促甲状腺激素对大鼠心肌成纤维细胞影响的研究

目的:探讨促甲状腺激素(TSH)对SD乳鼠心肌成纤维细胞(CFs)的影响及机制。方法:新生1-3 d的SD乳鼠15只,处死后进行CFs的分离与培养,倒置显微镜对原代培养细胞形态进行鉴定,免疫组织化学染色法对CFs胞浆内波动蛋白进行染色并鉴定。后续实验过程中,按照使用b TSH工作液的稀释浓度分为A组(1μmol·L~(-1))、B组(2μmol·L~(-1))、C组(4μmol·L~(-1))。MTT法检测心肌成纤维细胞增殖率。酶联免疫吸附法(ELISA)检测培养细胞的上清液中Ⅰ、Ⅲ型胶原蛋白含量。荧光定量PCR反应测定培养细胞上清液中基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)m RNA的相对表达量。Western-blot法检测培养细胞上清液中MMP-2、MMP-9的蛋白表达水平。结果:CFs的形态学变化及纯度鉴定结果显示乳鼠CFs培养成功。MTT试验结果显示,24 h时后,A、B、C组CFs的增殖率分别为112.91±10.23、123.22±9.34、132.56±9.36;48 h时后,A、B、C组CFs的增殖率分别为124.4±8.34、133.5±9.02、139.6±11.36。随着b TSH浓度的增加,CFs增殖率呈逐渐增加趋势。ELISA检测结果显示,A、B、C组I型胶原蛋白表达水平分别为(2.61±0.31)μg·L~(-1)、(5.53±0.66)μg·L~(-1)、(7.91±0.74)μg·L~(-1);A、B、C组Ⅲ型胶原蛋白表达水平分别为(3.96±0.25)μg·L~(-1)、(6.72±0.52)μg·L~(-1)、(8.11±0.65)μg·L~(-1)。随着b TSH浓度的升高,I型和Ⅲ型胶原蛋白表达水平呈升高趋势。荧光定量PCR法检测结果显示,A、B、C组MMP-2的m RNA表达量分别为9.124±1.021、15.223±1.536、18.678±1.742;A、B、C组MMP-9的m RNA表达量分别为16.447±1.664、17.402±1.881、24.247±2.364。随着b TSH浓度的增加,MMP-2、MMP-9的m RNA相对表达量呈增加趋势。Western-blot法检测结果显示,A、B、C组MMP-2灰度值分别为165.41±16.57、198.25±21.34、223.41±19.08;A、B、C组MMP-9灰度值分别为140.30±15.09、190.47±20.06、230.14±21.45。随着b TSH浓度的增加,MMP-2、MMP-9的蛋白表达水平呈增加趋势。结论:TSH能够促进乳鼠CFs细胞增殖及I、Ⅲ型胶原蛋白表达增加,导致细胞外基质成分降解及合成过程失调。     

阿胶对COPD模型小鼠的保护作用以及对MMP-2、MMP-9、TGF-β1水平的影响

利用香烟烟雾暴露法建立慢性阻塞性肺疾病(COPD)小鼠模型,观察阿胶对模型小鼠气道重塑及基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、转化生长因子-β1(TGF-β1)因子表达的影响,初步探讨其治疗慢性阻塞性肺疾病的作用机制。将24只雄性C57BL/6小鼠随机分为3组,分别是对照组、模型组和阿胶组,模型组利用香烟烟雾暴露法建立COPD小鼠模型,阿胶组在模型组基础上每日按照3 g/kg体重给予0.2 m L阿胶烊化液灌胃。12周后采用ELISA法检测肺泡灌洗液中MMP-2、MMP-9、TGF-β1蛋白水平,利用RT-PCR法检测肺组织中MMP-2、MMP-9、TGF-β1 m RNA水平。结果表明,模型组小鼠气道壁和平滑肌明显增厚,气道管腔狭窄,黏膜下层稍变宽,有部分上皮细胞脱落,气道周围有炎性细胞浸润,肺间质明显增厚,阿胶组上述改变较模型组明显改善;模型组肺脏MMP-2、MMP-9、TGF-β1 m RNA水平和蛋白水平均显著升高(p0.01),阿胶能显著降低肺泡灌洗液和肺脏组织中MMP-2、MMP-9、TGF-β1的表达水平(p0.01)。本研究表明香烟烟雾暴露会导致模型小鼠呼吸道和肺脏组织细胞外基质的降解和沉积失衡,造成气道重塑和肺实质破坏及间质增生,MMP-2、MMP-9、TGF-β1的异常表达在这一过程中发挥了重要作用,阿胶能通过调节MMP-2、MMP-9、TGF-β1的异常表达,有效抑制气道炎症和气道重塑的发生。     

