Expression of transcription factors Slug in the lens epithelial cells undergoing epithelial-mesenchymal transition induced by connective tissue growth factor

AIMTo investigate the expression of transcription factors Slug in human lens epithelial cells (HLECs) undergoing epithelial-mesenchymal transition (EMT) induced by connective tissue growth factor (CTGF).METHODSHLECs were treated with CTGF of different concentrations (20, 50 and 100 ng/mL) or without CTGF (control) for 24h. The morphological changes of HLECs were analysed by microscopy. The expression and cellular localization of Slug was evaluated by immumo-fluorescence. Expressions of Slug, E-cadherin and alpha smooth muscle actin (α-SMA) were further determined by Western blot analysis.RESULTSHLECs showed spidle fibrolasts-like characteristics and loosely connected each other after CTGF treatment. The immuno-fluorescence staining indicated that Slug was localized in the nuclei and its expression was induced by CTGF. The relative expressions of Slug protein were 1.64±0.11, 1.96 ±0.03, 3.12 ±0.10, and 4.08±0.14, respectively, in response to control group and treatment with CTGF of 20, 50 and 100 ng/mL (F=443.86, P<0.01). The increased Slug protein levels were correlated well with up-expression of α-SMA (0.78±0.05, 0.85±0.06, 2.17±0.15, 2.86±0.10; F=449.85, P<0.01) and down-expression of E-cadherin (2.50±0.11, 1.79±0.26, 1.05±0.14, 0.63±0.08; F=101.55, P<0.01).CONCLUSIONTranscription factor Slug may be involved in EMT of HLECs induced by CTGF in vitro.

Research progress on the role of connective tissue growth factor in fibrosis of diabetic retinopathy

Diabetic retinopathy（DR） is one of the most important types of diabetic microangiopathy, which is a specific change of fundus lesions and is one of the most serious complications of diabetes. When DR develops to proliferative DR, the main factors of decreasing vision, and even blindness, include retinal detachment and vitreous hemorrhage caused by contraction of blood vessels by fiber membrane. Recent studies reported that the formation of fiber vascular membrane is closely related to retinal fibrosis. The connective tissue growth factor（CTGF） is a cytokine that is closely related to DR fibrosis. However, its mechanism is poorly understood. This paper summarizes the recent studies about CTGF on DR fibrosis for a comprehensive understanding of the role and mechanism of CTGF in PDR.

Effects of transforming growth factor β2 and connective tissue growth factor on induction of epithelial mesenchymal transition and extracellular matrix synthesis in human lens epithelial cells

AIM:To Investigate the effects of transforming growth factorβ2(TGF-β2)and connective tissue growth factor(CTGF)on transdifferentiation of human lens epithelial cells(HLECs)cultured in vitro and synthesis of extracellular matrix(ECM).METHODS:HLECs were treated with TGF-β2(0,0.5,1.0,5,10μg/L)and CTGF(0,15,30,60,100μg/L)for different times(0,24,48,72h)in vitro and the expression ofα-smooth muscle actin(α-SMA),the main component of the extracellular matrix typeⅠcollagen(Col-1)and fibronectin(Fn)were measured by using real-time polymerase chain reaction(PCR)and western-blot.RESULTS:TGF-β2 and CTGF significantly increased expression ofα-SMA mRNA and protein(P【0.05,P【0.001),Fn mRNA and protein(P【0.001),Col-1 mRNA and protein(P【0.001).TGF-β2 could induce HLECs expression of CTGF mRNA and protein in dosedependent manner(P【0.05,P【0.001).TGF-β2 and CTGF could induce HLECs to expressα-SMA,Fn and Col-1 in time-dependent manner.Each time of TGF-β2and CTGF induced HELCs expression ofα-SMA,Fn,Col-1 mRNA and protein was significant increase compared with control(P【0.05,P【0.001).CONCLUSION:TGF-β2 and CTGF could induce HLECs epithelial mesenchymal transition and ECM synthesis.

