Keratinocyte growth factor gene transduction ameliorates pulmonary fibrosis induced by bleomycin in mice

Pulmonary fibrosis has high rates of mortality and morbidity, but there is no established therapy at present. We demonstrate here that bleomycin-induced pulmonary fibrosis in mice is ameliorated by intratracheal administration of keratinocyte growth factor (KGF)-expressing adenovirus vector. Progressive pulmonary fibrosis was created by continuous subcutaneous administration of 120 mg/kg of bleomycin subcutaneously using an osmotic pump twice from Day 1 to 7 and Day 29 to 35. The mice initially exhibited subpleural fibrosis and then exhibited advanced fibrosis in the parenchyma of the lungs. These histopathological changes were accompanied by reduced lung compliance (0.041 ± 0.011 versus 0.097 ± 0.004; P < 0.001), reduced messenger expression of surfactant proteins, and reduced KGF messenger expression in the lungs at 4 weeks compared with naive group. Intratracheal instillation of Ad-KGF at 1 week after the first administration of bleomycin increased KGF mRNA expression in the lungs compared with the fibrosis-induced mice that received saline alone. The phenotype was associated with alveolar epithelial cell proliferation, increased pulmonary compliance (0.062 ± 0.005 versus 0.041 ± 0.011; P = 0.023), and decreased mortality (survival rate on Day 56: 68.8% versus 0%; P = 0.002), compared with mice receiving only the saline vehicle. These observations suggest the therapeutic utility of a KGF-expressing adenoviral vector for pulmonary fibrosis.     

博莱霉素肺纤维化小鼠血管内皮细胞的研究

目的:研究C57BL/6小鼠肺微小血管内皮细胞血栓调节蛋白(TM)与因子Ⅷ相关抗原(vWf)的分布及博莱霉素(BLM)致肺纤维化过程中,血管内皮细胞亚型的转变。方法:采用双重免疫荧光染色及荧光强度定量分析法。结果:①正常C57BL/6小鼠肺泡毛细血管内皮细胞表面显示多数连续性线样TM荧光而vWf较少阳性,肺微小血管内皮细胞呈现vWf阳性。②BLM组小鼠内皮细胞TM荧光明显弱于正常,而vWf荧光显著强于正常水平。结论:①正常C57BL/6小鼠肺泡毛细血管内皮细胞以TM表达为主型,而非vWf表达为主型。②BLM致肺纤维化过程中,肺血管内皮细胞由以TM表达为主型转变为以vWf表达为主型,两抗原可被认为是内皮细胞损伤的标志物。     

Granulocyte colony-stimulating factor exacerbates the acute lung injury and pulmonary fibrosis induced by intratracheal administration of bleomycin in rats

We investigated the effects of granulocyte colony-stimulating factor (G-CSF) on lung injury induced by intratracheal administration of bleomycin (BLM, 2 mg/200 micro1) in rats. In experiment 1, G-CSF (10, 30 and 100 microg/kg/day, s.c.) was administered to rats treated with BLM or saline for 7 days starting immediately after BLM administration. In rats receiving G-CSF alone, a large number of neutrophils were noted in the pulmonary capillaries, although there were no lung lesions. In rats receiving BLM alone, diffuse alveolar damage was observed. The administration of G-CSF to BLM-treated rats increased the total lung lesion per unit of pulmonary parenchyma (total lung lesion %) along with increases in the peripheral neutrophil count and the number of neutrophils infiltrating in the pulmonary lesion in a dose-dependent fashion. In experiment 2, 100 microg/kg/day of G-CSF was administered to rats treated with BLM or saline for up to 28 days starting immediately after BLM administration. The administration of 100 microg/kg/day of G-CSF to BLM-treated rats showed no effects at 14 days but it increased the lung lesion % and the score of lung fibrosis along with the increase in the number of neutrophils infiltrating in the pulmonary lesion at 28 days. These findings suggest that G-CSF administration to BLM-treated rats influenced and exacerbated the BLM-induced acute lung injury, and also exacerbated pulmonary fibrosis in a dose-dependent fashion. The exacerbation of lung injury coincided with the marked increase in the peripheral neutrophil count and the number of neutrophils infiltrating in the pulmonary lesion.     

