STAT1反义寡核苷酸雾化吸入对肺纤维化大鼠肺组织STAT1、ICAM-1及PDGF-A表达的影响

目的:观察信号转导子与转录活化子1(STAT1)反义寡核苷酸(ASON)雾化吸入对博莱霉素(BLM)致肺纤维化大鼠肺组织STAT1、细胞粘附分子-1(ICAM-1)和血小板衍生生长因子(PDGF)表达的影响.方法:取Wistar大鼠45只,随机分成生理盐水(NS)组、BLM组和ASON组,NS组气管内灌注NS,BLM组和ASON组气管内灌注BLM,随后NS组和BLM组雾化吸入NS,ASON组雾化吸入STAT1 ASON,隔天雾化一次,共4次,分别于雾化后第7天、第14天、第28天处死大鼠各5只,右肺行支气管肺泡灌洗(BAL)进行细胞分类、计数;左肺免疫组化法测定肺组织中STAT1、ICAM-1和PDGF-A蛋白的表达.结果:①与BLM组比较,ASON组各时间点肺泡炎和肺纤维化程度明显减轻(P＜0.05).②与NS组比较,BLM组、ASON组肺组织STAT1、ICAM-1 和PDGF-A表达水平均显著升高(P＜0.01).BLM组第7天STATl、ICAM-1 、PDGF-A在肺组织中表达水平显著升高,随后下降,第28天时仍高于NS组(P＜0.05).与BLM组比较,ASON组各时间点STAT1、ICAM-1 和PDGF-A表达水平降低(P＜0.05).结论:STAT1 ASON雾化吸入能减轻BLM致肺纤维化大鼠肺泡炎和肺纤维化,其机制可能与STAT1被抑制后,炎症细胞在肺部浸润减轻,致炎因子和致纤维化因子分泌减少有关.     

博来霉素诱导肺纤维化小鼠中差异表达的lncRNA筛选及生物信息学分析

目的:探讨博来霉素(BLM)诱导的肺纤维化小鼠模型肺组织中差异表达的长链非编码RNA(lncRNA),并对其进行生物信息学分析。方法:将12只6～8周龄的C57BL/6小鼠随机分为对照组和实验组,每组6只,实验组小鼠气管内滴注BLM构建肺纤维化模型,取小鼠肺组织进行芯片微阵列分析,筛选出差异表达的lncRNA,对差异表达的lncRNA进行靶基因预测,并对靶基因进行GO(gene ontology)富集与KEGG(Kyoto Encyclope-dia of Genes and Genomes)信号通路分析。结果:与对照组相比,肺纤维化模型组小鼠肺组织识别出差异表达的lncRNA共1 384个,其中表达上调的lncRNA有645个,表达下调的lncRNA有739个,并通过对差异表达lncRNA的靶基因预测,对靶基因进行GO富集与KEGG信号通路分析提示差异表达的lncRNA可能通过钙信号通路、PI3KAKT信号通路、Ras信号通路、Wnt信号通路、JAK-STAT信号通路和TGF-β信号通路等对肺纤维化进行调控。结论:基于芯片微阵列分析技术,lncRNA在BLM诱导的肺纤维化模型小鼠与正常小鼠肺组织中的表达存在差异,并在调控肺纤维化过程中可能涉及多个信号通路。     