外源性精胺对糖尿病肾病小鼠肾纤维化的影响及其机制

目的: 观察外源性精胺对糖尿病肾病(DN)肾纤维化的保护作用,并探讨其机制。方法: 24 只雄性 C57 小鼠随机分为正常组(Control)、糖尿病组(T1D)和精胺预处理组(T1D+Sp,每组 n=8)。一次性注射 STZ(60 mg/kg)复制 1 型糖尿病小鼠模型,精胺预处理组在 STZ 注射前两周每天腹腔注射精胺(Sp,5 mg/(kg·d)),随后隔天注射精胺,第 12 周处死小鼠,检测血清肌酐、尿素氮判断肾功能变化,HE、PAS 和 Masson 染色观察肾组织损伤和纤维化水平。Western blot 法检测小鼠肾组织中基质金属蛋白酶(MMP-2、MMP-9)、IV型胶原(Coll-IV)蛋白的表达。结果: 与 Control 相比,T1D 组血糖(5.67±0.22 vs 28.40±0.57 mmol/L)、肌酐(14.33±1.22 vs 30.67±4.73 μmol/L)、尿素氮(6.93±4.94 vs 22.00±1.04 mmol/L)明显升高(P＜0.05),肾组织基底膜增厚,胶原含量明显增加,MMP-2、MMP-9 和 Coll-IV 蛋白表达均升高(分别为 0.57±0.07 vs 1.06±0.20、47.00±0.04 vs 1.29±0.09和0.42±0.16 vs 0.95±0.18,P＜0.05),精胺预处理明显减轻上述变化。结论: 外源性精胺预处理通过调节 MMPs 与胶原的平衡减轻 DN 小鼠的肾纤维化。     

地塞米松联合缬沙坦对慢性阻塞性肺疾病小鼠保护作用及机制探讨*

目的: 评估地塞米松联合缬沙坦对香烟所致慢性阻塞性肺疾病(COPD)小鼠的保护作用。方法: 40只C57BL/6小鼠随机分为(n=8):对照组、COPD组、地塞米松组、缬沙坦组和地塞米松+缬沙坦联合处理组。COPD组小鼠持续8周进行香烟暴露;在香烟暴露基础上,地塞米松组小鼠在5~8周香烟暴露前腹腔注射地塞米松(2 mg/kg);缬沙坦组小鼠在1~8周香烟暴露前腹腔注射缬沙坦(30 mg/kg);地塞米松+缬沙坦联合处理组小鼠腹腔注射地塞米松(2 mg/kg)和缬沙坦(30 mg/kg)。8周后收集各组小鼠肺组织及支气管肺泡灌洗液(BALF),评估肺组织病理学评分及BALF中超氧化物歧化酶(SOD)和基质金属蛋白酶9(MMP-9)活性,以及丙二醛(MDA)、细胞内黏附分子1(ICAM-1)、C反应蛋白(CRP)和一氧化氮(NO)含量。结果: 与对照组相比,COPD组小鼠存在肺气肿和肺泡充血,BALF中MDA、ICAM-1、MMP-9、CRP和淋巴细胞升高,SOD、巨噬细胞和NO降低(P均＜0.05)。与COPD组相比,地塞米松或缬沙坦组小鼠肺气肿和肺泡充血无明显改善,BALF中SOD 和NO升高,MDA、淋巴细胞和巨噬细胞降低(P均＜0.05)。与地塞米松或缬沙坦组相比较,地塞米松+缬沙坦联合处理组能更有效预防香烟引起的肺气肿和肺泡充血,降低BALF中MDA、ICAM-1、MMP-9、CRP和淋巴细胞,升高SOD、巨噬细胞和NO(P均＜ 0.05)。结论: 地塞米松联合缬沙坦通过抑制氧化应激和炎症,可以更有效在COPD小鼠中发挥保护作用。     