Expression of pulmonary aquaporin 1 is dramatically upregulated in mice with pulmonary fibrosis induced by bleomycin

Introduction

Pulmonary fibrosis occurs due to fibroblast proliferation and collagen production in the lung and begins with alveolar inflammatory edema. Aquaporins (AQPs) play pivotal roles in lung fluid transport. In this study we establish the experimental model for pulmonary fibrosis in C57BL/6 mice to investigate expressions of AQP1 and AQP5 in lung tissue.

Material and methods

Mice in model groups were treated intratracheally with bleomycin with the dose of 5 mg/kg body weight. The mice were sacrificed at 1 week, 2 weeks and 3 weeks respectively. The left upper lungs were harvested for histopathologic H-E and Masson''s staining. The mRNAs of AQP1 and AQP5 were analyzed by real-time polymerase chain reaction (real-time PCR) and the proteins of AQP1 and AQP5 were analyzed by western blotting.

Results

Real-time PCR showed that AQP1 mRNA in bleomycin 1 w, 2 w, and 3 w groups increased by 377%, 880% and 823% respectively compared to that in the control group (p < 0.01). Western blotting showed that the expression of AQP1 protein in bleomycin 1 w, 2 w, and 3 w groups increased by 53%, 144%, and 141%, respectively (p < 0.05). AQP5 mRNA in bleomycin 1 w and 2 w group decreased by 78% and 66%, respectively (p < 0.05). In bleomycin 2 w and 3 w groups it decreased by 69% and 80% (p < 0.05).

Conclusions

The expression of AQP1 dramatically increased in pulmonary fibrosis. AQP1 plays an important role in the progress of pulmonary fibrosis.     

Overexpression of tumor necrosis factor-alpha diminishes pulmonary fibrosis induced by bleomycin or transforming growth factor-beta

Tumor necrosis factor-alpha (TNF-alpha) is thought to be important in the development of pulmonary fibrosis. However, surfactant protein-C/TNF-alpha transgenic mice do not spontaneously develop pulmonary fibrosis but instead develop alveolar enlargement and loss of elastic recoil. We hypothesized that overexpression of TNF-alpha in the lung requires an additional insult to produce fibrosis. In this study we evaluated whether TNF-alpha overexpression altered the development of pulmonary fibrosis due to bleomycin or transforming growth factor-beta (TGF-beta). Either 0.2 U bleomycin or saline was administered into left lung of TNF-alpha transgenic mice and their transgene-negative littermates. To overexpress TGF-beta, an adenovirus vector containing either active TGF-beta (AdTGF-beta) or LacZ was administered at a dose of 3 x 108 plaque-forming units per mouse. Fibrosis was assessed histologically and by measurement of hydroxyproline. TNF-alpha transgenic mice tolerated bleomycin or AdTGF-beta, whereas the transgene-negative littermates demonstrated severe pulmonary fibrosis after either agent. An increase in prostaglandin E2 and downregulation of TNF receptor I expression were observed in the TNF-alpha transgenic mice. In addition, recombinant human TNF-alpha attenuated bleomycin-induced pulmonary fibrosis. TNF-alpha has a complex role in the development of pulmonary fibrosis. Endogenous TNF-alpha may be important in the development of fibrosis as indicated in other reports, but overexpression of TNF-alpha or exogenous TNF-alpha limits pulmonary fibrosis in mice.     

