Keratinocyte growth factor protects against elastase-induced pulmonary emphysema in mice

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The keratinocyte growth factor (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg x kg(-1) x day(-1) KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, 2) a reduction in the pulmonary expression of the chemokines CCL2 (or monocyte chemoattractant protein-1) and CXCL2 (or macrophage inflammatory protein-2alpha) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-2 and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL2, CXCL2, and ICAM-1 mRNA in the MLE-12 murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.     

VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice

To determine whether increased levels of VEGF disrupt postnatal lung formation or function, conditional transgenic mice in which VEGF 164 expression was enhanced in respiratory epithelial cells were produced. VEGF expression was induced in the lungs of VEGF transgenic pups with doxycycline from postnatal day 1 through 2 and 6 wk of age. VEGF levels were higher in bronchoalveolar lavage fluid (BALF) and lung homogenates of VEGF transgenic mice compared with endogenous VEGF levels in controls. Neonatal mortality was increased by 50% in VEGF transgenic mice. Total protein content in BALF was elevated in VEGF transgenic mice. Surfactant protein B protein expression was unaltered in VEGF transgenic mice. Although postnatal alveolar and vascular development were not disrupted by VEGF expression, VEGF transgenic mice developed pulmonary hemorrhage, alveolar remodeling, and macrophage accumulation as early as 2 wk of age. Electron microscopy demonstrated abnormal alveolar capillary endothelium in the VEGF transgenic mice. In many locations, the endothelium was discontinuous with segments of attenuated endothelial cells. Large numbers of hemosiderin-laden macrophages and varying degrees of emphysema were observed in adult VEGF transgenic mice. Overexpression of VEGF in the neonatal lung increased infant mortality and caused pulmonary hemorrhage, hemosiderosis, alveolar remodeling, and inflammation.     

烟雾加气管内滴入PPE 诱发非均质性肺气肿模型

目的:探讨烟雾与弹性蛋白酶(PPE)联合应用在兔阻塞性肺气肿模型形成过程中对肺组织X线、动脉血气、肺组织形态学结构的影响以及如何在较短时间内诱发出类似于人类疾病的非均质性肺气肿模型。方法:将40只小白兔随机分为吸烟组、注酶组、联合组及对照组4组,分别给予单纯香烟熏吸、气管内注入猪胰弹性蛋白酶、烟熏加气管内注入弹性蛋白酶及单纯气管内注入生理盐水作为对照组。7周后分别进行肺组织X线、动脉血气和肺组织形态学检查。结果:注酶组和联合组的平均肺泡数(Na)、肺泡隔面密度(Ds)及PaO2减少,PaCO2、肺总容积(TLC)和肺泡直径(Da)增加,与对照组比较,差异有显著性(P<0.05)。注酶组和联合组大鼠平均肺泡数(MAN)与正常对照组大鼠相比明显减少(P<0.05),平均肺泡面积(MAA)、平均内衬间隔(Lm)明显大于正常对照组(P<0.05),而吸烟组与正常对照组无统计学差异(P>0.05)。结论:烟雾可强化弹性蛋白酶在诱发肺气肿模型过程中的作用,二者联合应用可加快兔肺气肿模型的诱导过程。     

Thioredoxin prevents the development and progression of elastase-induced emphysema

Thioredoxin 1 (TRX1) is a redox (reduction/oxidation)-active protein that scavenges reactive oxygen species. Here we examined whether endogenous or exogenous administration of TRX1 prevented the development and progression of elastase-induced pulmonary emphysema. Mice were treated with intratracheal elastase via microspray on day 0, and were given recombinant human TRX1 (rhTRX1) every other day from days -1 to 21. To determine the effects of TRX1 on the progression of established emphysema, mice were treated intratracheally with elastase on day 0, and rhTRX1 was administered from days 14 to 21. Histopathologic examination was performed on day 21. TRX1-transgenic but not transgene-negative mice demonstrated a decrease in the physiological indicators of elastase-induced emphysema. TRX1 administration from days -1 to 19 significantly decreased the signs of elastase-induced emphysema. Moreover, TRX1 administration beginning 14 days after elastase treatment significantly slowed the progression of emphysema. TRX1 may be of clinical benefit for the treatment of COPD.     

Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×10⁵), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-β levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.     