不同周龄Balb/c小鼠脏器质量及其变化趋势分析

目的:测定不同周龄Balb/c小鼠主要脏器质量、脏器系数,并进行比较。方法:取120只3周龄、5周龄、7周龄的Balb/c小鼠,雌雄各半,精确测量小鼠体重和主要脏器质量,计算脏器系数。结果:①雌性与雄性Balb/c小鼠脏器质量相比较:3周龄时肝、脾有显著差异(P0.05);5周龄时肝有非常显著差异(P0.01),脾、肺有显著差异(P0.05);7周龄时肝、肺及双肾有非常显著差异(P0.01),心、脾有显著差异(P0.05)。②雌性与雄性Balb/c小鼠脏器系数相比较:3周龄时肝、脾有显著差异(P0.05);5周龄时肝、脾有非常显著差异(P0.01),膀胱有显著差异(P0.05);7周龄时肺、双肾有非常显著差异(P0.01),脾、膀胱有显著差异(P0.05)。结论:随着周龄的增长,Balb/c雌、雄性小鼠之间,存在差异的脏器也在增多。     

2 型糖尿病动物模型KKAy小鼠肝、肾的病变

目的 研究2型糖尿病动物模型KKAy小鼠的糖尿病肝、肾的病变过程,探索KKAy小鼠作为2型糖尿病并发多脏器病变模型的价值.方法 9～11周龄雄性KKAy小鼠(n=20)和对照组的雄性C57BL/J小鼠(n=25),测量14、16、20、24、28周龄小鼠空腹血糖和体重.两组动物分别于24、28周处死,测定血清肌酐、尿素;胆固醇、甘油三酯;丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST).光镜观察两组小鼠肾脏、肝脏的病理改变.结果 ①KKAy小鼠体重、血糖比同龄对照组C57BL/J小鼠高(P<0.01).②24、28周龄KKAy小鼠血脂均较对照组高(P<0.05);24周龄KKAy小鼠肌酐,ALT比对照组高(P<0.05);28周龄KKAy小鼠的肌酐、ALT及AST比对照组高(P<0.05).③24、28周龄KKAy小鼠肾脏系膜基质增多,肾小管上皮细胞胞质出现明显空泡,肾问质有纤维化;肝脏结构紊乱,肝细胞胞质有明显空泡,肝细胞脂肪变.结论 KKAy小鼠14周龄以后可出现明显肥胖,高血糖,24周龄、28周龄出现高脂血症,有肝、肾形态和功能的损害,是较好的研究2型糖尿病病变过程及并发症的动物模型.     

黄芪甲苷在正常小鼠与2型糖尿病肾病 db/db小鼠体内分布差异的比较研究

为研究黄芪甲苷(Astragaloside IV,AS-Ⅳ)在正常小鼠(db/m)和2型糖尿病肾病(db/db)小鼠体内的组织分布差异,为AS-Ⅳ抗2型糖尿病肾病的临床运用及新药研发提供实验依据。以尾静脉注射给予2型糖尿病肾病(db/db)和正常小鼠(db/m)小鼠AS-Ⅳ,剂量为8 mg/kg。于15 min、2 h、4 h时处死小鼠,收集心、肝、脾、肺、肾、胃、小肠、脑、肌肉组织,采用HPLC-MS/MS法测定各组织中AS-Ⅳ含量,对比AS-Ⅳ在正常及病理状态下,在各组织中的分布差异。在2型糖尿病肾病状态下,AS-Ⅳ在肝、脾、肺、肾组织中浓度分别为349.72±70.72、370.69±45.46、5 830.65±581.75、4 290.63±485.34 ng/mL;正常状态下,AS-Ⅳ在肝、脾、肺、肾组织中浓度分别为202.47±47.96、267.92±41.24、4 725.80±867.51、2 354.55±256.11 ng/mL,在2型糖尿病肾病状态下,AS-Ⅳ在肝、脾、肺、肾组织中浓度显著高于其在正常组织中浓度(P0.05)。静脉注射给药后,肺、肾、心、胃、脾是其主要分布器官,其中在肺、肾组织中浓度最高;病理状态下,AS-Ⅳ组织分布发生了一定改变,为AS-Ⅳ防治2型糖尿病肾病的临床合理应用及开发提供实验依据。     

