adiposity and insulin sensitivity; (3) localization of CTGF protein in subcutaneous adipose tissue; and (4) effect of progressive (5%, 11%, and 16%) weight loss on adipose tissue CTGF gene expression. CTGF was highly expressed in preadipocytes, not adipocytes. Adipose tissue CTGF expression was strongly correlated with body fat mass and both skeletal muscle and liver insulin sensitivity, and CTGF-positive cells were predominantly found in areas of fibrosis. Progressive weight loss caused a stepwise decrease in adipose tissue CTGF expression. It was concluded that increased CTGF expression is associated with adipose tissue expansion, adipose tissue fibrosis, and multi-organ insulin resistance in people with obesity.

PTGFR activation promotes the expression of PTGS-2 and growth factors via activation of the PKC signaling pathway in bovine endometrial epithelial cells

The endometrium of domestic animals has a remarkable capacity to self-repair. Prostaglandin F2α (PGF2α) is one of the major prostaglandins secreted from the endometrium. The role of PGF2α in endometrial repair, however, is still unknown. In the present study, it was investigated whether prostaglandin F2α receptor (PTGFR) activation could induce expression of prostaglandin-endoperoxide synthase 2 (PTGS-2) and growth factors associated with endometrial repair via activation of protein kinase C (PKC) signaling in endometrial epithelial cells (bEECs) of cattle. Results of the present study indicated that the treatment with the PTGFR agonist, fluprostenol, resulted in an increase in abundance of proteins for PTGS-2, vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), transforming growth factor beta 1 (TGF-β1), and interleukin-8 (IL-8). The increased abundances of these proteins were suppressed by the treatment with the PTGFR antagonist, AL8810.Furthermore, fluprostenol treatment also induced PKC phosphorylation. Subsequently, treatment with AL8810 inhibited the fluprostenol-induced PKC phosphorylation. Additionally, treatment with the PKC inhibitor, chelerythrine, reduced the fluprostenol-induced increase in the relative abundance of VEGF, CTGF, TGF-β1, and IL-8 mRNA in bEECs. Taken together, these results suggest that PTGFR activation may induce endometrial repair by upregulating PTGS-2 gene expression and stimulating VEGF, CTGF, TGF-β1, and IL-8 gene expression via activation of the PKC signaling pathway.

Effects of connective tissue growth factor on prostate cancer bone metastasis and osteoblast differentiation.

Previous studies have demonstrated that connective tissue growth factor (CTGF) is expressed at increased levels in prostate cancer bone metastasis mouse models and patients with prostate cancer which metastasizes to the bone; however, the underlying molecular mechanism(s) remain unknown. The present study investigated the function of CTGF in osteoblast differentiation and its effect on prostate cancer bone metastasis by analyzing CTGF gene expression and transcription at different levels of invasion, metastasis of prostate cancer cells, and the influence of CTGF on proliferation and xenotransplantation. A mouse model demonstrating bone metastasis was used to investigate the function(s) of CTGF in bone metastasis and osteoblast differentiation. Results demonstrated that CTGF expression was increased in association with high bone metastasis in prostate cancer cells, and its expression was significantly decreased in whole cell lysates. CTGF expression in prostate cancer cells with high levels of bone metastasis was increased 1.9-fold compared with prostate cancer cells with low levels of bone metastasis. The expression of CTGF in mesenchymal cells was markedly increased compared with epithelial cells. Results indicated that the increased expression of CTGF does not affect the proliferation of tumor cells and possesses no influence on tumor volume. Control and CTGF plasmids were transfected into RM1 cells and led to 4 and 17% bone lesions, respectively. Increased expression of CTGF significantly enlarged the tumor area in the bone metastatic position compared with the control. Positive areas of alkaline phosphatase were significantly decreased as the concentration of CTGF increased. The results of the present study demonstrated that CTGF promotes prostate carcinoma to metastasize in the bone by dysregulating osteoblast differentiation.

Unique mechanisms of connective tissue growth factor regulation in airway smooth muscle in asthma: Relationship with airway remodelling.