穿心莲内酯对肺纤维化大鼠BALF中细胞因子和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响

目的:探讨穿心莲内酯灌胃对博来霉素(BLM)致肺纤维化大鼠支气管肺泡灌洗液(BALF)中TNF-α、TGF-β1浓度和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响。方法:取健康雄性SD大鼠90只,随机分为生理盐水(NS)组、BLM组、泼尼松(Pred)组、不同剂量穿心莲内酯组(即穿A组62.5mg/kg、穿B组125 mg/kg、穿C组250 mg/kg),各组大鼠15只,分别气管内灌注BLM(BLM组、Pred组、穿A组、穿B组、穿C组)或NS(NS组)后,每天给予NS(NS组、BLM组)、Pred(Pred组)或穿心莲内酯(穿A组、穿B组、穿C组)灌胃,各组分别于气管内灌注药物后第7、14、28天处死大鼠5只。用HE、Masson染色观察肺泡炎症和纤维化改变;实时荧光定量逆转录-聚合酶链反应检测肺组织Ⅰ、Ⅲ型胶原mRNA表达;酶联免疫吸附试验测定BALF中TNF-α、TGF-β1浓度;同时监测肾功能指标血尿素氮(BUN)、肌酐(Cr)及肝功能指标血丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)。结果:肝肾功能监测显示:不同剂量穿心莲内酯组、NS组、BLM组、Pred组所监测的AST、ALT、BUN、Cr比较,差异无统计学意义(P>0.05)。NS组大鼠肺组织未发现肺泡间隔水肿、炎性细胞浸润和纤维化形成。BLM组第7天时肺泡腔内可见大量炎性细胞浸润,第14天时肺泡炎仍存在,但炎症细胞明显减少,肺泡间隔内成纤维细胞明显增多,肺泡结构破坏,肺泡隔增宽,第28天时炎症较前减轻,肺纤维化程度加重,部分肺泡腔消失,形成严重纤维化。穿A组病理形态改变与BLM组相似。Pred组、穿B组、穿C组大鼠第7天有较多的炎症细胞浸润及局部聚积,第14天和第28天的纤维化病理改变均较BLM组、穿A组明显减轻。NS组各个时间点BALF中TGF-β1、TNF-α含量均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组BALF中的浓度(P<0.05)。BLM组3个时间点BALF中TGF-β1、TNF-α含量较Pred组、穿B、穿C组高(P<0.05)。与BLM组比较,穿A组BALF中TGF-β1、TNF-α含量无统计学意义。NS组各个时间点肺组织Ⅰ、Ⅲ型胶原mRNA表达均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组肺组织中的表达(P<0.05)。BLM组3个时间点Ⅰ、Ⅲ型胶原mRNA表达较Pred组、穿B组、穿C组高(P<0.05)。与BLM组比较,穿A组肺组织Ⅰ、Ⅲ型胶原mRNA表达无统计学差异。结论:穿心莲内酯灌胃可减轻BLM致肺纤维化大鼠肺泡炎和肺纤维化程度,降低肺组织Ⅰ、Ⅲ型胶原mRNA表达,降低BALF中TNF-α、TGF-β1浓度,且对肝肾无明显毒副作用。     

穿心莲内酯对肺纤维化大鼠BALF中细胞因子和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响

目的:探讨穿心莲内酯灌胃对博来霉素(BLM)致肺纤维化大鼠支气管肺泡灌洗液(BALF)中TNF-α、TGF-β1浓度和肺组织Ⅰ、Ⅲ型胶原mRNA表达的影响.方法:取健康雄性SD大鼠90只,随机分为生理盐水(NS)组、BLM组、泼尼松(Pred)组、不同剂量穿心莲内酯组(即穿A组62.5mg/kg、穿B组125 mg/kg、穿C组250 mg/kg),各组大鼠15只,分别气管内灌注BLM(BLM组、Pred组、穿A组、穿B组、穿C组)或NS(NS组)后,每天给予NS(NS组、BLM组)、Pred(Pred组)或穿心莲内酯(穿A组、穿B组、穿C组)灌胃,各组分别于气管内灌注药物后第7、14、28天处死大鼠5只.用HE、Masson染色观察肺泡炎症和纤维化改变;实时荧光定量逆转录-聚合酶链反应检测肺组织Ⅰ、Ⅲ型胶原mRNA表达;酶联免疫吸附试验测定BALF中TNF-α、TGF-β1浓度;同时监测肾功能指标血尿素氮(BUN)、肌酐(Cr)及肝功能指标血丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST).结果:肝肾功能监测显示:不同剂量穿心莲内酯组、NS组、BLM组、Pred组所监测的AST、ALT、BUN、Cr比较,差异无统计学意义(P＞0.05).NS组大鼠肺组织未发现肺泡间隔水肿、炎性细胞浸润和纤维化形成.BLM组第7天时肺泡腔内可见大量炎性细胞浸润,第14天时肺泡炎仍存在,但炎症细胞明显减少,肺泡间隔内成纤维细胞明显增多,肺泡结构破坏,肺泡隔增宽,第28天时炎症较前减轻,肺纤维化程度加重,部分肺泡腔消失,形成严重纤维化.穿A组病理形态改变与BLM组相似.Pred组、穿B组、穿C组大鼠第7天有较多的炎症细胞浸润及局部聚积,第14天和第28天的纤维化病理改变均较BLM组、穿A组明显减轻.NS组各个时间点BALF中TGF-β1、TNF-α含量均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组BALF中的浓度(P＜0.05).BLM组3个时间点BALF中TGF-β1、TNF-α含量较Pred组、穿B、穿C组高(P＜0.05).与BLM组比较,穿A组BALF中TGF-β1、TNF-α含量无统计学意义.NS组各个时间点肺组织Ⅰ、Ⅲ型胶原mRNA表达均明显低于同时间点BLM组、Pred组、穿A组、穿B组、穿C组肺组织中的表达(P＜0.05).BLM组3个时间点Ⅰ、Ⅲ型胶原mRNA表达较Pred组、穿B组、穿C组高(P＜O.05).与BLM组比较,穿A组肺组织Ⅰ、Ⅲ型胶原mRNA表达无统计学差异.结论:穿心莲内酯灌胃可减轻BuM致肺纤维化大鼠肺泡炎和肺纤维化程度,降低肺组织Ⅰ、Ⅲ型胶原mRNA表达,降低BALF中TNF-α、TGF-β1浓度,且对肝肾无明显毒副作用.     