小鼠心肌纤维化模型的制备及其胶原沉积的机制

目的 建立缺血性心肌纤维化小鼠模型并探讨其胶原沉积机制.方法 将BALB/c小鼠随机分为实验组和对照组,每组10只.实验组予以腹部皮下注射异丙肾上腺素50 ms/kg,每天2次,连续10 d.对照组同法注射生理盐水.对比体表心电图,45 d后处死小鼠,天狼猩红染色观察心脏Ⅰ、Ⅲ型胶原纤维含量,荧光定量PCR检测心脏基质金属蛋白酶9(MMP-9)、基质金属蛋白酶组织抑制剂一1(TIMP-1)基因表达,免疫组化染色分析心、肝、肾组织层粘连蛋白(LN)的表达.结果 实验组小鼠室性心律失常增多,心率加快(P<0.05);心脏胶原沉积较多,MMP-9、TIMP-1、LN表达上调(P<0.05),肝、肾组织LN无明显改变(P>0.05).结论 异丙肾上腺素能制备缺血性心肌纤维化小鼠模型,其机制与MMP-TIMP失衡有关.     

中国南方灌丛优势木本植物叶的氮、磷含量及其影响因素

探索植物器官中氮(N)、磷(P)含量沿环境梯度的分异规律,有助于揭示陆地生态系统物质循环和植物养分适应策略的过程和机制。该文采用分层随机抽样法,在中国南方12个省市区布设462个灌丛样点,对其中193种优势木本植物叶N、P含量进行调查取样。结果表明:1)南方灌丛优势木本植物叶的N、P含量几何均值分别为16.57 mg·g~(–1)和1.02 mg·g~(–1);其中,落叶木本植物(17.91 mg·g~(–1)、1.14 mg·g~(–1))显著高于常绿木本植物(15.19 mg·g~(–1)、0.89 mg·g~(–1));叶P含量较N含量具有更大的变异性和环境依赖性。2)随年平均气温(MAT)的升高,常绿木本植物叶N、P含量降低,落叶木本植物叶N、P含量呈逐渐增加的趋势;随年降水量(MAP)的增加,常绿木本植物叶N含量降低,落叶木本植物叶N含量增加,两者P含量下降。3)土壤N含量的增加对两者叶的N含量无显著影响;但随土壤P含量的增加,常绿和落叶木本植物叶P含量均显著增加。4)广义线性回归显示,植物生活型分别可解释叶N、P变异的7.6%和14.4%,MAP和土壤P含量分别解释了0.8%和16.4%的叶P变异。结果表明,中国南方灌丛中优势植物叶的N含量主要受不同生活型植物生长需求所决定,而P含量则受气候、土壤和植物生活型共同决定。     

基质金属蛋白酶及其抑制剂在肝纤维化中的表达变化

目的 观察肝纤维化形成过程中基质金属蛋白酶MMP-1及其抑制剂TIMP-1的表达变化,从细胞外基质降解代谢的角度研究四氯化碳(CCl4)中毒性肝纤维化发生的机制.方法 雄性Wistar大鼠20只,分为正常组和肝纤维化模型组.肝纤维化组采用CCl4、饮酒、高脂低蛋白饮食等复合病因刺激制备肝纤维化动物模型,造模时间为8周.实验结束后测定肝脏指数、血清透明质酸(HA)、谷丙转氨酶(ALT)及尿羟脯氨酸(HYP)排出量,光镜下观察肝组织纤维化程度,并用免疫组化SABC法检测肝组织中Ⅰ、Ⅲ型胶原蛋白及MMP-1、TIMP-1的表达,同时用荧光实时定量PCR(RT-PCR)的方法检测肝组织中MMP-1、TIMP-1 mRNA的表达.结果 与正常对照组比较,肝纤维化模型组大鼠肝脏指数、血清HA及ALT显著增高,尿羟脯氨酸的排出量明显增加,病理组织学检查发现肝组织内纤维结缔组织增生明显,有假小叶形成;免疫组化的结果显示肝组织内Ⅰ、Ⅲ型胶原蛋白、MMP-1及TIMP-1的表达较正常组显著增加.结论 肝组织中MMP-1及TIMP-1的表达变化可能是导致肝纤维化的重要机制之一.     