Neovascularization, increased basal membrane thickness and increased airway smooth muscle (ASM) bulk are hallmarks of airway remodelling in asthma. In this study, we examined connective tissue growth factor (CTGF) dysregulation in human lung tissue and animal models of allergic airway disease. Immunohistochemistry revealed that ASM cells from patients with severe asthma (A) exhibited high expression of CTGF, compared to mild and non‐asthmatic (NA) tissues. This finding was replicated in a sheep model of allergic airways disease. In vitro, transforming growth factor (TGF)‐β increased CTGF expression both in NA‐ and A‐ASM cells but the expression was higher in A‐ASM at both the mRNA and protein level as assessed by PCR and Western blot. Transfection of CTGF promoter‐luciferase reporter constructs into NA‐ and A‐ASM cells indicated that no region of the CTGF promoter (−1500 to +200 bp) displayed enhanced activity in the presence of TGF‐β. However, in silico analysis of the CTGF promoter suggested that distant transcription factor binding sites may influence CTGF promoter activation by TGF‐β in ASM cells. The discord between promoter activity and mRNA expression was also explained, in part, by differential post‐transcriptional regulation in A‐ASM cells due to enhanced mRNA stability for CTGF. In patients, higher CTGF gene expression in bronchial biopsies was correlated with increased basement membrane thickness indicating that the enhanced CTGF expression in A‐ASM may contribute to airway remodelling in asthma.

Connective tissue growth factor dependent collagen gene expression induced by MAS agonist AR234960 in human cardiac fibroblasts.

Perspectives on whether the functions of MAS, a G protein-coupled receptor, are beneficial or deleterious in the heart remain controversial. MAS gene knockout reduces coronary vasodilatation leading to ischemic injury. G protein signaling activated by MAS has been implicated in progression of adaptive cardiac hypertrophy to heart failure and fibrosis. In the present study, we observed increased expression of MAS, connective tissue growth factor (CTGF) and collagen genes in failing (HF) human heart samples when compared to non-failing (NF). Expression levels of MAS are correlated with CTGF in HF and NF leading to our hypothesis that MAS controls CTGF production and the ensuing expression of collagen genes. In support of this hypothesis we show that the non-peptide MAS agonist AR234960 increases both mRNA and protein levels of CTGF via ERK1/2 signaling in HEK293-MAS cells and adult human cardiac fibroblasts. MAS-mediated CTGF expression can be specifically blocked by MAS inverse agonist AR244555 and also by MEK1 inhibition. Expression of CTGF gene was essential for MAS-mediated up-regulation of different collagen subtype genes in HEK293-MAS cells and human cardiac fibroblasts. Knockdown of CTGF by RNAi disrupted collagen gene regulation by the MAS-agonist. Our data indicate that CTGF mediates the profibrotic effects of MAS in cardiac fibroblasts. Blocking MAS-CTGF-collagen pathway should be considered for pharmacological intervention for HF.

HIF-1α mediates visfatin-induced CTGF expression in vascular endothelial cells.

Visfatin is an adipokine that functions as a mediator of endothelial dysfunction and cardiovascular diseases. Connective tissue growth factor (CTGF) is a key factor in vascular remodeling and atherosclerosis. However, the association between visfatin and CTGF is unclear. Therefore the study was to test the hypothesis that visfatin could modulate the expression of CTGF in vascular endothelial cells. In our study, cultured endothelial cell line EA.Hy926 cells were treated with different concentrations of visfatin for different times. The CTGF gene expression was analyzed by real-time PCR, and the protein expression of CTGF was assessed by Western Blot. The results showed that 100ng/mL concentration of visfatin could induce CTGF mRNA expression after 6 hours treatment, which peaked at 24 hours. And 100ng/mL concentration of visfatin also increased CTGF protein production after 12 hours treatment in EA.Hy926 cells. The expression of transforming growth factor-β1 (TGF-β1) mRNA was almost unaffected in cells treated with visfatin, whereas the expression of hypoxia inducible factor-1α (HIF-1α) was increased significantly. Moreover, knockdown of HIF-1α by its specific shRNA inhibits the effect of visfatin on CTGF expression. In conclusion, the up-regulation of CTGF expression by visfatin might be mediated via HIF-1α -dependent pathway, but not the TGF-β1 pathway in EA.Hy926 cells.

