慢性心力衰竭大鼠肾组织中血小板源性生长因子及结缔组织生长因子的表达

目的:探讨血小板源性生长因子BB(PDGF-BB)及结缔组织生长因子(CTGF)在慢性心力衰竭大鼠肾组织的表达及意义。方法:采用肾上腹主动脉缩窄法制作Wistar大鼠慢性心力衰竭模型,随机分为假手术组,心衰10 d、20 d、30 d组。观察及比较各组大鼠的血流动力学数值;Masson染色方法观察大鼠慢性心衰时肾组织结构变化;免疫组织化学方法检测各组大鼠肾组织的PDGF-BB及CTGF的表达部位;免疫印迹半定量检测各组大鼠肾组织的PDGF-BB、CTGF的表达水平。结果:心衰模型各组大鼠PDGF-BB、CTGF的蛋白表达均较假手术组高,且随心衰程度的加重而表达升高。结论:慢性心衰可引起肾间质纤维化,其发生发展与心衰的严重程度密切相关。     

Induction of myocardial connective tissue growth factor in pacing-induced heart failure in pigs

AIMS: Connective tissue growth factor (CTGF) is a secreted, heparin-binding, and extracellular matrix associated protein shown to stimulate many of the cellular events underlying fibrosis. Previous investigations have revealed that myocardial CTGF is substantially induced in ischaemic heart failure, particularly in the ischaemic and peri-ischaemic region. The purpose of the present study was to investigate to what extent myocardial induction of CTGF is a general response to congestive heart failure (CHF) and to what extent CTGF is a decisive effector of fibrosis. METHODS: Experimental heart failure in pigs was induced by rapid pacing at 220-240 beats min(-1) for 3 weeks (CHF pigs; n = 12). RESULTS: The CHF pigs exhibited significant left ventricular (LV) dilatation, reduced contractility, and increased cardiac filling pressures. Northern blot analysis demonstrated that myocardial CTGF mRNA levels in CHF pigs were fivefold higher (P < 0.05) than those in control pigs (n = 10). Similar elevations of immunoreactive CTGF (sixfold; P < 0.05) were observed in myocardial tissue samples prepared for Western blot analysis. Immunohistochemical analysis of myocardial tissue sections revealed predominant expression in interstitial and perivascular fibroblasts and endothelial cells. Myocardial procollagen alpha1(I) mRNA levels were also significantly elevated (sixfold; P < 0.05) in CHF pigs compared with controls, whereas myocardial tissue contents of collagen were not statistically different between the groups. CONCLUSION: Induction of myocardial CTGF in heart failure is not just a response to ischaemia, but rather a general response to evolving heart failure. Yet, induction of myocardial CTGF was clearly not a sufficient effector of fibrosis.     

CTGF在胃癌中的表达及其促进血管生成的机制

目的研究CTGF在人胃癌标本中表达情况及其促进血管生成的机制。方法应用Real Time PCR和免疫组织化学染色检测胃癌标本中CTGF mRNA,VEGF mRNA及其在组织中蛋白表达情况;应用pcDNA3.1质粒上调AGS细胞中CTGF的表达,并用Real Time PCR和Western blot检测VEGF mRNA水平和蛋白水平表达变化;AGS稳定株细胞接种于裸鼠,成瘤后肿瘤组织切片,免疫组化染色。结果与正常组织相比,胃癌组织中CTGF mRNA,VEGF mRNA表达水平均显著增加,免疫组化亦显示CT-GF、VEGF在胃癌的标本中显色比正常对照组织强。统计显示两者mRNA水平及免疫组化表达有相关性（P〈0.05）。转染pcD-NA3.1 CTGF质粒后,AGS细胞中CTGF蛋白的表达上调,Real Time PCR及Western blot检测显示过表达CTGF后,VEGF表达上调。与对照细胞相比,过表达CTGF的AGS的肿瘤形成和生成速度明显加快（P〈0.05）。免疫组化染色显示VEGF的染色明显强于对照组。结论在胃癌中CTGF,VEGF表达明显增加,CTGF可能通过上调VEGF表达促进肿瘤血管生成。     