MMP mediated degradation of type VI collagen is highly associated with liver fibrosis--identification and validation of a novel biochemical marker assay

Background and Aims

During fibrogenesis, in which excessive remodeling of the extracellular matrix occurs, both the quantity of type VI collagen and levels of matrix metalloproteinases, including MMP-2 and MMP-9, increase significantly. Proteolytic degradation of type VI collagen into small fragments, so-called neo-epitopes, may be specific biochemical marker of liver fibrosis. The aim of this study was to develop an ELISA detecting a fragment of type VI collagen generated by MMP-2 and MMP-9, and evaluate this assay in two preclinical models of liver fibrosis.

Methods

Mass spectrometric analysis of cleaved type VI collagen revealed a large number of protease-generated neo-epitopes. A fragment unique to type VI collagen generated by MMP-2 and MMP-9 was selected for ELISA development. The CO6-MMP assay was evaluated in two rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetrachloride (CCl4)-treated rats.

Results

Intra- and inter-assay variation was 4.1% and 10.1% respectively. CO6-MMP levels were significantly elevated in CCl₄-treated rats compared to vehicle-treated rats at weeks 12 (mean 30.9 ng/mL vs. 12.8 ng/mL, p = 0.002); week 16 (mean 34.0 ng/mL vs. 13.7 ng/mL, p = 0.0018); and week 20 (mean 35.3 ng/mL vs. 13.3 ng/mL, p = 0.0033) with a tight correlation between hepatic collagen content and serum levels of CO6-MMP (R² = 0.58, p<0.0001) in CCl₄- treated rats. In BDL rats, serum levels of CO6-MMP were significantly elevated compared to the levels in sham-operated animals both at 2 weeks (mean 29.5 ng/mL vs. 14.2 ng/mL, p = 0.0001) and 4 weeks (mean 33.0 ng/mLvs. 11.8 ng/mL, p = 0.0003).

Conclusions

This novel ELISA is the first assay enabling assessment of MMP degraded type VI collagen, allowing quantification of type VI collagen degradation, which would be relevant for different pathologies. The marker was highly associated with liver fibrosis in two liver fibrosis animal models, suggesting type VI turnover to be a central player in fibrogenesis.     

Three novel collagen VI chains, alpha4(VI), alpha5(VI), and alpha6(VI)

We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the alpha4(VI), alpha5(VI), and alpha6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (alpha4 and alpha6 chains) or three (alpha5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the alpha6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse alpha4(VI)N6-C2 chain co-assembled with endogenous alpha1(VI) and alpha2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, alpha5(VI)N5-C1 and alpha6(VI)N6-C2 chains did not assemble with alpha1(VI) and alpha2(VI) chains and accumulated intracellularly. We conclude that the alpha4(VI)N6-C2 chain contains all the elements necessary for trimerization with alpha1(VI) and alpha2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.     

Continuing Treatment with Salvia miltiorrhiza Injection Attenuates Myocardial Fibrosis in Chronic Iron-Overloaded Mice