Per2-Mediated Vascular Dysfunction Is Caused by the Upregulation of the Connective Tissue Growth Factor (CTGF).

Period 2-mutant mice (Per2m/m), which possess a circadian dysfunction, recapitulate the retinal vascular phenotype similar to diabetic retinopathy (DR). The vascular dysfunction in Per2m/m is associated with an increase in connective tissue growth factor (CTGF/CCN2). At the molecular level, CTGF gene expression is dependent on the canonical Wnt/β-catenin pathway. The nuclear binding of β-catenin to a transcription factor, lymphoid enhancer binding protein (Lef)/ T-cell factor (TCF/LEF), leads to downstream activation of CTGF. For this study, we hypothesized that the silencing of Per2 results in nuclear translocation and subsequent transactivation of the CTGF gene. To test this hypothesis, we performed immunofluorescence labeling for CTGF in retinal sections from wild-type (WT) and Per2m/m mice. Human retinal endothelial cells (HRECs) were transfected with siRNA for Per2, and the protein expression of CTGF and β-catenin was evaluated. The TCF/LEF luciferase reporter (TOPflash) assay was performed to validate the involvement of β-catenin in the activation of CTGF. Per2m/m retinas exhibited an increased CTGF immunostaining in ganglion cell layer and retinal endothelium. Silencing of Per2 using siRNA resulted in an upregulation of CTGF and β-catenin. The TOPflash assay revealed an increase in luminescence for HRECs transfected with Per2 siRNA. Our studies show that loss of Per2 results in an activation of CTGF via nuclear entry of β-catenin. Our study provides novel insight into the understanding of microvascular dysfunction in Per2m/m mice.

Is there any role of epithelial to mesenchymal transition in the pathogenesis of contrast nephropathy?

Aim: Contrast medium-induced nephropathy is one of the major complications of intravenous contrast medium use. But its pathogenesis is unclear. Epithelial mesenchymal transition (EMT) is defined as the transformation of the primer epithelial cells to mesenchymal cells. EMT in tubular cells might cause tubulointerstitial damage. In this study, we investigated whether or not EMT has a role in radiocontrast-induced nephropathy. Radiocontrast medium might be triggering reversible EMT via serum and glucocorticoid-regulated kinase 1 (SGK 1). We investigated the effect of different concentrations of the contrast agent iopromide on human proximal tubule cell (HK-2) culture by measuring the level of SGK1, snail family zinc finger 1 (SNAIL1), connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL1A1).Methods: We conducted a scratch assay and qPCR. HK-2 cells were cultured in the petri dishes/flasks and starved with serum-free medium. The 40, 20, and 10 mg/mL doses of iopromide were administrated to cells. The scratches were photographed immediately and again at the 20th hour. The levels of gene expression of SGK1, SNAIL1, CTGF, and COL1A1 were measured using the real-time qPCR system at the end of the 24th hour.Results: Iopromide caused the breaking of intercellular connections, the disappearance of the cobblestone appearance of cells, and the migration of cells at the 20th hour in the scratch assay. It also increased the expression of SGK1, SNAIL1, CTGF, and COL1A1 genes.Conclusion: Our study concluded that certain important markers of EMT increase in different concentrations of the contrast agent. High osmolality might trigger EMT. The relationship between contrast agent and EMT has not been defined before. Further in vivo and in vitro studies are required.

Increased Expression of Connective Tissue Growth Factor (CTGF) in Multiple Organs After Exposure of Non-Human Primates (NHP) to Lethal Doses of Radiation.

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

Inhibition of connective tissue growth factor attenuates paraquat-induced lung fibrosis in a human MRC-5 cell line.