Role of Connective Tissue Growth Factor in Extracellular Matrix Degradation in Renal Tubular Epithelial Cells

In order to investigate the effects of connective tissue growth factor （CTGF） antisense oligodeoxynucleotide （ODN） on plasminogen activator inhibitor-1 （PAI-1） expression in renal tubular cells induced by transforming growth factor β1 （TGF-β1） and to explore the role of CTGF in the degradation of renal extracellular matrix （ECM）, a human proximal tubular epithelial cell line （HKC） was cultured in vitro. Cationic lipid-mediated CTGF antisense ODN was transfected into HKC. After HKC were stimulated with TGF-β1 （5 μg/L）, the mRNA level of PAI-1 was detected by RT-PCR. Intracellular PAI-1 protein synthesis was assessed by flow cytometry. The secreted PAI-1 in the media was determined by Western blot. The results showed that TGF-β1 could induce tubular CTGF and PAI-1 mRNA expression. The PAI-1 mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense ODN. CTGF antisense ODN also inhibited intracellular PAI-1 protein synthesis and lowered the levels of PAI-1 protein secreted into the media. It was concluded that CTGF might play a crucial role in the degradation of excessive ECM during tubulointerstitial fibrosis, and blocking the biological effect of CTGF may he a novel way in preventing renal fibrosis.

Expression of Connective Tissue Growth Factor in Renal Tubulointerstitial Fibrosis in Rats and Its Pathogenic Role

Summary： In order to explore the role of connective tissue growth factor （CTGF） in the pathogenesis of renal tubulointerstitial fibrosis, 48 Wistar rats were randomly divided into sham-operated and unilateral ureteral obstruction （UUO） group. On the postoperative day 1, 3, 7 and 14, the rats were killed and the kidneys were removed. The renal tubulointerstitial injury index was evaluated according to the MASSON staining. The mRNA levels of CTGF, transforming growth factor β1 （TGF-β1）. collagen [ （col I ）, and plasminogen activator inhibitor-1 （PAI 1） were detected using rexerse transcriptional-polymerase chain reaction （RT PCR）. Immunohistochemistry was performed to evaluale the protein expression of the above factors, and the relations among them were analyzed. Quantitative expression of CTGF protein in the kidneys was also assessed using Western blot. The results showed that TGF-β1 mRNA level was increased at first day after UUO, followed by a marked elevation of CTGF mRNA level, which began to increase 3 days after UUO （P〈0.01）. With the progression of the disease, the mRNA expression of CTGF, col I and PAI-1 was increased progressively. Immunohistochemistry revealed that the CTGF protein expression was significantly increased in fibrotic areas and tubular epithelial cells 3 days after UUO. On the post-UUO day 7, the protein level of CTGF was positively related to the renal tubulointerstitial injury index （r =0.62, P〈0.01）, the expression of TGF-β1 （r=0.85, P〈0.01）, colI （r=0.78, P〈0.01）, and PAI-1（r=0.76, P〈0.01）. Upon Western blot analysis, CTGF protein expression began to increase 3 days after UUO, and appeared progressively throughout the time course （P〈0.01, as compared with sham-operated group）. It is concluded that CTGF can be induced by TGF-β and mediate various profibrotic actions of this cytokine, such as increasing extracellular matrix （ECM） synthesis and decreasing ECM degradation. The increased expression of CTGF may play a crucial role in the development and progression of tubulointerstitial fibrosis.