结缔组织生长因子在慢性肝病诊断中的应用研究

探讨结缔组织生长因子(CTGF)在慢性肝病诊断中的应用价值。运用ELISA方法检测198例研究对象,观察血清CTGF水平的变化,分析CTGF水平与临床特征及实验室指标的相关性;运用实时荧光定量RT-PCR方法检测组织中CT-GF mRNA的表达量,比较各组间表达量的变化。乙肝后重度肝纤维化组、肝硬化组、原发性肝细胞癌(HCC)组的血清CTGF水平均显著高于正常对照组,且具有统计学意义(P<0.05);血清CTGF水平与部分临床实验室特征存在相关性,在区分早晚期肝纤维化上具有诊断提示作用;癌旁组织的CTGF mRNA相对表达量显著高于正常对照和癌组织的表达量且具有统计学差异(P<0.05)。血清CTGF水平与肝纤维化、肝硬化及肝癌的发生发展关系密切,有望成为一个新的无创性肝纤维化检测诊断指标,为临床诊断提供依据。     

结缔组织生长因子在人及大鼠肝纤维化组织中表达增强

目的观察结缔组织生长因子(CTGF)在人及大鼠肝纤维化组织中的表达。方法雄性SD大鼠32只,皮下注射CCl4后1、4、8周收集肝组织标本;44例人肝组织,其中包括12例正常肝组织、32例慢性病毒性肝炎和肝硬化组织。用免疫组化方法检测CTGF的表达及分布。结果CTGF主要表达于大鼠肝星状细胞及肝细胞胞质中。注射CCl4后,大鼠肝组织中CTGF呈时间依赖性表达增强(P<0.01或P<0.05)。CTGF在人肝纤维化组织中的表达与大鼠相类似,表达水平显著高于正常人(P<0.01)。结论CTGF作为一种促纤维化因子,其过表达可促进肝星状细胞的增殖活化,促进细胞外基质的形成,从而促进肝纤维化的发生、发展。     

Fell‐Muir lecture: connective tissue growth factor (CCN2) – a pernicious and pleiotropic player in the development of kidney fibrosis

Connective tissue growth factor (CTGF, CCN2) is a member of the CCN family of matricellular proteins. It interacts with many other proteins, including plasma membrane proteins, modulating cell function. It is expressed at low levels in normal adult kidney cells but is increased in kidney diseases, playing important roles in inflammation and in the development of glomerular and interstitial fibrosis in chronic disease. This review reports the evidence for its expression in human and animal models of chronic kidney disease and summarizes data showing that anti‐CTGF therapy can successfully attenuate fibrotic changes in several such models, suggesting that therapies targeting CTGF and events downstream of it in renal cells may be useful for the treatment of human kidney fibrosis. Connective tissue growth factor stimulates the development of fibrosis in the kidney in many ways including activating cells to increase extracellular matrix synthesis, inducing cell cycle arrest and hypertrophy, and prolonging survival of activated cells. The relationship between CTGF and the pro‐fibrotic factor TGFβ is examined and mechanisms by which CTGF promotes signalling by the latter are discussed. No specific cellular receptors for CTGF have been discovered but it interacts with and activates several plasma membrane proteins including low‐density lipoprotein receptor‐related protein (LRP)‐1, LRP‐6, tropomyosin‐related kinase A, integrins and heparan sulphate proteoglycans. Intracellular signalling and downstream events triggered by such interactions are reviewed. Finally, the relationships between CTGF and several anti‐fibrotic factors, such as bone morphogenetic factor‐4 (BMP4), BMP7, hepatocyte growth factor, CCN3 and Oncostatin M, are discussed. These may determine whether injured tissue heals or progresses to fibrosis.     