层黏连蛋白受体1单克隆抗体抑制博莱霉素诱导的大鼠肺纤维化

目的 研究层黏连蛋白受体1(LAMRl)单克隆抗体(mAb)干预博莱霉素诱导的大鼠肺纤维化.方法 雄性SD大鼠72只,随机分为LAMRl mAb组(L)、对照组(C)和模型组(M)3组,气管内滴入博莱霉素(5 mg/kg体质量)制备肺纤维化模型.3组分别给予LAMRl mAb、地塞米松、生理盐水3次/周,腹腔注射,于第7、14、28天处死8只大鼠.HE染色观察肺纤维化程度;免疫组化方法检测肺组织中表面活性物质A(SP-A)的含量;ELISA测定血清中羟脯氨酸(Hyp)的含量;PCR方法检测肺组织中基质金属蛋白酶9(MMP-9)及转化生长因子β1(TGF-β1) mRNA的含量.结果 各时间段,M组肺纤维化程度及Hyp含量均高于C组和L组,SP-A的含量均低于其余两组,M组肺组织的MMP-9和TGF-β1的mRNA的含量明显高于其余两组.结论 LAMRl mAb可明显减轻博莱霉素诱导的肺纤维化程度.     

Morphologic and biochemical study of pulmonary changes induced by bleomycin in mice.

Pulmonary changes induced by bleomycin were investigated in mice by using light and electron microscopy and by phospholipid analysis of the alveolar washings. The damage began in the endothelium of capillaries and was accompanied by interstitial edema and an appearance of enlarged macrophages along with hypertrophy of type II epithelial cells. This condition was followed by degeneration of type I cells. The denuded epithelium appeared to be repaired by two different mechanisms. In areas away from the bronchioles, division of existing type II cells and subsequent transformation into type I cells appeared to be the pattern of epithelial repair. In areas near the bronchiole, downgrowth of bronchiolar undifferentiated cells and subsequent maturation of these cells to type II and type I cells appeared to be prominent. Biochemically, total phospholipids and disaturated lecithin in the alveolar wash increased along with the increase of the alveolar lining layer and the hypertrophy of type II cells. This was considered to be consistent with the view that the hyperactive type II cells secreted more surfactant in the early phase of the experiment.     

Murine candidate bleomycin induced pulmonary fibrosis susceptibility genes identified by gene expression and sequence analysis of linkage regions

Background: Pulmonary fibrosis is a complex disease for which the predisposing genetic variants remain unknown. In a prior study, susceptibility to bleomycin induced pulmonary fibrosis was mapped to loci Blmpf1 and Blmpf2 on chromosomes 17 and 11, respectively, in a C57BL/6J (B6, susceptible) and C3Hf/KAM (C3H, resistant) mouse cross. Methods: Herein, the genetic basis of bleomycin induced pulmonary fibrosis was investigated in an approach combining gene expression and sequencing data with previously mapped linkage intervals. Results: In this study, gene expression analysis with microarrays revealed 1892 genes or ESTs (expressed sequence tags) to be differentially expressed between bleomycin treated B6 and C3H mice and 67 of these genetic elements map to Blmpf1 or Blmpf2. This group included genes involved in an oxidative stress response, in apoptosis, and in immune regulation. A comparison of the B6 and C3H sequence, for Blmpf1 and Blmpf2, made using the NCBI database and available C3H sequence, revealed approximately 35% of the genes in these regions contain non-synonymous coding sequence changes. An assessment of genotype/phenotype correlation among other inbred strains revealed 36% of these B6/C3H sequence variations predict for the known bleomycin induced fibrosis susceptibility of the DBA (susceptible) and A/J (resistant) mouse strains. Conclusions: Combining genomics approaches of differential gene expression and sequence variation potentially identifies approximately 5% the linked genes as fibrosis susceptibility candidate genes in this mouse cross.     

Inhibition of bleomycin-induced pulmonary fibrosis by cobra venom factor.

A single dose of cobra venom factor, a known, potent depletor of serum complement, has recently been reported to be highly effective in inhibiting lung collagen deposition at 7 days after bleomycin treatment. The present study shows that this is associated with suppression of the bleomycin-induced increase in collagen synthesis, down to virtually normal levels. There is also a concomitant reduction in tissue free proline pool size. No significant effects are noted in noncollagenous protein synthetic rates. This inhibitory effect is not seen at 30 days after bleomycin and is correlated with a return to normal levels of serum complement hemolytic activity. These data suggest an important role for complementary activity in the lung's fibrotic response to bleomycin.     