Association of Plasma Connective Tissue Growth Factor Levels with Hyperthyroid Heart Disease

Hyperthyroid heart disease(HHD)is one of the most severe complications of overt hyperthyroidism and increases the risk of mortality in affected patients.Early identification of patients at a higher risk of developing HHD can improve clinical outcomes through active surveillance and management.Connective tissue growth factor(CTGF),a secreted extracellular protein,plays a significant role in cardiac remodeling and dysfunction.We aimed to investigate the association between plasma CTGF level and the risk of HHD in this study.A total of 142 overt hyperthyroid patients without HHD and 99 patients with HHD were included.The plasma CTGF levels were measured using ELISA kits.Routine clinical medical data and echocardiography parameters were recorded for analysis.The plasma CTGF level was significantly higher in patients with HHD than in those without HHD(P=0.002).The plasma CTGF level was positively correlated with free triiodothyronin,tryrotropin receptor antibody,troponin I and lactate dehydrogenase levels and the left atrium diameters,right atrium diameters,and right ventricular end-diastolic diameters(all P<0.05).Logistic regression analysis showed that quartiles 3 and 4 of plasma CTGF levels were significantly associated with the increased risk of HHD(crude OR:2.529;95%CI:1.188-5.387).However,after adjustment for the potentially confounding variables,quartile 4 alone was significantly associated with the higher risk of HHD relative to quartile I.Hyperthyroid patients with HHD display higher plasma CTGF levels.Furthermore,CTGF is an independent risk factor for HHD.Therefore,the plasma CTGF level may be a potential biomarker for the risk of HHD.

The Effect of Connective Tissue Growth Factor on Human Renal Tubular Epithelial Cell Transdifferentiation

To investigate the role of connective tissue growth factor (CTGF) in transdifferentiation of human renal tubular epithelial cell (HKC), in vitro cultured HKC cells were divided into 3 groups: negtive control, low dose CTGF-treated group (rh CTGF, 2.5 ng/ml) and high dose CTGF-treated (rhCTGF, 5.0 ng/ml). Then the expression of α-smooth muscle actin (α-SMA) were assessed by indirect immuno-fluorescence, and the percentage of α-SMA positive cells were assessed by flow cytometry. RT-PCR were also performed to examine the mRNA level of α-SMA. Upon the stimulation of different concentrations of rhCTGF, the expression of α-SMA were markedly stronger than that in negative controls. The percentages of α-SMA positive cells were significantly higher in the stimulated groups than that of negative controls (38.9 %, 65.5 % vs 2.4 %, P<0.01) .α-SMA mRNA levels were also up-regulated by the stimulation of rhCTGF (P<0.01). These results suggest that CTGF can promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblast (Myo-F).

Connective tissue growth factor expression hints at aggressive nature of colorectal cancer

BACKGROUND Connective tissue growth factor(CTGF)is a mediator of transforming growth factor-beta signaling and plays a key role in connective tissue remodeling,inflammatory processes and fibrosis in various illnesses including cancer.AIM To investigate the role of CTGF in colorectal cancer(CRC)progression and to compare the CTGF expression with different clinicopathological parameters.METHODS Real-time polymerase chain reaction,immunohistochemistry and Western blotting was performed to evaluate the CTGF expression and the results were statistically analyzed against the clinicopathological variables of patient data using STATA software version 16.RESULTS CTGF expression levels in tumor specimens were significantly higher than their paired normal specimens.The higher protein expression levels showed a significant association with smoking,staging,tumor grade,invasion depth,necrosis of tumor tissue,and both lymphovascular and perineural invasion.As per the cox regression model and classification tree analysis,tumor-nodemetastasis stage and perineural invasion were important predictors for CTGF expression and prognosis of CRC patients.Survival analysis indicated that CTGF overexpression was associated with poorer overall and disease-free survival.CONCLUSION Expression of CTGF was increased in CRC and was linked with poor overall and disease-free survival of CRC patients.These findings support prior observations and thus CTGF may be a possible prognostic marker in CRC.