百令对慢性肾衰竭模型大鼠肾脏保护和肾结缔组织生长因子表达的影响

目的 观察百令对5/6肾切除大鼠的肾脏保护作用及对肾结缔组织生长因子(CTGF)表达的影响,探讨其延缓肾衰竭进展及抗纤维化的相关机制. 方法 50只SD大鼠随机取8只为假手术组,其余行5/6肾切除术.根据术后3周血肌酐(Scr)值分为模型组、天然虫草组(2.0 g·kg-1·d-1)、百令治疗组(2.0 g·kg-1·d-1)和百令高剂量组(3.0 g·kg-1·d-1).术后4周给药.治疗1个月后检测Scr、尿素氮(BUN)浓度;光镜下观察肾脏病理改变,免疫组化方法检测肾组织CTGF、α平滑肌肌动蛋白(α-SMA)的表达水平,采用图像分析系统进行定量分析. 结果 治疗后模型组大鼠Scr、BUN明显高于假手术组(P<0.01),肾小球与肾小管间质均有明显病理改变,CTGF、α-SMA的表达明显上调;而药物治疗组的Scr、BUN明显低于模型组(P<0.05),肾脏病理损伤减轻,CTGF、α-SMA的表达降低(P<0.05). 结论 百令能改善5/6肾切除大鼠的肾功能,减轻肾脏病理损害,其机制可能与下调肾组织CTGF的表达有关.     

结缔组织生长因子促进肾小球系膜细胞合成RANTES部分依赖于p42/44 MAPK

检测结缔组织生长因子(CTGF)是否诱导肾小球系膜细胞分泌正常T细胞表达分泌的活化调节因子(RANTES),并探讨其作用机制。应用CTGF刺激静息的培养大鼠肾小球系膜细胞,在刺激后不同时间点应用RT-PCR方法测定RANTES的mRNA表达,应用酶联免疫吸附试验(ELISA)测定上清液中RANTES。应用趋化试验测定上清液对单核细胞(THP-1)的趋化作用。应用Westernblot测定CTGF对丝裂原激活的蛋白激酶(p42/44MAPK)磷酸化的作用。应用磷酸化p42/44MAPK抑制剂PD98059预处理,观察CTGF对上清液中RANTES分泌的影响。结果显示,应用CTGF(100ng/ml)刺激后,系膜细胞的RANTES的mRNA表达上升,上清液中RANTES分泌量增加。RANTES抗体可部分阻止上清液对单核细胞的趋化作用。CTGF诱导p42/p44MAPK磷酸化,而PD98059可抑制这一作用,并部分抑制CTGF诱导的上清液中RANTES的分泌。研究表明,CTGF可引起系膜细胞分泌RANTES,其作用机制部分依赖于p42/p44MAPK的磷酸化。     

Regulation of pancreatic inflammation by connective tissue growth factor (CTGF/CCN2)

Pancreatitis is caused by long‐term heavy alcohol consumption, which results in injury and death of pancreatic acinar cells (PAC). The PAC play a pivotal role in mediating early inflammatory responses but the underlying mechanisms remain poorly understood. Treatment of C57BL/6 mice with ethanol and cerulein resulted in increased staining for acinar interleukin‐1β (IL‐1β), chemokine (C‐C motif) ligand 3 (CCL3), or connective tissue growth factor (CTGF/CCN2) by Day 16 and this was associated with increased infiltration of F4/80‐positive macrophages and increased expression of pancreatic CTGF/CCN2 mRNA. Compared with wild‐type Swiss Webster mice, ethanol treatment of pan‐green fluorescent protein (GFP)‐CTGF/CCN2 transgenic mice caused enhanced acinar staining for GFP or CTGF/CCN2 and a significant increase in pancreatic infiltration of F4/80‐positive macrophages or NIMP‐R14‐positive neutrophils. Treatment of primary mouse PAC or the rat AR42J PAC line with ethanol or CTGF/CCN2 resulted in enhanced expression of IL‐1β or CCL3. Conditioned medium from CTGF/CCN2‐treated AR42J cells induced chemotaxis in NR8383 macrophages and this response was abrogated in a dose‐dependent manner by addition of BX471, an inhibitor of chemokine (C‐C motif) receptor 1. These results reveal that acinar CTGF/CCN2 plays a novel role in alcohol‐induced inflammatory processes in the pancreas by increasing infiltration of macrophages and neutrophils and increasing acinar production of inflammatory mediators such as IL‐1β or CCL3. The early production of CTGF/CCN2 by PAC to drive inflammation is distinct from its previously reported production by pancreatic stellate cells to drive fibrosis at later stages of pancreatic injury.     