Chronic exposure to Paraquat (PQ) may result in progressive pulmonary fibrosis and subsequent chronic obstructive pulmonary malfunction. Connective tissue growth factor (CTGF) has been proposed as a key determinant in the development of lung fibrosis. We investigated thus whether knock down of CTGF can prevent human lung fibroblasts (MRC-5) activation and proliferation with the subsequent inhibition of PQ-induced fibrosis. MRC-5 was transfected with CTGF-siRNAs and exposed to different concentrations of PQ. The siRNA-silencing efficacy was evaluated using western blotting analyses, qRT-PCR and flow cytometry. Next, the viability and migration of MRC-5 was determined. MMP-2, MMP-9, and TIMP-1 accumulation were quantified to evaluate the lung fibrosis exposure to PQ. Over expression of CTGF mRNA was observed in human MRC-5 cell as early as 6 h following PQ stimulation. CTGF gene expression in MRC-5 cells was substantially reduced by RNAi, which significantly suppressed the expression of the lung fibrosis markers such as tissue inhibitor of metalloproteinase-2 (TIMP-2), Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9) that were stimulated by PQ. Inhibition of CTGF expression suppressed impeded the proliferation and migration ability of MRC-5 cells and resulted in cell-extracellular matrix (ECM) protein accumulation in cells. Our results suggest that CTGF promoted the development of PQ-induced lung fibrosis in collaboration with transforming growth factor β1 (TGFβ1). Furthermore, the observed arresting effects of CTGF knock down during this process suggested that CTGF is the potential target site for preventing PQ-induced pulmonary fibrosis. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1620-1626, 2016.

Effect of melatonin on the expression of connective tissue growth factor of fibroblasts from human hypertrophic scar

Objective To investigate the effect of melatonin on the expression of connective tissue growth factor(CTGF)of fibroblasts from human hypertrophic scar. Methods Cultured primary fibroblasts cells by using enzyme digestion technique. Fibroblasts were cultured and incubated with melatonin, Luzindole, melatonin and Luzindole for 48 hours. Fluorescent quantitative polymerase chain reaction assay(FQ- PCR) were used to detect the gene expression of CTGF and α-smooth muscle actin(α- SMA). Immunofluorescence cytochemistry were used to quantify the expression of CTGF and collagen-Ⅰ(COL-Ⅰ). In order to study the extracellular matrix expression of CTGF, COL-Ⅰand COL-Ⅲ, enzyme linked immunosorbent assay(ELISA)was performed. Results The expression of CTGF mRNA in control group, melatonin group, luzindole group and luzindole+ melatonin group was 0.129 4±0.006 1, 0.086 9±0.003 1,0.123 5± 0.008 1,0.123 2±0.009 4(F= 30.130, P 0.05). Compared with control group, melatonin decreased the gene expression of CTGF,α- SMA and extracellular matrix expression of CTGF,COL-Ⅰ, and the effect of melatonin on fibroblasts was singnificantly by luzindole, while melatonin could not affect the expression of COL-Ⅲ. Conclusion Melatonin would down-regulate the gene expression of CTGF, α- SMA and extracellular matrix expression of CTGF, COL-Ⅰof hypertrophic scar through a receptor pathway. Key words: Melatonin; Hypertrophic scar fibroblast; Connective tissue growth factor

Inhibition of local aldosterone by eplerenone reduces renal structural damage in a novel model of chronic cyclosporine A nephrotoxicity.

The fact that mineralocorticoid receptor antagonists reduce structural and functional alterations induced by cyclosporine A (CsA) indicates that aldosterone plays a key role in chronic CsA nephrotoxicity. We and other researchers have reported local renal aldosterone synthesis. To investigate local renal aldosterone's role in chronic CsA nephrotoxicity, we evaluated the effect of eplerenone (Epl) on renal structural damage and renal dysfunction in adrenalectomized (ADX) rats, and assessed whether the therapeutic benefit was associated with reduction of transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), plasminogen activator inhibitor type 1 (PAI-1) and collagen I (COL-I) expression. Male Sprague-Dawley rats fed a normal-sodium diet were divided in four groups: sham-ADX, ADX, CsA, or Epl. Rats in the ADX, CsA and Epl groups were adrenalectomized first. Aldosterone, sodium and potassium levels in serum and urine were measured on the second day. Two weeks later, vehicle (sham-ADX and ADX group), CsA (25mg/kg/d), or CsA and Epl (100 mg/ kg/d) combination was administrated, respectively. After six weeks, urinary protein, creatinine clearance (Ccr), tubulointerstitial fibrosis (TIF), aldosterone level in kidney, and renal aldosterone synthase CYP11B2, COL-I, TGF-β1, CTGF and PAI-1 gene expression levels were determined. On the second day after surgery, adrenalectomized rats showed undetectable aldosterone with natriuresis, hyponatremia, decreased urinary potassium excretion and hyperpotassemia. CsA reduced Ccr, induced urinary proteins and up-regulated COL-I, TGF-β1, CTGF and PAI-1 gene expression with a significant development of TIF. Eplerenone administration prevented TIF and COL-I, TGF-β1 and PAI-1 up-regulation but did not improve renal function. Our results suggest local renal aldosterone is an important mediator of renal injury induced by CsA.