Activation of PPAR-γ Inhibits Differentiation of Rat Osteoblasts by Reducing Expression of Connective Tissue Growth Factor

Long-term treatment with an agonist of peroxisome proliferator-activated receptor （PPAR）-γ is associated with bone fractures in the clinical practice. However, the mechanisms underlying the frac- tures are not fully understood. This study was aimed to examine the effect of rosiglitazone （an agonist of PPAR-T） of different doses on the proliferation, differentiation, and transforming growth factor beta 1 （TGF-131）-induced expression of connective tissue growth factor （CTGF） in primary rat osteoblasts in vitro. Osteoblasts were isolated from newly born SD rats and treated with different doses of rosiglita- zone （0-20 gmol/L）. The proliferation and differentiation of osteoblasts were measured by MTT assay and NPP assay, respectively. The expression of CTGF was determined by RT-PCR and Western blotting. The results showed that most isolated osteoblasts displayed strong alkaline phosphatase （ALP） activity and treatment with different doses of rosiglitazone did not affect their proliferation, but significantly in- hibited the differentiation of osteoblasts in a dose-dependent manner. Moreover, treatment with different doses of rosiglitazone significantly reduced the TGF-131-induced CTGF mRNA transcription and protein expression in a dose-dependent manner in rat osteoblasts. It was concluded that the activation of PPAR-y may inhibit the differentiation of osteoblasts by reducing the TGF-131-induced CTGF expres- sion in vitro.

Connective Tissue Growth Factor Expression in Rabbit Corneal Fibroblast and Its Effect on Fibroblast Proliferation In Vitro

The connective tissue growth factor (CTGF) expression in cultured corneal fibroblasts and its effect on corneal fibroblasts proliferation in vitro were examined. Total RNA was extracted from early passaged rabbit corneal fibroblasts by guanidine isothiocyanate one-step method and mRNA was reversely transcripted into complementary DNA (cDNA). Specific CTGF primers were used in the PCR reaction and the products were analyzed by electrophoresis to determine the expression of mRNA for CTGF against DNA marker. House-keeping gene GAPDH was used as control. Different concentrations of CTGF (0.5, 5, 50, 500, 5 000, 50 000 ng/ml) were added into the third passaged corneal fibroblast culture system, and its effect on corneal fibroblast proliferation was measured by MTT method. The results showed that compared with a GAPDH 450 bp band, CTGF RT-PCR product showed a specific 120 bp band as expected. CTGF produced a dose-dependent increase in the proliferation of corneal fibroblasts but it inhibited fibroblast proliferation at higher concentrations (5 000 and 50 000 ng/ml). It was concluded that proliferating corneal fibroblasts produce CTGF and CTGF helps to promote corneal fibroblast proliferation. The results indicated that CTGF might be involved in the corneal wound healing after photorefractive keratectomy in which corneal fibroblasts are activated to proliferate and secrete growth factors that in turn promote corneal fibroblast proliferation.

Effects of connective tissue growth factor and collagen type Ⅰ scleroderma

Objective： To investigate the effects of connective tissue growth factor（CTGF） and collagen type I（COL-I） on the pathogenesis of scleroderma and explore the relationship between the level of COL-I and CTGF. Methods： 12 mice model of scleroderma was established by the injection of Bleomycin. The level of CTGF and COL-I were detected by immunohistochemical method. The relationship was analyzed between CTGF and COL-I level. As control group, 12 healthy mice were selected. Results： The levels of CTGF and COL-I in sclerotic models were higher than in normal controls （P 〈 0.05）. It was found that there was a correlation between the level of CTGF and COL-I. Conclusion： CTGF and COL-I played an important role in the hardening process of the skin lesions of the mice model, which may be involved in the pathogenesis of scleroderma.