甘草酸对博莱霉素诱导的实验性肺纤维化的干预作用

目的:探讨甘草酸(GA)对博莱霉素(BLM)诱导的小鼠肺纤维化的干预作用及其可能机制。方法:将160只雄性C57BL/6J小鼠随机分为生理盐水(NS)组、BLM组、BLM+NS组和BLM+GA组。通过口咽气管吸入法吸入博莱霉素(2.5 mg/kg)建立实验性肺纤维化模型,BLM+GA组及BLM+NS组每天给予40 mg/kg甘草酸或等体积的生理盐水灌胃,于术后第3、7、14、21天取材。采用HE染色和Masson染色观察肺组织病理学变化及纤维化程度,采用流式细胞术检测循环单核细胞和肺泡巨噬细胞的亚群比例变化,采用RT-qPCR检测肺组织中转化生长因子β1(TGF-β1)mRNA的表达水平,采用碱水解法检测肺组织中羟脯氨酸(HYP)含量。结果:与NS组相比,BLM组和BLM+NS组肺组织的炎症浸润及胶原含量明显增多,实验性肺纤维化模型制备成功。与BLM+NS组相比:BLM+GA组肺组织的炎症细胞浸润及胶原纤维沉积较少;第3、7天的Ly6C~(hi)单核细胞亚群比例和第7、14天肺泡巨噬细胞M2表型比例显著降低(P0.01);肺组织TGF-β1 mRNA表达量和HYP含量显著降低(P0.01)。结论:GA可以减轻博莱霉素诱导的小鼠肺组织的炎症反应和胶原纤维沉积,可能与GA对单核巨噬细胞的表型偏移和调控以及肺组织TGF-β1的表达下调有关。     

高氧对新生鼠肺组织VEGF蛋白表达及肺血管内皮细胞超微结构的影响

目的探讨高浓度氧对新生鼠肺组织血管内皮生长因子(VEGF)蛋白表达及肺血管内皮细胞超微结构影响的动态变化规律。方法建立高浓度氧诱导新生鼠CLD模型,60只新生鼠随机分为实验组和对照组,分别采用免疫组化和透射电镜技术,测定实验组和对照组在生后1d、3d、7d、14d和21dl肺组织内VEGF蛋白表达,同时观察肺血管内皮细胞超微结构变化。结果对照组肺组织VEGF蛋白表达1d主要以传导气道上皮为主,3d以后远端气道上皮表达增加,7d以后肺泡上皮和肺泡间隔明显增多,14d达高峰,以后持续高表达,实验组肺组织VEGF蛋白表达水平7d开始下降,14d以后未见阳性表达。高氧可引起肺血管内皮细胞肿胀、线粒体肿胀和毛细血管基底膜厚薄不均等各种损伤性形态变化,损伤程度随高氧时间延长而加重。结论肺血管的生长是正常肺泡发育重要环节,推测肺组织VEGF蛋白表达下降和肺血管内皮细胞的损伤在高氧诱导CLD肺血管发育障碍中可能发挥重要作用。     