Experimental pulmonary fibrosis induced by trisodium citrate and acid-citrate-dextrose

A single intrapulmonary injection of 3.8% trisodium citrate and acid-citrate-dextrose (ACD) into rabbits results in extensive degeneration and necrosis of alveolar pneumocytes, including the type II pneumocyte, and of bronchiolar or bronchial epithelial cells. Subsequently, the alveoli and alveolar ducts collapse, and the septa and ductal walls adhere to each other, accompanied by the proliferation of interstitial fibroblasts. These fibroblasts produce fibrous connective tissue which is followed by pulmonary fibrosis in 1 week. Epithelial regeneration, especially that resulting from the proliferation of immature type II pneumocytes, occurs around the periphery of the fibrous lesions. The synthesis and release of large amounts of surfactant materials by the proliferated type II pneumocytes may induce the surfactant materials to reopen the air spaces of the collapsed and adhesive alveoli. By 4 weeks those fibrous areas in the pathological lungs become smaller and/or appear normal. These results suggest that this is a useful experimental animal model for pulmonary fibrosis, and that epithelial cells, especially type II pneumocytes, are associated with both the induction of and the recovery from the disorder; in the early stage, interference by reepithelization resulting from type II pneumocyte proliferation may elicit the proliferation of fibroblasts, and in later stages, reepithelization and surfactant synthesis by newly proliferated type II pneumocytes may permit the reopening of collapsed and adhesive air spaces.     

博莱霉素诱导小鼠肺间质纤维化造模方式的选择

目的:建立博莱霉素导致肺间质纤维化小鼠动物模型,比较不同给药方式的成模差异。方法:利用8周龄雄性ICR小鼠,①随机分为腹腔给药组(P组)、气管内给药组(I组)、阴性对照组(C组),分别经腹腔注射BLM 40 mg/kg 5次、气管内滴入BLM 5 mg/kg 1次或气管内滴入生理盐水50μl。分别于14、28、40天处死,②小鼠随机分为4组,分别经腹腔注射BLM 40mg/kg3、4、5次或经腹腔给予生理盐水200μl。分别于28、40天处死。观察小鼠体重、咳嗽、挠鼻症状、肺系数及肺组织病理改变。结果:给予博莱霉素后①小鼠的体重均下降并出现咳嗽及挠鼻等呼吸障碍症状;处置后第14、28及40天处死小鼠,计算肺系数,P组较I组肺系数高;处死小鼠后,P组和I组小鼠均形成广泛、稳定的间质纤维化病理改变,P组主要分布在胸膜下及血管周围,而I组主要分布在肺门和支气管周围。P组较I组肺纤维化病理评分高。②不同腹腔给药次数模型小鼠体重变化以5次给药对体重影响最大;计算肺系数以给药5次肺系数变化最大。上述模型均成功建立。通过比较生存率、呼吸困难症状、组织病理变化等指标,选出腹腔给药5次相对于给药3次及4次为更好的造模方式。结论:利用BLM腹腔注射和气管内滴入制备了肺间质纤维化动物模型,纤维化形成的部位存在着一定的差异,腹腔给药5次方法制备肺间质纤维化模型的成功率更佳。     

Pathogenesis of pulmonary fibrosis induced by chrysotile asbestos

Summary A single instillation of 1 mg chrysotile B with a fiber length between 0.05 and 0.2 µm in 0.1 ml tricaprylin was made via a polyvinyl catheter into the lower lobe of the right lung of 120 six-week-old Wistar rats under anesthesia. The animals were killed at intervals between five minutes and two years. The lower lobes of the right lung were investigated by light and electron microscopy.The process of pulmonary fibrosis induced by asbestos can be subdivided into four phases: these are the phase of phagocytosis (five to 15 min), the phase of granuloma formation (between one and two weeks), the phase of septal fibrosis (between two and six months) and finally the scar stage (after one year). After instillation of small asbestos fibers into the alveoli, a major proportion of these fibers is phagocytosed by alveolar macrophages after five minutes and leaves the lungs via the airways. A proportion of the fibers penetrates through the alveolar wall (mostly conveyed by type I pneumocytes) and reaches the interstitium of the lungs. There, the fibers are taken up by pulmonary tissue macrophages and giant cells. Within the phagolysosomes, the fibers are broken down into fragments less than 0.01 µm in length. Type II pneumocytes produce surfactant in excess. These cells become necrotic, tubular myelin and lamellar bodies pass into the alveoli and into the interstitium. Surfactant is phagocytosed by resident macrophages. These macrophages phages can break down. Besides asbestos and surfactant, mediators of fibrillogenesis are released. Macrophages following up from blood monocytes ingest surfactant and asbestos. This process is perpetuated up to complete scarring. After two years, small asbestos fibers less than 0.01 µm long are present in fibroblasts and pleural mesothelia.     

Neoformation of blood vessels in association with rat lung fibrosis induced by bleomycin

We have used intratracheal instillation of bleomycin in rats to study the microanatomical changes of blood vessels associated with lung fibrosis. Bleomycin is a toxic cytostatic drug employed in classical models of lung fibrosis. Wistar rats were submitted to intratracheal injection of 1.5 units of bleomycin and sacrificed 2.5 months later, a timing when marked fibrosis of the lung is observed. We casted the vascular tree of the rat lungs by perfusion with a methacrylate resin. These caste were studied by scanning electron microscopy. Lung tissue was also studied by light microscopy and thin section electron microscopy. The major vascular modifications observed in the bleomycin-treated rats were: (1) neoformation of an elaborate network of vessels located in the peribronchial domains of the lung, and (2) distortion of the architecture of alveolar capillaries. By light microscopy, it was clear that the newly formed vascular network was located in regions of fibrosis (which in the resin casts were digested away). These neoformed vessels appeared to originate from bronchial arteries. Thin section electron microscopy revealed that endothelial cells of the neoformed vessels were plump, presented large nuclei, and showed numerous pinocytotic vesicles that were also observed in subendothelial pericytes. The alveoli of the bleomycin-treated rats were heterogeneous in size and shape in contrast with the homogeneity of alveoli of control animals. The alveolar capillaries of fibrotic lungs appeared to occupy a larger volume of the alveolar wall than alveolar capillaries of control rats. Our findings indicate that lung fibrosis encompasses marked changes of the vascular system, namely, the neoformation of vessels and the rearrangement of alveolar capillaries. These structural changes suggest that fibrotic transformation of the lung is associated with the local generation of angiogenic stimuli. © 1994 Wiley-Liss, Inc.     

Secretion of macrophage-derived growth factor during acute lung injury induced by bleomycin

Monocytes are capable of producing a growth factor which causes fibroblasts to replicate in vitro. This macrophage-derived growth factor (MDGF) may be particularly important in controlling the proliferation of mesenchymal cells in the pulmonary interstitium following acute lung injury. To evaluate the role of macrophages in modulating the proliferative capacity of fibroblasts, we added serum-free culture medium from lavage-derived rat lung macrophages to confluent monolayers of quiescent adult rat lung fibroblasts and monitored their return to the cell cycle both by 3H-thymidine incorporation and by increases in cell number. Growth factor activity secreted in 24 h by untreated macrophages was compared to that from normal macrophages stimulated in vitro by bacterial lipopolysaccharide (LPS) and from macrophages retrieved from lungs injured by intratracheal administration of bleomycin. Unstimulated macrophages from control animals secreted minimal amounts of MDGF; addition of LPS to normal macrophages increased MDGF secretion 3- to 4-fold. Adherent cells obtained by lung lavage of bleomycin-treated animals spontaneously secreted near maximal levels of nondialyzable MDGF with little further stimulation by LPS. The MDGF secreted by LPS-stimulated control cells and by macrophages retrieved from injured lungs share identical elution profiles from DEAE-Sephacel anionic exchange columns, and heat stability profiles. The substance acts as a competence factor when tested on rat lung fibroblasts. These shared physical and biologic properties suggest that the growth factor secreted by cells lavaged from injured lungs is identical to MDGF secreted in response to LPS. Because MDGF release could not be induced by direct exposure of freshly isolated macrophages to bleomycin, we suggest that augmented release of MDGF is an indirect feature of acute lung injury in this model.     