Extracellular matrix profiles in the progression to heart failure. European Young Physiologists Symposium Keynote Lecture-Bratislava 2007

The myocardial extracellular matrix (ECM), which preserves the geometry and integrity of the myocardium, is a dynamic structure whose component proteins are maintained by a finely controlled homeostatic balance between deposition and degradation. One of the key targets in cardiology is the elucidation of the molecular mechanisms which mediate pathological remodelling of this matrix causing the transition from compensatory hypertrophy to congestive decompensated heart failure. In response to injury or increased workload, cardiac remodelling including myocyte hypertrophy, develops as the heart attempts to compensate for increased wall stresses. Persistence of these stresses over extended time periods leads to disruption of ECM homeostasis resulting in irreversible maladaptive cardiac remodelling, ventricular dilatation and finally heart failure. ECM remodelling is regulated by the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). Clinical studies and experimental models of cardiac disease states have reported alterations in the balance between the MMPs and TIMPs in the failing heart and crucially at intermediate time points in the progression to failure. This article reviews the recent clinical, genetic and experimental approaches employed to compare ECM, MMP and TIMP profiles in healthy, compensated and failing hearts and identifies common themes in the perturbation of ECM homeostasis in the transition to heart failure.     

结缔组织生长因子在幼年兔关节软骨全层缺损修复中的表达及意义

目的:探讨结缔组织生长因子(CTGF)在幼年兔膝关节软骨全层缺损修复过程中的表达及意义。方法:采用兔膝关节股骨关节面全层软骨缺损自我修复模型,25只幼年新西兰白兔,随机分为5组(每组5只):对照组、损伤24 h组、1周组、4周组、8周组。应用RT-PCR技术和免疫组化技术分别检测CTGF mRNA和蛋白在修复过程中的表达。结果:幼年兔关节软骨全层损伤后自我修复良好。各组检测均见CTGF mRNA表达,损伤后各组表达水平显著高于对照组(P0.05)。免疫组化显示各组软骨细胞均有CTGF蛋白表达,阳性表达主要集中在软骨的中间带及深层带。结论:幼年兔关节软骨全层缺损自我修复过程伴有显著的CTGF表达上调,CTGF可能在关节软骨修复中发挥重要作用。     

The roles of connective tissue growth factor in the development of anastomotic esophageal strictures

Introduction

The aim of this study was to investigate the roles of connective tissue growth factor (CTGF) in the development of anastomotic strictures after surgical repair of the esophagus.

Material and methods

Tissues collected from the patients were divided into three groups based on the results of endoscopy and clinical grading. Patients without dysphagia after esophagectomy were used as the control population. The protein levels of CTGF, TGF-β1, Smad2, and Smad4 were determined by immunohistochemistry (IHC) and western blot analyses, while the mRNA levels of the two growth factors were evaluated by real-time polymerase chain reaction.

Results

Compared with the control group, significantly increased (p < 0.01) levels of CTGF and TGF-β1 protein were observed in the anastomotic stenosis (AS) group, and levels of the two proteins detected by the IHC and western blot analyses were also significantly increased with the increasing severity of stenosis (p < 0.05). The mRNA levels of CTGF and TGF-β1 in the tissues collected from the patients with stenosis were significantly up-regulated (p < 0.05) as compared with those from the control group. In addition, the levels of Smad2 and Smad4 protein were also significantly increased (p < 0.05) with the increasing severity of stenosis, and the protein levels were positively correlated with the levels of CTGF (r = 0.59, p < 0.05) and TGF-β1 (r = 0.63, p < 0.05).

Conclusions

Inhibition of CTGF protein or mRNA expression may be a distinctive and effective therapy for the treatment of postoperative anastomotic strictures.     

Inhibition of connective tissue growth factor (CTGF/CCN2) in gallbladder cancer cells leads to decreased growth in vitro

Gallbladder cancer (GBC) is an aggressive neoplasm associated with late diagnosis, unsatisfactory treatment and poor prognosis. Previous work showed that connective tissue growth factor (CTGF) expression is increased in this malignancy. This matricellular protein plays an important role in various cellular processes and its involvement in the tumorigenesis of several human cancers has been demonstrated. However, the precise function of CTGF expression in cancer cells is yet to be determined. The aim of this study was to evaluate the CTGF expression in gallbladder cancer cell lines, and its effect on cell viability, colony formation and in vitro cell migration. CTGF expression was evaluated in seven GBC cell lines by Western blot assay. Endogenous CTGF expression was downregulated by lentiviral shRNA directed against CTGF mRNA in G‐415 cells, and the effects on cell viability, anchorage‐independent growth and migration was assessed by comparing them to scrambled vector‐transfected cells. Knockdown of CTGF resulted in significant reduction in cell viability, colony formation and anchorage‐independent growth (P < 0.05). An increased p27 expression was observed in G‐415 cells with loss of CTGF function. Our results suggest that high expression of this protein in gallbladder cancer may confer a growth advantage for neoplastic cells.     