苦参碱通过调节单核吞噬细胞表型偏移改善博莱霉素诱导的肺纤维化

目的:研究苦参碱(matrine,MA)对博菜霉素(bleomycin,BLM)诱导的循环单核细胞和肺泡巨噬细胞表型偏移的调节作用。方法:160只C57BL/6雄性小鼠随机分为生理盐水(NS)组、BLM组、苦参碱干预组(BLM+MA组)及溶剂对照组(BLM+NS组)经口咽吸入法给予BLM(2.5 mg/kg)建立实验性肺纤维化模型,对照组给予等体积NS,BLM+MA组和BLM+NS组分别在术后每天灌胃给予MA(15 mg·kg~(-1)·d~(-1))或等量NS。术后第3、7、14和21天处死小鼠,采用HE染色和Masson染色观察肺组织病理学变化及纤维化程度,采用碱水解法测定肺组织羟脯氨酸的含量,用流式细胞术分别检测循环单核细胞亚群和支气管肺泡灌洗液细胞表型的变化。结果:与对照组相比,MA干预可以明显减轻BLM诱导的小鼠肺组织炎症反应及纤维化程度(P0.05);与NS组相比,BLM组的Ly6C~(hi)单核细胞比例升高,肺泡巨噬细胞表型由M1型向M2型偏移且与炎症反应和纤维化程度呈现一定的相关性;MA干预后可以部分逆转BLM诱导的循环单核细胞和肺泡巨噬细胞的表型偏移。结论:苦参碱可以减轻BLM诱导的急性肺泡炎症和肺纤维化程度,可能是部分通过逆转循环单核细胞和肺泡巨噬细胞表型的偏移而实现的。     

丹参联合川芎嗪对肺纤维化大鼠血清、支气管肺泡灌洗液中TNF-α和TGF-β1水平的影响

目的观察丹参联合川芎嗪腹腔注射对博来霉素(BLM)致肺纤维化大鼠血清、支气管肺泡灌洗液(BALF)中TNF-α、TGF-β1水平的影响。方法将90只健康雄性SD大鼠,随机分为生理盐水(NS)组、BLM组、地塞米松(DXM)组、不同剂量中药联合干预组(即C1组:小剂量、C2组:中剂量、C3组:大剂量)6组。NS组大鼠气管内灌注NS,其余各组大鼠气管内灌注BLM造模,每天给予NS(NS组、BLM组)、DXM(DXM组)或不同剂量中药联合(C1、C2、C3组)腹腔注射,各组分别于气管内灌注药物后第7、14、28天处死大鼠各5只。肺组织HE染色和Masson染色观察肺泡炎和肺纤维化改变;ELISA检测血清和BALF中TNF-α、TGF-β1的水平。结果与NS组相比,BLM组第7天时肺泡炎较明显,第14天时有所减轻,出现肺纤维化,第28天时肺泡炎继续减轻,肺纤维化程度加重;DXM组不同时间点肺泡炎及肺纤维化程度均较BLM组减轻;C1组与BLM组相比,肺纤维化程度没有明显减轻;C2组和C3组大鼠第14天时肺泡炎及肺纤维化程度较BLM组明显减轻;C2组和C3组大鼠第28天时肺纤维化程度较BLM组明显减轻。与NS组比较,各组大鼠血清和BALF中TNF-α、TGF-β1水平均明显增加;与BLM组比较,DXM组和C2组、C3组不同时间点大鼠血清和BALF中TNF-α、TGF-β1水平均降低;与DXM组比较,不同剂量中药联合干预组不同时间点大鼠血清和BALF中TNF-α、TGF-β1表达均降低;以上比较差异均有统计学意义(P<0.05);C2组和C3组不同时间点血清和BALF中TNF-α、TGF-β1水平差异无统计学意义(P>0.05)。各组大鼠血清和BALF中TNF-α、TGF-β1的水平呈高度一致性。结论中、大剂量丹参联合川芎嗪腹腔注射可减轻BLM致肺纤维化大鼠肺泡炎和肺纤维化程度,其机制可能与降低血清和BALF中TNF-α、TGF-β1的表达水平有关。     

不同发育期C57BL/6小鼠脑内TRPV1表达变化与热性惊厥的关系

<正>目的:检测不同发育阶段C57BL/6小鼠大脑TRPV1表达变化及其与热性惊厥的关系。方法:应用qRT-PCR和免疫组化方法检测2周龄、4周龄、8周龄和16周龄C57BL/6小鼠脑海马和皮质内TRPV1mRNA和蛋白表达变化,同时应用热气浴(43℃)方法诱导C57BL/6小鼠热性惊厥模型。结果:不同发育阶段C57BL/6小鼠海马和皮质中TRPV1mRNA和蛋白表达有明显差异,且热性惊厥模型鼠海马和皮质中TRPV1表达明显高于正常对照鼠。     

小鼠实验性肺纤维化上皮细胞-间充质细胞转变及骨桥蛋白的表达

目的从形态学上证实小鼠实验性肺纤维化上皮细胞-间充质细胞转变(EMT)及骨桥蛋白(OPN)的表达变化。方法将60只健康雄性C57BL/6小鼠随机分为生理盐水对照组和博来霉素(BLM)模型组,采用口咽抽吸法经咽部注入气管50μl博来霉素溶液,对照组注入50μl生理盐水。分别在造模后第3d、7d、14d、21d及28d 5个时间点处死对照组及模型组小鼠。取小鼠左肺制备石蜡切片,行苏木素-伊红(HE)染色及天狼猩红染色观察肺组织形态变化及胶原增生情况;行表面活性物质相关蛋白-C(SP-C)、上皮型钙黏蛋白(E-Cadherin)、波形蛋白(vimentin)、成纤维细胞特异蛋白1(FSP1)和骨桥蛋白(OPN)免疫组织化学染色,观察肺纤维化上皮细胞-间充质细胞转变(EMT)形态与OPN表达,利用图像分析软件Image-Pro Plus 6.0(IPP6.0)测量切片积分吸光度(IA),并进行统计分析。结果HE及天狼猩红染色显示出小鼠肺组织正常结构和纤维化形态变化,并且模型组小鼠肺组织的胶原生成量随时间推移而增加(P<0.05)。免疫组织化学结果显示,模型组小鼠肺OPN表达量随病程进展持续升高,在纤维化期达到峰值,而对照组表达量很少(P<0.05);对照组SP-C在Ⅱ型肺泡细胞恒定表达,模型组SP-C持续增加,且胞体增大;对照组E-Cadherin表达量多,而模型组在肺实变部位几乎不表达,但肺实变部位产生波形蛋白及FSP1,这些蛋白提示肺纤维化存在EMT。结论BLM诱导性肺纤维化中存在EMT,且与显著增加的OPN有潜在关系。     

Endothelial Fenestration of the Alveolar Capillaries in Interstitial Fibrotic Lung Diseases

A light and electron microscopy study was performed on endothelial cells of alveolar capillaries in biopsied lung tissues obtained from 28 patients with interstitial fibrotic lung diseases, including idiopathic pulmonary fibrosis, sar-coidosis, hypersensitivity pneumonitis, chronic eosinophilic pneumonia, collagen vascular diseases and acute interstitial pneumonia. In the relatively early stages of acute interstitial pneumonia, hypersensitivity pneumonitis and other diseases, the cytoplasms of the endothelial cells appeared swollen and electron-lucent and occasionally showed degeneration and necrosis. Although mitosis was not evident in the endothelium at any disease stage, some capillary endothelial cells showed regeneration. Furthermore, although rarely, they showed obvious phenotypic transformation into diaphragmed fenestrae in some limited segments of fibrotic lungs in the 20 of the 28 patients examined. The frequency of endothelial fenestration seemed to be correlated with the degree of interstitial fibrosis along the alveolar walls. In such fibrotic lung tissues, cuboidal metaplastic cells of bronchiolar origin proliferated on the luminal side. The mechanism of endothelial fenestration in the alveolar capillaries is assumed to be comparable with cuboidal metaplasia of alveolar epithelial cells. The alveolar capillary endothelium is recruited from the bronchiolar capillaries via bronchopul-monary anastomoses unless endothelial repair occurs in situ . Regenerating endothelial cells move into the alveolar capillary tubes along the remnant sleeves of the basement membrane. New endothelial processes finally display their original fenestrated structure while secreting irregular fragments of basement membrane during implantation in the capillary beds. Acta Pathol Jpn 42: 177– 184, 1992.     