Iron overload cardiomyopathy results from iron accumulation in the myocardium that is closely linked to iron-mediated myocardial fibrosis. Salvia miltiorrhiza (SM, also known as Danshen), a traditional Chinese medicinal herb, has been widely used for hundreds of years to treat cardiovascular diseases. Here, we investigated the effect and potential mechanism of SM on myocardial fibrosis induced by chronic iron overload (CIO) in mice. Kunming male mice (8 weeks old) were randomized to six groups of 10 animals each: control (CONT), CIO, low-dose SM (L-SM), high-dose SM (H-SM), verapamil (VRP) and deferoxamine (DFO) groups. Normal saline was injected in the CONT group. Mice in the other five groups were treated with iron dextran at 50 mg/kg per day intraperitoneally for 7 weeks, and those in the latter four groups also received corresponding daily treatments, including 3 g/kg or 6 g/kg of SM, 100 mg/kg of VRP, or 100 mg/kg of DFO. The iron deposition was estimated histologically using Prussian blue staining. Myocardial fibrosis was determined by Masson’s trichrome staining and hydroxyproline (Hyp) quantitative assay. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and protein expression levels of type I collagen (COL I), type I collagen (COL III), transforming growth factor-β1 (TGF-β₁) and matrix metalloproteinase-9 (MMP-9) were analyzed to investigate the mechanisms underlying the effects of SM against iron-overloaded fibrosis. Treatment of chronic iron-overloaded mice with SM dose-dependently reduced iron deposition levels, fibrotic area percentage, Hyp content, expression levels of COL I and COL III, as well as upregulated the expression of TGF- β₁ and MMP-9 proteins in the heart. Moreover, SM treatment decreased MDA content and increased SOD activity. In conclusion, SM exerted activities against cardiac fibrosis induced by CIO, which may be attributed to its inhibition of iron deposition, as well as collagen metabolism and oxidative stress.     

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1–100 ng for MT-2 and 10–1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our anti-serum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl₂ (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.     

Effects of basic fibroblast factor (bFGF) on MMP-2, TIMP-2, and type-I collagen levels in human lung carcinoma fibroblasts

Matrix metalloproteinases (MMP's) and tissue inhibitors of metalloproteinases (TIMP's) possess a preponderant role in the metabolism of the major extracellular matrix protein, collagen, and are thought to be important in the mechanism of tumor invasion. Lung cancer occupies the first position in mortality and the second position in incidence, among all cancers. In the present investigation, we studied the effect of basic fibroblast growth factor (bFGF) on collagen, matrix metalloproteinase-2 (MMP-2), and tissue metalloproteinase inhibitor-2 (TIMP-2) levels in normal and carcinoma lung tissue fibroblast cultures. MMP-2 was selected because of its high specificity in the degradation of type IV collagen, major component of the basal membrane. The effect of bFGF on MMP-2, TIMP-2, total collagen, and type I collagen levels of normal and carcinoma lung fibroblast cultures was investigated at 0, 10, and 100 ng/ml. Statistical analysis was carried out using the Mann-Whitney-U test and possible correlations were searched using the Spearman correlation analysis method. MMP-2, TIMP-2, total collagen, and type-1 collagen levels based on cell counts (10(3) cells) showed no statistically significant differences between the carcinoma and normal fibroblast cultures. However, positive correlations were found between MMP-2 and TIMP-2 in normal (P = 0.016) and carcinoma (P = 0.001) tissue fibroblast cultures. Positive correlations were also found between total collagen and TIMP-2 levels in normal and carcinoma tissue fibroblast cultures (P = 0.002 and P = 0.032). Total collagen and TIMP-2 levels also showed positive and strong correlations in all cultures except in 100 ng/ml bFGF concentrations. In addition, type I collagen and MMP-2 levels showed positive significant correlations only in normal and carcinoma control cultures, while type I collagen and TIMP-2 levels showed positive correlations in all cultures except carcinoma fibroblasts at 100 ng/ml bFGF. It may be concluded that bFGF does not affect MMP-2, TIMP-2, total collagen, and type-1 collagen levels in fibroblast cultures grown from human carcinoma and normal lung tissues. However, bFGF was noted, in vitro, to disturb the equilibrium which normally exists between the four parameters, both in normal and carcinoma tissue fibroblasts.     

Hypoxia modulates the effects of transforming growth factor-beta isoforms on matrix-formation by primary human lung fibroblasts

Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-beta (TGF-beta) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-beta on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-beta1, TGF-beta2 or TGF-beta3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-betas significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-beta and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-betas-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.     