Effects of shRNA-CTGF on the expression of collagen-I in keloid

Objective To explore the effects of recombinant plasmids of pGPU6/GFP/NeoshRNA-CTGF (shRNA-CTGF) on the type Ⅰ collagen (COL-Ⅰ) protein expression in keloid,through RNA interference on connective tissue growth factor (CTGF) in vivo and in vitro.Methods Recombinant plasmids were designed and constructed by specific shRNA-CTGF; After transfeced human keloid fibroblast with shRNA-CTGF in vitro,RT-PCR was used to detect the CTGF mRNA level,and Western blot to detect the secretion of COL-Ⅰ.After transfected the keloid of nude mice with shRNA-CTGF in vivo,RT-PCR was used to detect the CTGF and COL-Ⅰ mRNA level,and Western blot was used to detect the protein expression of COL-Ⅰ.Results Recombinant plasmids of CTGF were successfully constructed,and the CTGF gene expression was significantly decreased in vivo and in vitro by 86.8％ and 54.1 ％,respectively; Down-regulation of CTGF in vitro significantly inhibited the mRNA and protein level of COL-Ⅰ by 76.8％ and 65.6％,respectively; Down-regulation of CTGF in vivo significantly reduced the COL-Ⅰ mRNA and protein level by 52.7％ and 48.0％,respectively.Conclusions CTGF gene expression is successfully down-regulated by the recombinant plasmid of shRNA-CTGF in vivo and in vitro.shRNA-CTGF significantly reduces the COL-Ⅰ protein level in keloid.It implies that CTGF gene is a potential target in the therapy of pathological scar. Key words: Keloid; Connective tissue growth factor (CTGF) ; Fibroblast; RNA interference (RNAi)

The effect of connective tissue growth factor on renal fibrosis and podocyte injury in hypertensive rats

Objective: The aim of this study was to evaluate the roles of podocalyxin (PCX) and connective tissue growth factor (CTGF) in spontaneously hypertensive rats. Methods: Spontaneously hypertensive rats (SHR) and normotensive control Wistar–Kyoto (WKY) rats were divided into groups referred to as SHR 12W, SHR 24W, WKY 12W and WKY 24W. Systolic blood pressure and 24-hour total uric protein were measured every two weeks in the respective groups. CTGF, PCX, alpha-smooth muscle actin (α-SMA) and collagen-III were evaluated via immunohistochemical staining. In addition, CTGF, PCX, and α-SMA gene expression levels were determined by analyzing mRNA levels. Results: More kidney lesions occurred alongside foot processes effacement in SHR 24W rats than in SHR 12W rats. In SHR 12W rats, blood pressure and 24-hour total uric protein level were elevated and continued to increase thereafter. In the SHR 12W and SHR 24W groups, the expression of CTGF, α-SMA and collagen-III was significantly increased. Immunohistochemical staining showed that PCX expression was significantly reduced in the SHR group and CTGF expression was increased. A significant decrease in PCX mRNA and an increase in CTGF mRNA were observed in SHR 24W rats relative to SHR 12W rats. Conclusion: Both the overexpression of CTGF and the loss of podocalyxin reflect renal damage in spontaneously hypertensive rats. CTGF and PCX may be involved in the mechanisms of podocyte injury and apoptosis induced by hypertension.