Endothelial fenestration of the alveolar capillaries in interstitial fibrotic lung diseases.

A light and electron microscopy study was performed on endothelial cells of alveolar capillaries in biopsied lung tissues obtained from 28 patients with interstitial fibrotic lung diseases, including idiopathic pulmonary fibrosis, sarcoidosis, hypersensitivity pneumonitis, chronic eosinophilic pneumonia, collagen vascular diseases and acute interstitial pneumonia. In the relatively early stages of acute interstitial pneumonia, hypersensitivity pneumonitis and other diseases, the cytoplasms of the endothelial cells appeared swollen and electron-lucent and occasionally showed degeneration and necrosis. Although mitosis was not evident in the endothelium at any disease stage, some capillary endothelial cells showed regeneration. Furthermore, although rarely, they showed obvious phenotypic transformation into diaphragmed fenestrae in some limited segments of fibrotic lungs in the 20 of the 28 patients examined. The frequency of endothelial fenestration seemed to be correlated with the degree of interstitial fibrosis along the alveolar walls. In such fibrotic lung tissues, cuboidal metaplastic cells of bronchiolar origin proliferated on the luminal side. The mechanism of endothelial fenestration in the alveolar capillaries is assumed to be comparable with cuboidal metaplasia of alveolar epithelial cells. The alveolar capillary endothelium is recruited from the bronchiolar capillaries via bronchopulmonary anastomoses unless endothelial repair occurs in situ. Regenerating endothelial cells move into the alveolar capillary tubes along the remnant sleeves of the basement membrane. New endothelial processes finally display their original fenestrated structure while secreting irregular fragments of basement membrane during implantation in the capillary beds.     

T cell independence of bleomycin-induced pulmonary fibrosis

The role of T cells and cytokines in bleomycin (BLM)-induced fibrosis was evaluated in susceptible and resistant strains of normal and SCID mice. Histology and hydroxyproline analysis showed that BLM induced pulmonary fibrosis in C57BL/6 and (C57BL/6 x BALB/c)F1 mice, whereas BALB/c mice were resistant to the disease. To test whether lymphocytes were required for the induction of BLM-induced pulmonary fibrosis, SCID mice were injected intratracheally with BLM and evaluated for the development of pulmonary inflammation and fibrosis. Similar morphological changes and increases in hydroxyproline were observed in both C57BL/6 SCID and (C57BL/6 x CB.17)F1 SCID animals compared to those seen in wild-type C57BL/6 and (C57BL/6 x BALB/c)F1 mice. In contrast, CB.17 SCID mice, which are genetically similar to BALB/c mice, were resistant to disease induction. Analysis of the cellular infiltrate in BLM-treated C57Bl/6 SCID mice confirmed a lack of T cells in the lungs of SCID mice and demonstrated a pronounced accumulation of eosinophils in areas of developing pulmonary fibrosis. NK cells were significantly elevated in untreated SCID mice and did not increase further after BLM treatment. Analysis of selected cytokines 1 day after initiation of BLM-induced pulmonary fibrosis indicated that the levels of TNF-alpha and IFN-gamma appeared to segregate with fibrosis in both the SCID and wild-type mice. The data demonstrate that T cells are not required for the induction of fibrosis by BLM and suggest that responses by non-lymphoid cells may be sufficient for the induction of fibrosis.     

Effects of injury and repair of the pulmonary endothelium on lung metastasis after bleomycin

Acute endothelial injury induced by bleomycin has been shown to enhance the localization and metastasis of circulating tumour cells. In the present study we wished to determine whether increased metastases to the lung is related to the degree of endothelial damage as indicated by morphology and protein leakage to alveoli and whether the progression to repair with pulmonary fibrosis also effects metastatic tumour growth. C57b1/6 mice were injected with a single intravenous dose of bleomycin (120 mg/kg). After 5 days, severe enothelial injury was demonstrated by morphology and by increased levels of protein in lung lavage fluid. When [131I]-iododeoxyuridine labeled syngeneic fibrosarcoma cells were injected intravenously at this time, a 9-fold increase in their localization was detected 24 h later in bleomycin-treated lungs compared with saline controls. By electron microscopy tumour cells were observed at sites of denuded vascular basement membrane. There was also a significant increase in the number of gross metastases which developed subsequently and in the percentage of lung occupied by tumour in the bleomycin group. Animals examined 10 days after bleomycin showed less endothelial damage and a smaller increase in tumour cell localization and metastases. At 21 days, when endothelial structure and alveolar protein levels had returned to normal, and at 6 weeks, when there was focal fibrosis, no increase in tumour cell localization or metastases was found. It is concluded that damage to the pulmonary endothelium is a key factor in enhancing the trapping of circulating tumour cells and increasing metastatic tumour growth after bleomycin.     