Prostaglandin PGE2 at very low concentrations suppresses collagen cleavage in cultured human osteoarthritic articular cartilage: this involves a decrease in expression of proinflammatory genes, collagenases and COL10A1, a gene linked to chondrocyte hypertrophy

Suppression of type II collagen (COL2A1) cleavage by transforming growth factor (TGF)-β2 in cultured human osteoarthritic cartilage has been shown to be associated with decreased expression of collagenases, cytokines, genes associated with chondrocyte hypertrophy, and upregulation of prostaglandin (PG)E₂ production. This results in a normalization of chondrocyte phenotypic expression. Here we tested the hypothesis that PGE₂ is associated with the suppressive effects of TGF-β2 in osteoarthritic (OA) cartilage and is itself capable of downregulating collagen cleavage and hypertrophy in human OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with a wide range of concentrations of exogenous PGE₂ (1 pg/ml to 10 ng/ml). COL2A1 cleavage was measured by ELISA. Proteoglycan content was determined by a colorimetric assay. Gene expression studies were performed with real-time PCR. In explants from patients with OA, collagenase-mediated COL2A1 cleavage was frequently downregulated at 10 pg/ml (in the range 1 pg/ml to 10 ng/ml) by PGE₂ as well as by 5 ng/ml TGF-β2. In control OA cultures (no additions) there was an inverse relationship between PGE₂ concentration (range 0 to 70 pg/ml) and collagen cleavage. None of these concentrations of added PGE₂ inhibited the degradation of proteoglycan (aggrecan). Real-time PCR analysis of articular cartilage from five patients with OA revealed that PGE₂ at 10 pg/ml suppressed the expression of matrix metalloproteinase (MMP)-13 and to a smaller extent MMP-1, as well as the proinflammatory cytokines IL-1β and TNF-α and type X collagen (COL10A1), the last of these being a marker of chondrocyte hypertrophy. These studies show that PGE₂ at concentrations much lower than those generated in inflammation is often chondroprotective in that it is frequently capable of selectively suppressing the excessive collagenase-mediated COL2A1 cleavage found in OA cartilage. The results also show that chondrocyte hypertrophy in OA articular cartilage is functionally linked to this increased cleavage and is often suppressed by these low concentrations of added PGE₂. Together these initial observations reveal the importance of very low concentrations of PGE₂ in maintaining a more normal chondrocyte phenotype.     

Modified synthetic siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibrogenesis in rats

BACKGROUND/AIMS: Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the ECM turnover. We investigate the effect of modified synthetic small interfering RNA (siRNA) targeting TIMP-2 in rat model of liver fibrosis. METHODS: Rat hepatic fibrosis was induced by CCl4 for 8 weeks. After the 2-week CCl4 injection period, rats in the three siRNA groups simultaneously received a different dosage (0.05, 0.1 and 0.2 mg.kg(-1), respectively) of modified synthetic siRNA targeting TIMP-2 via the tail vein every 3 days for 6 weeks. The pathological changes in liver tissues were observed by light microscopy and transmission electron microscopy. Portal vein pressure and proliferating cell nuclear antigen were measured. Expression of TIMP-2, MMP-2, MT1-MMP, MMP-13, hepatocyte growth factor, collagen type I, collagen type III and alpha-SMA were evaluated by quantitative real-time polymerase chain reaction or Western blotting or gelatin zymography. RESULTS: Modified synthetic siRNA targeting TIMP-2 induced a dose-dependent inhibition of the TIMP-2 expression in the rat model of liver fibrosis with a similar trend in MMP-2 and MT1-MMP, but an increase in MMP-13. Rats administered siRNA targeting TIMP-2 showed promotion of ECM degradation, reduction in activated hepatic stellate cells and enhancement of hepatocyte regeneration. Furthermore, portal hypertension was also ameliorated after treatment with siRNA targeting TIMP-2. CONCLUSIONS: Knock-down of TIMP-2 expression attenuates CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.     

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

Background

Transforming growth factor β1 (TGF-β1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1α1 (COL1A1), CTGF and MMP-2 mRNA.

Methods

Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 μM) in the absence or presence of the PPARγ antagonist GW9662 (10 μM) before TGFβ-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR.

Results

TGFβ-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ.

Conclusions

RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPARγ-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-β1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis.