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.

Significance of changes in transforming growth factor-β mRNA levels in autogenous vein grafts

Background This study was designed to investigate changes in mRNA levels of transforming growth factor-β(TGF-β), collagen Ⅰ, and collagen Ⅲ in autogenous vein grafts. Methods Twenty-four New Zealand rabbits were randomly divided into 4 groups with 6 rabbits each. The external jugular veins of the New Zealand rabbits were harvested and grafted into the ipsilateral carotid artery. All rabbits were fed with a standard diet. After the operation, the rabbits were sacrificed at 1, 2, 3, or 4 weeks. TGF-β, collagen Ⅰ, and collagen Ⅲ mRNA levels in the venous grafts were measured by semiquantitative methods at every time point. The contralateral external jugular veins were also harvested and analyzed as controls. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard to normalize all samples for potential variations in mRNA content. In order to observe the expression of TGF-β protein, immunohistochemical SABC methods were used. Results One week postoperation, the mRNA level of TGF-β was upregulated to 1.73±0.19 in the vein graft and 1.21±0.16 in the control vein (P<0.01). High mRNA levels were maintained until week 4 postoperation. The mRNA levels of collagen Ⅰ and collagen Ⅲ were also significantly increased to 2.18±0.21 versus 1.12±0.24 and 1.08±0.13 versus 0.83±0.12, respectively (P<0.05). Immunohistochemical staining revealed a higher density of TGF-β expression in the vein grafts.Conclusions An uninterrupted increase in mRNA levels of TGF-β, collagen Ⅰ, and collagen Ⅲ is observed in autogenous vein grafts. This increase may be the major cause of intimal hyperplasia, sclerosis, and even graft failure.

Gardenia jasminoides attenuates hepatocellular injury and fibrosis in bile duct-ligated rats and human hepatic stellate cells

AIM:To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis.METHODS:Male Sprague-Dawley rats underwent common bile duct ligation(BDL) for 14 d and were treated with Gardenia jasminoides by gavage.The ef-fects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells(LX-2) in vitro.RESULTS:Treatment with Gardenia jasminoides decreased serum alanine aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,146.6 ± 15 U/L vs 77 ± 6.5 U/L,P = 0.0007) and aspartate aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,188 ± 35.2 U/L vs 128 ± 19 U/L,P = 0.005) as well as hydroxyproline(BDL vs BDL + 100 mg/kg Gardenia jasminoides,438 ± 40.2 μg/g vs 228 ± 10.3 μg/g liver tissue,P = 0.004) after BDL.Furthermore,Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor β1(TGF-β1),collagen type?Ⅰ?(Col?Ⅰ) and α-smooth muscle actin(α-SMA).Gardenia jasminoides significantly suppressed the upregulation of TGF-β1,Col?Ⅰand α-SMA in LX-2 exposed to recombinant TGF-β1.Moreover,Gardenia jasminoides inhibited TGF-β1-induced Smad2 phosphorylation in LX-2 cells.CONCLUSION:Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.

Differential Mechanisms of the Effect of Peroxisome Proliferator-Activated Receptor Gamma Agonists on Bleomycin-Induced Lung Fibrosis

Background and Objectives: Peroxisome proliferator-activated receptor-g (PPAR-g) is a nuclear receptor whose activation regulates inflammation and fibrosis in various organs. We aimed to investigate the effect of two PPAR-g ligands, telmisartan and rosiglitazone, on lung injury and fibrosis induced by intratracheal bleomycin (BLM). Methods: Lung injury and fibrosis was induced in female C57Bl/6 mice by intratracheal instillation of 1.0 mg/kg of BLM. Some of the animals received rosiglitazone intraperitoneally or telmisartan in drinking water. Bronchoalveolar lavage (BAL) was performed 2, 7, 14 or 21 days after BLM instillation for cell counting and measurement of mediators in the lung. In a separate series, the lungs were sampled for collagen assay and histopathological evaluation. Results: Treatment with rosiglitazone or telmisartan significantly attenuated the BLM-induced increases in lung collagen content, pathological score, and inflammatory cells in BAL fluid. Rosiglitazone significantly suppressed BLM-induced elevation of TGF-b1, MCP-1, and IL-6 levels in the lung. In contrast, telmisartan made no changes in these cytokines, whereas it mitigated the BLM-induced increase in prostaglandin F2a in the lung. Higher concentrations of rosiglitazone and telmisartan attenuated proliferation of lung fibroblasts in vitro. Conclusions: Two PPAR-g ligands, rosiglitazone and telmisartan, exert protective effects on BLM-induced lung fibrosis through the suppression of different profibrotic mediators.

TGF-β stimulation of EMT programs elicits non-genomic ER-α activity and anti-estrogen resistance in breast cancer cells

Aim:Estrogen receptor-α(ER-α)activation drives the progression of luminal breast cancers.Signaling by transforming growth factor-β(TGF-β)typically opposes the actions of ER-α;it also induces epithelial-mesenchymal transition(EMT)programs that promote breast cancer dissemination,stemness and chemoresistance.The impact of EMT programs on nongenomic ER-αsignaling remains unknown and was studied herein.Methods:MCF-7 and BT474 cells were stimulated with TGF-βto induce EMT programs,at which point ER-αexpression,localization,and nongenomic interactions with receptor tyrosine kinases and MAP kinases(MAPKs)were determined.Cell sensitivity to anti-estrogens both before and after traversing the EMT program was also investigated.Results:TGF-β-stimulated MCF-7 and BT474 cells to acquire EMT phenotypes,which enhanced cytoplasmic accumulation of ER-αwithout altering its expression.Post-EMT cells exhibited:(1)elevated expression of EGFR and IGF1R,which together with Src formed cytoplasmic complexes with ER-α;(2)enhanced coupling of EGF,IGF-1 and estrogen to the activation of MAPKs;and(3)reduced sensitivity to tamoxifen,an event reversed by administration of small molecule inhibitors against the receptors for TGF-β,EGF,and IGF-1,as well as those against MAPKs.Conclusion:EMT stimulated by TGF-βpromotes anti-estrogen resistance by activating EGFR-,IGF1R-,and MAPK-dependent nongenomic ER-αsignaling.