Experimental pulmonary fibrosis induced by soluble immune complex and 60% oxygen atmosphere.

In our previous papers we reported characteristic morphological changes of the capillary endothelium in the lung and of the arterial endothelium. These changes were induced by administration of various soluble immune complexes. In this paper we have induced pulmonary fibrosis in guinea pigs under allergic procedures. Administration of soluble immune complexes alone produces interstitial pneumonia. However, these changes would not normally result in diffuse pulmonary fibrosis. The administration of soluble immune complexes to guinea pigs plus exposure to a 60% oxygen-rich atmosphere yielded diffuse pulmonary fibrosis.     

Increased and prolonged pulmonary fibrosis in surfactant protein C-deficient mice following intratracheal bleomycin

Recent reports have linked mutations in the surfactant protein C gene (SFTPC) to familial forms of pulmonary fibrosis, but it is uncertain whether deficiency of mature SP-C contributes to disease pathogenesis. In this study, we evaluated bleomycin-induced lung fibrosis in mice with genetic deletion of SFTPC. Compared with wild-type (SFTPC+/+) controls, mice lacking surfactant protein C (SFTPC-/-) had greater lung neutrophil influx at 1 week after intratracheal bleomycin, greater weight loss during the first 2 weeks, and increased mortality. At 3 and 6 weeks after bleomycin, lungs from SFTPC-/- mice had increased fibroblast numbers, augmented collagen accumulation, and greater parenchymal distortion. Furthermore, resolution of fibrosis was delayed. Although remodeling was near complete in SFTPC+/+ mice by 6 weeks, SFTPC-/- mice did not return to baseline until 9 weeks after bleomycin. By terminal dUTP nick-end labeling staining, widespread cell injury was observed in SFTPC-/- and SFTPC+/+ mice 1 week after bleomycin; however, ongoing apoptosis of epithelial and interstitial cells occurred in lungs of SFTPC-/- mice, but not SFTPC+/+ mice, 6 weeks after bleomycin. Thus, SP-C functions to limit lung inflammation, inhibit collagen accumulation, and restore normal lung structure after bleomycin.     

Heparin attenuates bleomycin but not silica-induced pulmonary fibrosis in mice: possible relationship with involvement of myofibroblasts in bleomycin, and fibroblasts in silica-induced fibrosis

Pulmonary fibrosis was elicited in mice or rats by the intra-tracheal instillation of bleomycin or silica. Daily injections of heparin significantly reduced the collagen deposition in bleomycin, but not in silica, injected mice, as evaluated by the lung hydroxyproline content on day 15 after instillation. Heparin also reduced the bleomycin-induced morbidity and mortality. Study of the broncho-alveolar lavage fluid (BAL) detected no significant change in the number of leucocytes or the amount of protein in heparin treated mice. Histologies of bleomycin instilled mice suggested that heparin did reduce the alveolar remodelling but not the alveolitis, evidenced by leucocytic infiltration. As detected by electron microscopy (EM), bleomycin increased the number of leucocytes and platelets within the alveolar capillaries but this was not significantly reduced by heparin. The phenotype of the interstitial cell involved in these two types of pulmonary fibrosis was investigated by immunohistochemistry and EM. While in bleomycin injected animals the interstitial cells had the phenotype of an actin (α-actin in the rat) and lipid containing interstitial cell, with a poorly developed RE, in silica injected animals in contrast, the interstitial cells were without cytoplasmic actin or lipid but with a markedly developed endoplasmic reticulum (ER). Thus bleomycin and silica induced the growth of two different types of interstitial cells, the myofibroblast and the regular fibroblast, which might be a reason why heparin selectively inhibits bleomycin but not silica-induced fibrosis.