炔丙基甘氨酸增加高肺血流大鼠肺动脉结缔组织生长因子的表达

目的 探讨硫化氢(H2S)生成酶抑制剂炔丙基甘氨酸(PPG)对高肺血流大鼠肺动脉结缔组织生长因子(CTGF)表达的影响.方法 将32只雄性SD大鼠随机分为分流组、分流 炔丙基甘氨酸(PPG)组、假手术组和假手术 PPG组,每组8只.在大鼠高肺血流动物模型上,观察肺组织H2S含量、血浆ET-1含量、肺组织ET-1 mRNA的表达和肺动脉CTGF蛋白表达的变化.结果 分流4周后,肺组织H2S含量、血浆ET-1含量、肺组织ET-1 mRNA表达及肺动脉CTGF表达均明显升高(P<0.01);应用PPG干预后,分流 PPG组大鼠H2S含量明显降低;血浆ET-1含量、肺组织ET-1 mRNA的表达及肺动脉CTGF蛋白表达比分流组明显增加(P<0.05).结论 内源性H2S可能通过降低血管活性肽ET-1及CTGF在肺组织的表达参与对高肺血流时肺循环结构与功能的调节.     

Genistein ameliorates parathyroid hormone-induced epithelial-to-mesenchymal transition and inhibits expression of connective tissue growth factor in human renal proximal tubular cells

Introduction

Genistein, a soybean and soy-based product, has been reported to inhibit the growth of a wide range of cancer cells, but there is no evidence concerning its treatment of chronic kidney disease. The aim was to investigate whether genistein has potential to inhibit parathyroid hormone (PTH)-induced renal interstitial fibrosis.

Material and methods

Using human renal tubular epithelial HK-2 cells, α-smooth muscle actin (α-SMA) was assessed by using immunofluorescence detection. α-Smooth muscle actin, E-cadherin and connective tissue growth factor (CTGF) were measured by Western blot analysis. The promoter activity of the CTGF gene was examined by the luciferase reporter assay.

Results

When cells were treated with PTH (0.1 nM) for 48 h, α-SMA protein expression was induced significantly, the protein expression of E-cadherin decreased substantially, and the promoter activity of the CTGF gene as well as its mRNA and protein expression levels increased (p < 0.01). Interestingly, genistein effectively inhibited PTH-induced α-SMA expression, restored E-cadherin expression, decreased mRNA and protein expression of CTGF, and suppressed the promoter activity of CTGF in a dose-dependent manner.

Conclusions

Genistein has the ability to block the biomarker for renal transdifferentiation and epithelial-to-mesenchymal transition, α-SMA, following PTH treatment and inhibit CTGF expression in human renal tubular epithelial cells; these might be important modes of actions that contribute to genistein anti-fibrogenic effects and may have great implications for its potential in clinical treatment of renal interstitial fibrosis.     