Vascular endothelial growth factor/connective tissue growth factor and proteomic analysis of aqueous humor after intravitreal conbercept for proliferative diabetes retinopathy

AIM:To investigate the role of connective tissue growth factor(CTGF)and vascular endothelial growth factor(VEGF)in the protein profile of the aqueous humor in patients with proliferative diabetic retinopathy(PDR)following intravitreal injection of conbercept.METHODS:This study included 72 PDR patients and 8 cataract patients as controls.PDR patients were divided into 3 groups according to the intervals of 3,5,and 7d between intravitreal conbercept(IVC,0.5 mg/0.05 mL)injection and pars plana vitrectomy(PPV)performed.Aqueous humor samples were collected before and after IVC and PPV for VEGF and CTGF levels detected with enzyme-linked immunosorbent assay(ELISA).The differential proteomics of 10 patients who underwent PPV surgery 5d after IVC and 8 normal controls was studied,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed on the data,and the protein interaction network of 23 differential proteins was studied.RESULTS:Post-IVC,VEGF levels decreased and CTGF levels increased significantly in aqueous humor,with the CTGF/VEGF ratio rising significantly at all intervals.Liquid chromatography tandem mass spectrometry(LC-MS/MS)identified differentially expressed proteins between preand post-IVC samples.GO and KEGG analyses revealed involvement in immune response,stress response,complement and coagulation cascades,ferroptosis,and PPAR signaling pathways.PPI analysis highlighted key proteins like APOA1,C3,and transferrin(TF).ELISA assay confirmed the differential expression of proteins such as HBA1,SERPINA1,COL1A1,and ACTB,with significant changes in the IVC groups.CONCLUSION:The study demonstrates that IVC effectively reduces VEGF levels while increasing CTGF levels,thereby modifying the CTGF/VEGF ratio,and IVC significantly alters the protein profile in the aqueous humor of patients with PDR.Proteomic analysis reveals that these changes are associated with critical biological pathways and protein interactions involved in immune response,stress response,and cellular metabolism.

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

AIM: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.METHODS:UMB-containing niosomes were created,optimized,and characterized.RPE-like cells were treated with free UMB and UMB-containing niosomes.The IC_(50)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 IC_(50)of 96.2μg/mL,while UMB-containing niosomes had an IC_(50)of 25μg/mL.RESULTS: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.CONCLUSION: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.

Interference of Y-27632 on the signal transduction of transforming growth factor beta type 1 in ocular Tenon capsule fibroblasts

AIM: To investigate the interfering effect of Y-27632, a ROCK-I selective inhibitor, on the signal transduction pathway of transforming growth factor-beta 1 (TGF-beta 1) in ocular Tenon capsule fibroblasts (OTFS) in vitro. METHODS: After OTFS from passages 4 to 6 47 vitro were induced by TGF-beta 1 and then treated by Y-27632, the changes of the OTFS cell cycles were analyzed via flow cytometry, and the proteins expression of the alpha -smooth muscular actin (alpha -SMA), connective tissue growth factor (CTGF), collagen I were calculated by Western blot. After OTFS treated by the different concentrations of Y-27632, the expression levels of the alpha -SMA, CTGF and collagen I mRNA were assayed by RT-PCR. RESULTS: Y-27632 had no markedly effect on the OTFS cell cycles. After treated by TGF-beta 1, OTFS in G1 period significantly increased. The cell cycles distribution by both TGF-beta 1 and Y-27632 had no remarkable difference from that in control group. Y-27632 significantly inhibited the proteins expressions of both alpha -SMA and CTGF, while to some extent inhibited that of collagen I. TGF-beta 1 significantly promoted the proteins expressions of alpha -SMA, CTGF and collagen I. After OTFS treated by both TGF-beta 1 and Y-27632, of alpha -SMA, the protein expression was similar with that in control group (P=0.066>0.05), but the protein expression of CTGF or collagen I, respectively, was significantly different from that in control group (P=0.000<0.01). The differences of expressions of the alpha -SMA, CTGF and collagen I mRNA in 30, 150, 750 mu mol/L Y-27632 group were statistically significant, compared with those in control group, respectively (alpha -SMA, P=0.002, 0.000, 0.000; CTGF, P=0.014, 0.002, 0.001; collagen I,P=0.003, 0.002, 0.000). CONCLUSION: Blocking the Rho/ROCK signaling pathway by using of Y-27632 could inhibit the cellular proliferation and the expression of both CTGF and alpha -SMA whatever OTFS induced by TGF-beta 1 or not. Y-27632 suppressed the expression of collagen I mRNA without induction.

Effect of high glucose, angiotensin Ⅱ and receptor antagonist Losartan on the expression of connective tissue growth factor in cultured mesangial cells

To observe the effect of high glucose, angiotensin Ⅱ (AngⅡ) and Losartan on the expression of connective tissue growth factor (CTGF) mRNA in cultured mesangial cells (MCs) Methods MCs of SD rats were isolated and cultured High glucose (30 mmol/L) and AngⅡ (10 -9 , 10 - 7 , and 10 -5 mol/L) were added to the medium for 72 hours to observe the influence on CTGF mRNA expression Losartan of 10 -5 mol/L and AngⅡ of 10 -5 mol/L were added to the medium to observe the effects of Losartan on CTGF mRNA expression stimulated by AngⅡ The expressions of CTGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) Results RT-PCR showed that high glucose and AngⅡ up-regulated the expression of CTGF mRNA, and AngⅡ stimulated the expression in a dose-dependent manner Expression of CTGF mRNA induced by AngⅡwas partially suppressed by 10 -5 mol/L Losartan (P<0 05) Conclusions High glucose and AngⅡ can enhance the expression of CTGF mRNA and thus be involved in the process of renal fibrosis Losartan can have a partial fibrogenesis-inhibiting effect, with implications for the treatment of renal fibrosis

Connective tissue growth factor is associated with the early renal hypertrophy in uninephrectomized diabetic rats

Background Renal hypertrophy has been regarded as the early feature of diabetic nephropathy （DN）, which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor （CTGF） with renal hypertrophy in uninephrectomized diabetic rats. Methods Seventy-two Sprague-Dawley （SD） rats were randomly divided into two groups： control group （group C, n=32） and diabetic nephropathy （group DN, n=40）. Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin （STZ） after rats had received uninephrectomy. Blood glucose （BG）, body weight （BW）, 24-h urinary albumin excretion （24hUalb）, kidney weight （KW）, KW/BW, glomerular tuft area （AG）, glomerular tuft volume （VG）, proximal tubular area （AT） at each time point, the width of glomerular basement membrane （GBM） and tubular basement membrane （TBM） at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kipl were detected by immnnohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed. Results There was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C （P〈0.05, respectively）, diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [（245.7±103.0） nm vs （121.8±19.1） nm, P〈0.01] and TBM [（767.7±331.1） nm vs （293.0±110.5） nm, P〈0.01] at week 8. There was a weak expression for CTGF and p27kipl in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kipl was found in glomeruli and tubuli in diabetic kidney from week 1 onward （P〈0.05, respectively）, and the extent of CTGF expression was positively correlated with AG （r=0.92, P〈0.05）, VG （r=0.86, P〈0.05）, AT （r=0.94, P〈0.01） and positively correlated with the expression of p27kipl （r=0.96, P〈0.01）. Conclusion The expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.

Inhibition of connective tissue growth factor overexpression decreases growth of hepatocellular carcinoma cells in vitro and in vivo

