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.

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.

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.

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.

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.

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).

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 (&#x003b1;-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&#x000b1;0.11, 1.96 &#x000b1;0.03, 3.12 &#x000b1;0.10, and 4.08&#x000b1;0.14, respectively, in response to control group and treatment with CTGF of 20, 50 and 100 ng/mL (F=443.86, P&#x0003c;0.01). The increased Slug protein levels were correlated well with up-expression of &#x003b1;-SMA (0.78&#x000b1;0.05, 0.85&#x000b1;0.06, 2.17&#x000b1;0.15, 2.86&#x000b1;0.10; F=449.85, P&#x0003c;0.01) and down-expression of E-cadherin (2.50&#x000b1;0.11, 1.79&#x000b1;0.26, 1.05&#x000b1;0.14, 0.63&#x000b1;0.08; F=101.55, P&#x0003c;0.01).CONCLUSIONTranscription factor Slug may be involved in EMT of HLECs induced by CTGF in vitro.

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.

Finite Element Simulation of In-Stent Restenosis with Tissue Growth Model

In this study, a finite element simulation of in-stent restenosis (ISR) is conducted to simulate the deployment and expansion of a stent in an occluded artery with a contact model and a mechanics-based growth model. A tissue growth model based on the multiplicative decomposition of deformation is applied to investigate the growth of the plaque and artery wall upon the stent’s implantation. Due to the high stresses at the contact points between the stent struts and the tissue, further tissue injury or restenosis is observed. The simulation results show that after the stent deployment, the von Mises stress is significantly larger in the plaque compared to the artery wall, especially in the region that is in contact with the stent. However, the growth of the plaque and artery tends to even out the stress concentration over time. The tissue growth is found to be more significant near the inner wall than the outer layer. A 0.77 mm restenosis is predicted, which agrees with published clinical observations. The features of the artery growth are carefully analyzed, and the underlying mechanism is discussed. This study is the first attempt to apply finite element analysis to artery restenosis, which establishes a framework for predicting ISR’s occurrence and severity. The results also provide insights into understanding the underlying mechanism of in-stent restenosis.

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.

Role of connective tissue growth factor in experimental radiation nephropathy in rats

Background Connective tissue growth factor （CTGF） is a potent fibrogenic cytokine which has been associated with progressive glomerulosclerosis and tubulointerstitial fibrosis. We investigated the role of CTGF on the progression of a rat model of radiation nephropathy. Methods The model of radiation nephropathy in rats was established as follows： control group （n=12）, underwent only laparotomy; irradiated group （n=-20）, underwent a laparotomy, then the rats were subjected to a single dose 25 Gy X-ray to the kidneys. The rats were followed up one, three, six and nine months after renal exposure to radiation. Results Renal dysfunction was noted early in irradiated rats. Histological analysis showed focal glomerular sclerotic lesions at an early stage after irradiation. Radiation-induced glomerular and tubulointerstitial injuries were particularly severe the sixth month after irradiation as compared to the control group （P 〈0.01）. By immunohistochemistry, increased expression of CTGF was noted in the irradiated kidneys, which began to increase from the first month after irradiation, and remained significantly higher at the sixth and ninth month after irradiation （P 〈0.01）. Upon Western blot analysis CTGF protein expression showed an increase in the radiation treated kidneys compared with the control rats. The expression of CTGF closely correlated with the progression of radiation nephropathy. The expression of α-smooth muscle actin, vimentin, type Ⅲ collagen and type Ⅳ collagen was also high in the irradiated kidney as compared to control rat kidneys （P 〈0.05）, and was most severe at the sixth and ninth month after irradiation （P 〈0.01）. By double immunostaining, CTGF expressing cells were found to be α-SMA-positive myofibroblasts and vimentin-positive tubular epithelial cells. Glomerular expression of CTGF closely correlated with the glomerular expression of a-SMA （r=0.628, P 〈0.01）, vimentin （r=0.462, P 〈0.05） and accumulation of type IV collagen （r=0.584, P 〈0.01） in the irradiated kidney. Similarly, the expression of CTGF was positively correlated with the expression of α-SMA （r=0.613, P 〈0.01）, vimentin （r=0.629, P 〈0.01）, deposition of type Ⅲ collagen （r=0.741, P 〈0.001） and type Ⅳ collagen （r=0.799, P 〈0.0001） in the tubulointerstitium of the irradiated kidney. Finally the expression of CTGF after the irradiation of the kidney positively correlated with the levels of blood urea nitrogen and serum creatinine. Conclusion Overexpression of CTGF may play an important role in the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in radiation nephropathy.

Effects of Ganfukang(肝复康) on Expression of Connective Tissue Growth Factor and Focal Adhesion Kinase/Protein Kinase B Signal Pathway in Hepatic Fibrosis Rats

Objective： To investigate the effect of Ganfukang （肝复康, GFK） on connective tissue growth factor （CTGF） and focal adhesion kinase （FAK）/protein kinase B （PKB or Akt） signal pathway in a hepatic fibrosis rat model and to explore the underlying therapeutic molecular mechanisms of GFK. Methods： Fifty SD rats were randomly divided into five groups as follows： the control group, the model group （repeated subcutaneous injection of CCl4）, and the three GFK treatment groups （31.25, 312.5, and 3125 mg/kg, intragastric administration）. Reverse transcriptase-polymerase chain reaction （RT-PCR）, Western blotting, and immunohistochemistry were used to examine the expression of CTGF, integrin α 5, integrin β 1, FAK/Akt signal pathway, cyclinD1, and collagen in the different-treated rats. Results： GFK attenuated the up-regulation of CTGF, integrin α 5, and integrin β 1 in hepatic fibrosis rats and suppressed both the phosphorylation of FAK and the phosphorylation of Akt simultaneously （P〈0.01）. At the same time, the expression of cyclinD1, collagen Ⅰ, and collagen Ⅲ was decreased by GFK significantly （P〈0.01）. Conclusions： CTGF and FAK/Akt signal pathway were activated in the CCl4-induced hepatic fibrosis rats, which contribute to increased expression of cyclinD1 and collagen genes. The mechanisms of the anti-fibrosis activity of GFK may be due to its effects against CTGF and FAk/Akt signal pathway.