Bone marrow origin of myofibroblasts in irradiation pulmonary fibrosis

There is a rapid onset of organizing alveolitis/fibrosis at 120-140 d after whole lung irradiation of C57BL/6J mice. To test the hypothesis that circulating cells of bone marrow origin contribute to irradiation fibrosis, irradiated chimeric green fluorescent protein (GFP)+ C57BL/6J mice were followed for GFP+ cells in areas of lung fibrosis. In a second experimental model, C57BL/6J female mice received 20 Gy total lung irradiation, and after 60 or 80 d were intravenously injected with cells from a clonal GFP+ male bone marrow stromal cell line or male GFP+ whole bone marrow, respectively. The mice were then followed for the development of pulmonary fibrosis, and the contribution of Y-probe-positive, GFP+ cells to fibrotic areas was quantitated. Bromodeoxyuridine labeling of developing fibrotic areas showed that the cell division occurred predominantly in GFP+, Y-probe-positive, and vimentin-positive cells. Immunohistochemistry demonstrated that these cells were macrophages and fibroblasts, not endothelial cells. Mice that received manganese superoxide dismutase-plasmid/liposome intratracheal injection 24 h before total lung irradiation demonstrated a decrease in GFP+ fibroblastic cells in the lung. Thus, pulmonary irradiation fibrosis contains proliferating cells of bone marrow origin, and gene therapy prevention of this condition acts in part by decreasing the migration and proliferation of marrow origin cells.     

Differences in the fibrogenic response after transfer of active transforming growth factor-beta1 gene to lungs of "fibrosis-prone" and "fibrosis-resistant" mouse strains

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the interstitium, resulting in impaired lung function and respiratory failure. Investigation of the differences in individual susceptibility to the development of fibrosis may help to detect patients that are at risk to fibrosis when exposed to fibrogenic stimuli. In this study we used adenoviral gene transfer to transiently expose a fibrosis-prone (C57BL/6) and a fibrosis-resistant (Balb/c) mouse strain to high levels of active transforming growth factor (TGF)-beta1, a key profibrotic cytokine. Balb/c mice developed significantly less fibrosis compared with C57BL/6 mice in response to active TGF-beta1 despite higher levels of the transgene protein in the lung. This was not due to a general unresponsiveness of cells to TGF-beta1, because primary fibroblasts of both strains increased collagen synthesis upon stimulation with TGF-beta1 in vitro to the same degree. However, TGF-beta1 induced a strong upregulation of tissue inhibitor of metalloprotease-1 gene in pulmonary fibroblasts as well as in lungs of C57BL/6 mice, in contrast to a weak induction in Balb/c mice. These findings suggest that the differences in susceptibility to pulmonary fibrosis are downstream from TGF-beta1 and that fibrosis-prone individuals may have an altered collagen metabolism in the lungs that is balanced toward a "nondegrading" environment.     

Susceptibility of Hermansky-Pudlak mice to bleomycin-induced type II cell apoptosis and fibrosis