Background We have previously found that connective tissue growth factor （CTGF） is highly expressed in a rat model of liver cancer. Here, we examined expression of CTGF in human hepatocellular carcinoma （HCC） cells and its effect on celt growth. Methods Real-time PCR was used to observe expression of CTGF in human HCC cell lines HepG2, SMMC-7721, MHCC-97H and LO2. siRNA for the CTGFgene was designed, synthesized and cloned into a PIk0.1-GFP-SP6 vector to construct a lentivirus-mediated shRNA/CTGF. CTGF mRNA and protein expression in HepG2 cells treated by CTGF-specific shRNA was evaluated by real-time PCR and Western blotting. 3-（4,5-dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide （MTT） assay was utilized to evaluate the growth effect, and a colony formation assay was used for observing clonogenic growth. In vivo, tumor cell proliferation was evaluated in a nude mouse model of xenotransplantation. Statistical significance was determined by t test for comparison between two groups, or analysis of variance （ANOVA） for multiple groups. Results Immunohistochemical staining of CTGF was seen in 35 of 40 HCC samples （87.5%）. CTGF was overexpressed 5-fold in 20 HCC tissues, compared with surrounding non-tumor liver tissue. CTGF mRNA level was 5-8-fold higher in HepG2, SMMC-7721 and MHCC-97H than in LO2 cells. This indicated that the inhibition rate of cell growth was 43% after knockdown of CTGF expression （P 〈0.05）. Soft agar colony formation assay showed that siRNA mediated knockdown of CTGF inhibited colony formation in soft agar of HepG2 cells （P 〈0.05）. The volume of tumors from CTGF-shRNA-expressing cells only accounted for 35% of the tumors from the scrambled control-infected HepG2 cells （P 〈0.05）. Conclusions CTGF was overexpressed in human HCC cells and downregulation of CTGF inhibited HCC growth in vitro and in vivo. Knockdown of CTGFmay be a potential therapeutic strategy for treatment of HCC.

Parathyroid hormone-mitogen-activated protein kinase axis exerts fibrogenic effect of connective tissue growth factor on human renal proximal tubular cells

Background Enhanced and prolonged expression of connective tissue growth factor （CTGF） is associated with kidney fibrosis. Parathyroid hormone （PTH） is involved in the genesis of disturbed calcium/phosphate metabolism and ostitis fibrosa in renal failure. PTH activated mitogen-activated protein kinase （MAPK） signaling pathway is present in renal tubular cells. The aim of this study was to identify the mechanism how the signal is transduced to result in extracellular signal-regulated protein kinase （ERK） activation, leading to upregulation of CTGF.Methods The levels of CTGF mRNA and protein in human kidney proximal tubular cells （HK-2） treated with PTH in the presence or absence of the MAPK inhibitor PD98059 were analyzed by quantitative real-time polymerase chain reaction （RT-PCR） and immunoblotting assay. The activation of the CTGF promoter in HK-2 cells was determined by the dual-luciferase assay. The effects of the protein kinase A （PKA） activator 8-Br-cAMP and protein kinase C （PKC） activator phorbol 12-myristate 13-acetate （PMA） on MAPK phosphorylation, and the effects of the PKA inhibitor H89 and PKC inhibitor calphostin C on MAPK phosphorylation and CTGF expression were detected by immunoblotting assay.Results PD98059 inhibited the PTH stimulated expression of CTGF, which strongly suggested that the MAPK signaling pathway plays an important role in the PTH-induced CTGF upregulation in renal tubular cells. A PKA activator as well as PKC activators induced MAPK phosphorylation, and both PKA and PKC inhibitors antagonized PTH-induced MAPK phosphorylation and CTGF expression.Conclusion CTGF expression is upregulated by PTH through a PKC/PKA-ERK-dependent pathway.

Enhancement of meniscal repair in the avascular zone using connective tissue growth factor in a rabbit model