Intratracheally administered titanium dioxide or carbon black nanoparticles do not aggravate elastase-induced pulmonary emphysema in rats

ABSTRACT: BACKGROUND: Titanium dioxide (TiO2) and carbon black (CB) nanoparticles (NPs) have biological effects that could aggravate pulmonary emphysema. The aim of this study was to evaluate whether pulmonary administration of TiO2 or CB NPs in rats could induce and/or aggravate elastase-induced emphysema, and to investigate the underlying molecular mechanisms. METHODS: On day 1, Sprague-Dawley rats were intratracheally instilled with 25 U kg1 pancreatic porcine elastase or saline. On day 7, they received an intratracheal instillation of TiO2 or CB (at 100 and 500 mug) dispersed in bovine serum albumin or bovine serum albumin alone. Animals were sacrificed at days 8 or 21, and bronchoalveolar lavage (BAL) cellularity, histological analysis of inflammation and emphysema, and lung mRNA expression of heme oxygenase-1 (HO-1), interleukin-1beta (IL-1beta), macrophage inflammatory protein-2, monocyte chemotactic protein-1, and matrix metalloprotease (MMP)-1, and -12 were measured. In addition, pulmonary MMP-12 expression was also analyzed at the protein level by immunohistochemistry. RESULTS: TiO2 NPs per se did not modify the parameters investigated, but CB NPs increased perivascular/peribronchial infiltration, and macrophage MMP-12 expression, without inducing emphysema. Elastase administration increased BAL cellularity, histological inflammation, HO-1, IL-1beta and macrophage MMP-12 expression and induced emphysema. Exposure to TiO2 NPs did not modify pulmonary responses to elastase, but exposure to CB NPs aggravated elastase-induced histological inflammation without aggravating emphysema. CONCLUSIONS: TiO2 and CB NPs did not aggravate elastase-induced emphysema. However, CB NPs induced histological inflammation and MMP-12 mRNA and protein expression in macrophages.     

Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice.

To test the hypothesis that VEGF is important for the maintenance of alveolar structure and elastic properties in adult mice, lung-targeted ablation of the VEGF gene was accomplished through intratracheal delivery of an adeno-associated cre recombinase virus (AAV/Cre) to VEGFloxP mice, and the effects were followed for 8 wk. Control mice were similarly treated with AAV/Cre. Pulmonary VEGF levels were reduced by 86% at 5 wk postinfection but returned to normal levels by 8 wk. VEGF receptor VEGFR-2 levels were also reduced at 5 wk (by 51%) and returned to control values by 8 wk. However, alveolar septal wall destruction (increased mean linear intercept) and loss of lung elastic recoil (increased compliance) persisted for 8 wk. No decrease in alveolar cell proliferation was detected by Western blot or immunohistochemical analysis of proliferating cell nuclear antigen. Increased alveolar septal cell and bronchial epithelial cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis at 5 wk. Total lung caspase-3 levels and enzyme activity were also increased at 5 wk. No obvious accumulation of inflammatory cells was observed at any time after tracheal instillation of AAV/Cre. Thus a transient decrease in pulmonary VEGF leads to increased alveolar and bronchial cell apoptosis, air space enlargement, and changes in lung elastic recoil (processes that are characteristic of emphysema) that persist for at least 8 wk.     

Elastase induces lung epithelial cell autophagy through placental growth factor: A new insight of emphysema pathogenesis

Chronic obstructive pulmonary disease (COPD) is a devastating disease, which is associated with increasing mortality and morbidity. Therefore, there is a need to clearly define the COPD pathogenic mechanism and to explore effective therapies. Previous studies indicated that cigarette smoke (CS) induces autophagy and apoptosis in lung epithelial (LE) cells. Excessive ELANE/HNE (elastase, neutrophil elastase), a factor involved in protease-antiprotease imbalance and the pathogenesis of COPD, causes LE cell apoptosis and upregulates the expression of several stimulus-responsive genes. However, whether or not elastase induces autophagy in LE cell remains unknown. The level of PGF (placental growth factor) is higher in COPD patients than non-COPD controls. We hypothesize that elastase induces PGF expression and causes autophagy in LE cells. In this study, we demonstrated that porcine pancreatic elastase (PPE) induced PGF expression and secretion in LE cells in vitro and in vivo. The activation of MAPK8/JNK1 (mitogen-activated protein kinase 8) and MAPK14/p38alpha MAPK signaling pathways was involved in the PGF mediated regulation of the TSC (tuberous sclerosis complex) pathway and autophagy in LE cells. Notably, PGF-induced MAPK8 and MAPK14 signaling pathways mediated the inactivation of MTOR (mechanistic target of rapamycin), the upregulation of MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 β) and the increase of autophagosome formation in mice. Furthermore, the PPE-induced autophagy promotes further apoptosis in vitro and in vivo. In summary, elastase-induced autophagy promotes LE cell apoptosis and pulmonary emphysema through the upregulation of PGF. PGF and its downstream MAPK8 and MAPK14 signaling pathways are potential therapeutic targets for the treatment of emphysema and COPD.     

CD40 amplifies Fas-mediated apoptosis: a mechanism contributing to emphysema

Excessive apoptosis and prolonged inflammation of alveolar cells are associated with the pathogenesis of pulmonary emphysema. We aimed to determine whether CD40 affects alveolar epithelial cells and endothelial cells, with regard to evoking apoptosis and inflammation. Mice were repeatedly treated with agonistic-anti CD40 antibody (Ab), with or without agonistic-anti Fas Ab, and evaluated for apoptosis and inflammation in lungs. Human pulmonary microvascular endothelial cells and alveolar epithelial cells were treated with agonistic anti-CD40 Ab and/or anti-Fas Ab to see their direct effect on apoptosis and secretion of proinflammatory molecules in vitro. Furthermore, plasma soluble CD40 ligand (sCD40L) level was evaluated in patients with chronic obstructive pulmonary disease (COPD). In mice, inhaling agonistic anti-CD40 Ab induced moderate alveolar enlargement. CD40 stimulation, in combination with anti-Fas Ab, induced significant emphysematous changes and increased alveolar cell apoptosis. CD40 stimulation also enhanced IFN-γ-mediated emphysematous changes, not via apoptosis induction, but via inflammation with lymphocyte accumulation. In vitro, Fas-mediated apoptosis was enhanced by CD40 stimulation and IFN-γ in endothelial cells and by CD40 stimulation in epithelial cells. CD40 stimulation induced secretion of CCR5 ligands in endothelial cells, enhanced with IFN-γ. Plasma sCD40L levels were significantly increased in patients with COPD, inversely correlating to the percentage of forced expiratory volume in 1 s and positively correlating to low attenuation area score by CT scan, regardless of smoking history. Collectively CD40 plays a contributing role in the development of pulmonary emphysema by sensitizing Fas-mediated apoptosis in alveolar cells and increasing the secretion of proinflammatory chemokines.     

烟雾暴露所致肺气肿小鼠模型的建立与评价

目的探讨单纯烟雾暴露所致肺气肿小鼠模型建立及病理学、气道炎症及肺功能评价,并进行支气管肺泡灌洗(bronchoalveolar lavage,BAL)技术的改进。方法 20只C57BL/6J小鼠随机分为正常对照及烟雾暴露组,烟雾暴露90d并观察30d后行小鼠肺功能检查、应用改进方法留取BALF行细胞计数及行肺组织病理切片观察,并与正常对照组进行比较。结果烟雾暴露组小鼠气道阻力(Raw)较正常对照组增高,动态肺顺应性(Cdyn)降低;BALF中细胞总数高于正常对照组,巨噬细胞数(AM)、中性粒细胞数(N)、中性粒细胞所占比例(N%)也高于对照组,差异均有统计学意义;病理学观察示烟雾暴露组肺泡腔扩大、部分肺泡间隔断裂、肺泡腔融合、肺气肿形成,气道上皮排列紊乱、部分气道上皮增生、周围炎症细胞浸润并伴有平滑肌增生;形态学计量分析示烟雾暴露组平均内衬间隔(MLI)及肺泡破坏指数(DI)较正常对照组增加。应用改进技术行BAL成功率100%,回收率高达90%。结论单纯烟雾暴露可以成功建立小鼠肺气肿模型且稳定可靠,与人类慢性阻塞性肺病相似性好,经BAL技术改进后该模型可行性高。     

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.     