Pulmonary inflammation, abnormalities in type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS), a recessive disorder associated with intracellular trafficking defects. We have previously reported that "Pearl" (HPS2) and "Pale Ear" (HPS1) mouse models have pulmonary inflammatory dysregulation and constitutive alveolar macrophage (AM) activation (Young LR et al., J Immunol 2006;176:4361-4368). In the current study, we used these HPS models to investigate mechanisms of lung fibrosis. Unchallenged HPS1 and HPS2 mice have subtle airspace enlargement and foamy AMs, but little or no histologic evidence of lung fibrosis. Seven days after intratracheal bleomycin (0.025 units), HPS1 and HPS2 mice exhibited increased mortality and diffuse pulmonary fibrosis compared to strain-matched C57BL/6J wild-type (WT) mice. HPS mice had significantly increased collagen deposition, and reduced quasi-static and static compliance consistent with a restrictive defect. The early airway and parenchymal cellular inflammatory responses to bleomycin were similar in HPS2 and WT mice. Greater elevations in levels of TGF-beta and IL-12p40 were produced in the lungs and AMs from bleomycin-challenged HPS mice than in WT mice. TUNEL staining revealed apoptosis of type II cells as early as 5 h after low-dose bleomycin challenge in HPS mice, suggesting that type II cell susceptibility to apoptosis may play a role in the fibrotic response. We conclude that the trafficking abnormalities in HPS promote alveolar apoptosis and pulmonary fibrosis in response to bleomycin challenge.     

Role of the SDF-1/CXCR4 axis in the pathogenesis of lung injury and fibrosis

Stromal cell-derived factor-1 (SDF-1) participates in mobilizing bone marrow-derived stem cells, via its receptor CXCR4. We studied the role of the SDF-1/CXCR4 axis in a rodent model of bleomycin-induced lung injury in C57BL/6 wild-type and matrix metalloproteinase (MMP)-9 knockout mice. After intratracheal instillation of bleomycin, SDF-1 levels in serum and bronchial alveolar lavage fluid increased. These changes were accompanied by increased numbers of CXCR4(+) cells in the lung and a decrease in a population of CXCR4(+) cells in the bone marrow that did not occur in MMP-9(-)/(-) mice. Both SDF-1 and lung lysates from bleomycin-treated mice induced migration of bone marrow-derived stem cells in vitro that was blocked by a CXCR4 antagonist, TN14003. Treatment of mice with TN14003 with bleomycin-induced lung injury significantly attenuated lung fibrosis. Lung tissue from patients with idiopathic pulmonary fibrosis had higher numbers of cells expressing both SDF-1 and CXCR4 than did normal lungs. Our data suggest that the SDF-1/CXCR4 axis is important in the complex sequence of events triggered by bleomycin exposure that eventuates in lung repair. SDF-1 participates in mobilizing bone marrow-derived stem cells, via its receptor CXCR4.     

In situ evidence of collagen V and signaling pathway of found inflammatory zone 1 (FIZZ1) is associated with silicotic granuloma in lung mice

Inhalation of silica particles causes silicosis: an occupational lung disease characterized by persistent inflammation with granuloma formation that leads to tissue remodeling and impairment of lung function. Although silicosis has been studied intensely, little is known about the crucial cellular mechanisms that initiate and drive the process of inflammation and fibrosis. Recently, found in inflammatory zone 1 (FIZZ1) protein, produced by alveolar macrophages and fibroblasts have been shown to induce the proliferation of myofibroblasts and their transdifferentiation, causing tissue fibrosis. Moreover, autoimmunogenic collagen V, produced by alveolar epithelial cells and fibroblasts, is involved in the pathophysiology of interstitial pulmonary fibrosis and bleomycin-induced lung fibrosis. Based on the aforementioned we hypothesized that FIZZ1 and collagen V may be involved in the silicotic granuloma process in mice lungs. Male C57BL/6 mice (N = 20) received intratracheal administration of silica particles (Silica; 20 mg in 50 μL saline) or saline (Control; 50 μL). After 15 days, the lung histology was performed through immunohistochemistry and morphometric analysis. Within silicotic granulomas, collagen V and FIZZ1 increased, while peroxisome proliferator-activated receptor gamma (PPARγ) positive cells decreased. In addition, the expression of proteins Notch-1, alpha smooth muscle actin (α-SMA) and macrophages163 (CD163) were higher in silicotic granulomas than control lungs. A significant positive correlation was found between collagen V and FIZZ1 (r = 0.70; p < 0.05), collagen V and Notch-1 (r = 0.72; p < 0.05), whereas Collagen V was inversely associated with peroxisome proliferator-activated receptor gamma (r=-0.69; p < 0.05). These findings suggested that collagen V association with FIZZ1, Notch-1 and PPARγ might be a key pathogenic mechanism for silicotic granulomas in mice lungs.