Background Connective tissue growth factor （CTGF） is a secreted protein containing several domains that mediate interactions with growth factors, integrins and extracellular matrix components. CTGF plays an important role in extracellular matrix production by its ability to mediate collagen deposition during wound healing. CTGF also induces neovascularization in vitro, suggesting a role in angiogenesis in vivo. We herein evaluated whether CTGF was required for extracellular matrix synthesis of meniscal fibrochondrocytes and/or angiogenesis during the repair of meniscal tears. Methods Meniscal fibrochondrocytes were isolated from the inner-I/2 of rabbit meniscus by trypsin collagenase treatment and further treated with 100 ng/ml CTGF in vitro. Characterization of fibrochondrocytes was identified by flow cytometry analyzing CD31, CD44, CD45 and CD105, and was further tested by type II collagen immunocytochemistry. Changes in gene expression of meniscal fibrochondrocytes were monitored by quantitative real-time polymerase chain reaction. Histological sections prepared from a 3-mm portion of a longitudinal tearing defect in the middle of the rabbit meniscus were subjected to fluorescence-immunohistochemistry analysis at 1, 4 and 10 weeks following surgical treatment with 1.5 IJg of CTGF/fibrin-glue composites. Results Quantitative RT-PCR assay showed that types I and II collagen and vascular endothelial growth factor mRNA expression in the 100 ng/ml CTGF group were remarkably enhanced as compared to levels in the no-dose group at 14 days （（2.38±0.63） fold, （2.96±0.87） fold, （2.14±0.56） fold, respectively）. Likewise, fluorescence-immunohistochemical analysis revealed that in the group implanted with CTGF-fibrin glue, types Ⅰand Ⅱcollagen, as well as the capillaries, completely filled the defect by 10 weeks, postoperatively. In contrast, only soft tissue repair occurred when PBS-fibrin glue was implanted. Conclusions These findings suggest that CTGF can significantly promote extracellular matrix deposition （types Ⅰ and Ⅱcollagen） within the meniscal avascular zone; CTGF can greatly heighten the expression of vascular endothelial growth factor activity simultaneously in vivo, further enhancing the repair of meniscal tears in the avascular zone.

Full composition granules of Huanglian (Rhizoma Coptidis) decrease the serum monocyte chemotactic protein-1 and connective tissue growth factor levels and inhibit kidney nuclear factor-κB expression in rats with high-fat diet-induced diabetes

OBJECTIVE: To investigate the efficacy of the full composition granules of Huanglian(Rhizoma Coptidis)(FGC) on the serum monocyte chemotactic protein-1(MCP-1) and connective tissue growth factor(CTGF) levels and kidney nuclear factor-κB(NF-κB) expression in rats with high-fat diet-induced diabetes.METHODS: Diabetes was induced in rats by feeding a high-fat chow combined with intravenous streptozotocin injection. Forty diabetic SpragueDawley rats were randomly assigned to a normal group(NG), model group(MG), irbesartan group(IG), and low-, middle-, and high-dosage FGC groups(LFGC, MFGC, HFGC), with eight rats per group. The IG rats received 31.25 mg·kg^(-1)·d^(-1) irbesartan tablets, whereas those in the LFGC, MFGC,and HFGC were administered 52, 312.5, and 625 mg·kg^(-1)·d^(-1) FGC, respectively. After 12 weeks,bodyweight(BW), left kidney weight(KW), hemoglobin A1c(HbA1c), serum creatinine(Scre), blood urea nitrogen(BUN), and serum MCP-1 and CTGF levels were determined, pathological changes of the kidney were recorded, and kidney NF-κB p65(A) expression was measured.RESULTS: The 24-h urine albumin and levels of HbA1c, Scre, BUN, and serum MCP-1 and CTGF were significantly increased in in the MG compared with the NG, as was the kidney NF-κB(p65) expression(P < 0.05). Furthermore, clear pathological changes in kidney fibrosis were observed in the MG rats. Following irbesartan and FGC administration,the 24-h urine albumin and the levels of HbA1c,Scre, and serum MCP-1 and CTGF were significantly decreased in FCG groups compared with those in the MG, which is in agreement with the change in the kidney NF-κB(p65) expression, whereas the similarly significant decrease only exist in 24-h urine albumin and the levels of serum CTGF after irbesartan administration. Hematoxylin-eosin(HE)staining results indicated that the fibrosis observed in the MG samples was alleviated through FGC treatment.CONCLUSION: FGC may alleviate potential kidney injury by decreasing the serum MCP-1 and CTGF levels and inhibiting NF-k B expression in diabetic nephropathy in rats with high-fat diet-induced diabetes.