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。     

Pneumonitis and emphysema in sp-C gene targeted mice

SP-C-deficient (SP-C -/-) mice developed a severe pulmonary disorder associated with emphysema, monocytic infiltrates, epithelial cell dysplasia, and atypical accumulations of intracellular lipids in type II epithelial cells and alveolar macrophages. Whereas alveolar and tissue surfactant phospholipid pools were increased, levels of other surfactant proteins were not altered (SP-B) or were modestly increased (SP-A and SP-D). Analysis of pressure-volume curves and forced oscillatory dynamics demonstrated abnormal respiratory mechanics typical of emphysema. Lung disease was progressive, causing weight loss and cardiomegaly. Extensive alveolar remodeling was accompanied by type II cell hyperplasia, obliteration of pulmonary capillaries, and widespread expression of alpha-smooth muscle actin, indicating myofibroblast transformation in the lung parenchyma. Dysplastic epithelial cells lining conducting airways stained intensely for the mucin, MUC5A/C. Tissue concentrations of proinflammatory cytokines were not substantially altered in the SP-C (-/-) mice. Production of matrix metalloproteinases (MMP-2 and MMP-9) was increased in alveolar macrophages from SP-C (-/-) mice. Absence of SP-C caused a severe progressive pulmonary disorder with histologic features consistent with interstitial pneumonitis.     

The effect of hyaluronan on elastic fiber injury in vitro and elastase-induced airspace enlargement in vivo

This laboratory has previously described a method of preventing air-space enlargement in experimental pulmonary emphysema using aerosolized hyaluronan (HA). Although it was found that HA preferentially binds to elastic fibers (which undergo breakdown by elastases in emphysema), it remains to be shown that such attachment actually prevents damage to the fibers. In the current study, cell-free radiolabeled extracellular matrices, derived from rat pleural mesothelial cells, were used to test the ability of low molecular weight ( approximately 100 kDa) streptococcal HA to prevent elastolysis. Coating the matrices with HA significantly decreased elastolysis (P<0.05) induced by porcine pancreatic elastase (43%), human neutrophil elastase (53%), and human macrophage metalloelastase (80%). Concomitant in vivo studies examined the ability of an aerosol preparation of the streptococcal HA to prevent experimental emphysema induced by intratracheal administration of porcine pancreatic elastase. As seen with earlier studies involving bovine tracheal HA, a single aerosol exposure significantly decreased elastase-induced airspace enlargement, as measured by the mean linear intercept (107.5 vs 89.6 microm; P < 0. 05). Furthermore, repeated exposure to the HA aerosol for 1 month did not reveal any morphological changes in the lung. The results provide further evidence that aerosolized HA may be an effective means of preventing pulmonary emphysema and perhaps other lung diseases that involve elastic fiber injury.     

Effect of pancreatic and leukocyte elastase on hydraulic conductivity in lung interstitial segments.

Elastase-induced changes in flow were used to quantify the degradation of lung interstitial elastin. Degassed rabbit lungs were inflated with silicon rubber via airways and vessels. The lungs were cut into 1-cm-thick sections. Two chambers were bonded to each section to enclose the interstitium surrounding an arterial segment. Flow of albumin solution (0-5 g/dl) between the chambers was followed by that of the albumin solution with 0.25 g/dl pancreatic elastase solution. Driving pressure was 5 cmH(2)0, and mean interstitial pressure was either 0 or 10 cmH(2)O. Elastase caused an increase in flow in approximately 70% of the interstitial segments and a reduction in flow in the remaining segments. The elastase-induced response in flow was independent of both albumin concentration and mean interstitial pressure. Leukocyte elastase (5 units/dl) produced flow responses similar to those of 0.25 g/dl pancreatic elastase. The increased flow of leukocyte elastase was reduced by a subsequent flow with 0.25 g/dl pancreatic elastase but enhanced by a subsequent flow with a 10-fold lower concentration. A change in the order of the elastase flows reversed the concentration-dependent responses. This behavior suggests a complex interaction among the interstitial fibers after degradation by pancreatic and leukocyte elastase. Endogenous elastase-induced increases in interstitial permeability might affect blood-lymph barrier permeability, whereas elastase-induced cessation of flow might be related to the alveolar septal wall destruction observed in emphysema.