Epithelial and connective tissue cell CTGF/CCN2 expression in gingival fibrosis

Gingival overgrowth is a side effect of certain medications and occurs in non-drug-induced forms either as inherited (human gingival fibromatosis) or idiopathic gingival overgrowth. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin; the least fibrotic lesions are caused by cyclosporin A; and intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Connective tissue growth factor (CTGF/CCN2) expression is positively related to the degree of fibrosis in these tissues. The present study has investigated the hypothesis that CTGF/CCN2 is expressed in human gingival fibromatosis tissues and contributes to this form of non-drug-induced gingival overgrowth. Histopathology/immunohistochemistry studies showed that human gingival fibromatosis lesions are highly fibrotic, similar to phenytoin-induced lesions. Connective tissue CTGF/CCN2 levels were equivalent to the expression in phenytoin-induced gingival overgrowth. The additional novel observation was made that CTGF/CCN2 is highly expressed in the epithelium of fibrotic gingival tissues. This finding was confirmed by in situ hybridization. Real-time polymerase chain reaction (PCR) analyses of RNA extracted from drug-induced gingival overgrowth tissues for CTGF/CCN2 were fully consistent with these findings. Finally, normal primary gingival epithelial cell cultures were analysed for basal and transforming growth factor beta1 (TGF-beta1) or lysophosphatidic acid-stimulated CTGF/CCN2 expression at protein and RNA levels. These data indicate that fibrotic human gingival tissues express CTGF/CCN2 in both the epithelium and connective tissues; that cultured gingival epithelial cells express CTGF/CCN2; and that lysophosphatidic acid further stimulates CTGF/CCN2 expression. These findings suggest that interactions between epithelial and connective tissues could contribute to gingival fibrosis.     

成纤维细胞生长因子10在前列腺增生组织中的表达

目的 检测前列腺增生组织中成纤维细胞生长因子10（FGF－10）的表达，探讨其在前列腺增生中的作用。方法 应用半定量R－TPCR技术和免疫组织化学方法，检测20例前列腺增生组织中FGF－10的表达，并与正常的前列腺组织进行比较。结果 前列腺增生组织中FGF－10 mRNA的水平高于正常前列腺组织，前列腺增生组织中FGF－10 mRNA的水平高于正常前列腺组织；前列腺组织中均见FGF－10阳性染色，前列腺增生组织中表达显著高于正常的前列腺组织。结论 前列腺增生组织中FGF－10的表达高于正常前列腺组织，提示FGF－10对促进前列腺的增生起重要作用。     

Concentrations of connective tissue growth factor in patients with nonalcoholic fatty liver disease: Association with liver fibrosis

Aim: In this study, we aimed to investigate the relationship between the histological fibrosis stage of nonalcoholic fatty liver disease (NAFLD) and serum connective tissue growth factor (CTGF) to determine the usefulness of this relationship in clinical practice. Methods: Serum samples were collected from 51 patients with biopsy-proven NAFLD and 28 healthy controls, and serum levels of CTGF were assayed by ELISA. Results: Levels of CTGF were significantly higher in patients with NAFLD compared with controls (P=0.001). The serum CTGF levels were significantly increased, that correlated with histological fibrosis stage, in patients with NAFLD [in patients with no fibrosis (stage 0) 308.2 ± 142.9, with mild fibrosis (stage 1-2) 519.9 ± 375.2 and with advanced fibrosis (stage 3-4) 1353.2 ± 610 ng/l, P < 0.001]. Also serum level of CTGF was found as an independent predictor of histological fibrosis stage in patients with NAFLD (β = 0.662, t=5.6, P <0.001). The area under the ROC curve was estimated 0.931 to separate patients with severe fibrosis from patients with other fibrotic stages. Conclusion: Serum levels of CTGF may be a clinical utility for distinguishing NAFLD patients with and without advanced fibrosis.     

Expression of connective tissue growth factor is in agreement with the expression of VEGF, VEGF-C, -D and associated with shorter survival in gastric cancer

Connective tissue growth factor (CTGF) is believed to be a multifunctional signaling modulator involved in a wide variety of biological or pathological processes including carcinogenesis. The role of CTGF in gastric cancer (GC) has not been reported so far. In the present study the expression of CTGF, vascular endothelial growth factor (VEGF), VEGF-C and VEGF-D on immunohistochemistry in GC and the correlation between the expression of CTGF and VEGF, VEGF-C, VEGF-D were examined, along with the correlation between the expression of CTGF and clinicopathological parameters, as well as survival of the patients with GC. The expression of CTGF was significantly in agreement with expression of VEGF, VEGF-C and VEGF-D (kappa and P, respectively: 0.538, P < 0.001; 0.502, P < 0.001; 0.558, P < 0.001). High CTGF expression was significantly associated with lymph nodes metastasis (P = 0.038) and lower postoperative 5 year overall survival rates (23.9%) compared with those patients with low CTGF expression (48.4%, P = 0.0035). The present findings suggest that CTGF is a useful prognostic marker for GC. High CTGF expression is associated with the risk of lymph nodes metastasis and a poor survival time in GC.