Keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice

At present there is no known effective pharmacological therapy for acute lung injury (ALI). Because keratinocyte growth factor (KGF) promotes epithelial cell growth, intratracheal administration of KGF has the possibility of restoring lung tissue integrity in injured lungs and improving patient outcomes. However, treatment using recombinant KGF protein is limited by its short effective duration. Thus, we investigated the effectiveness of intratracheal KGF gene transduction using adenoviral vector in ALI. We constructed an adenoviral vector expressing mouse KGF (mKGF), and 1.0 x 10(9 ) plaque-forming units of mKGF cDNA-expressing (Ad-KGF) and control (Ad-1w1) adenoviral vector was intratracheally instilled, using a MicroSprayer, into anesthetized BALB/c mice. Three days later, the mice were exposed to >90% oxygen for 72 hr, and the effect of KGF on hyperoxia-induced lung injury was examined. In the Ad-KGF group, KGF was strongly expressed in the airway epithelial cells, while peribronchiolar and alveolar inflammation caused by adenoviral vector instillation was minimal. The KGF overexpression not only induced proliferation of surfactant protein C-positive cuboidal cells, especially in the terminal bronchiolar and alveolar walls, but also prevented lung injury including intraalveolar exudation/hemorrhage, albumin permeability increase, and pulmonary edema. The arterial oxygen tension and the survival rate were significantly higher in the KGF-transfected group. These findings suggest that KGF gene transduction into the airway epithelium is a promising potential treatment for ALI.     

Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo.

Keratinocyte growth factor (KGF) administered as a single intratracheal injection causes a prominent dose-dependent proliferation of type II alveolar epithelial cells in the lungs of adult rats. The increase in mitotically active alveolar cells histologically appears as a micropapillary epithelial cell hyperplasia after 2 d and peaks after 3 d in the form of monolayers of cuboidal epithelial cells lining alveolar septae. Proliferating cell nuclear antigen immunohistochemistry confirmed the profound proliferative response induced by KGF. The hyperplastic alveolar lining cells contain immunoreactive surfactant protein B and are ultrastructurally noted to contain lamellar inclusions characteristic of surfactant-producing type II pneumocytes. Mild focal bronchiolar epithelial hyperplasia is noted but is much less striking than the proliferation of type II pneumocytes. Large airways are unaffected by KGF. Daily intravenous injection of KGF is also able to cause pneumocyte proliferation. The normal adult rat lung constitutively expresses both KGF and KGF receptor mRNA, suggesting that endogenous KGF may be implicated in the paracrine regulation of the growth of pneumocytes. In conclusion, KGF rapidly and specifically induces proliferation and differentiation of type II pneumocytes in the normal adult lung.     

Keratinocyte growth factor stimulates transduction of the respiratory epithelium by retroviral vectors

Cell proliferation is required for transduction by standard retrovirus vectors derived from viruses in the murine leukemia virus (MuLV) group. Since proliferation rates are low in the mature pulmonary epithelium, we tested the hypothesis that the efficiency of retrovirus-mediated transduction of respiratory epithelial cells can be enhanced by stimulation of cell proliferation with recombinant human keratinocyte growth factor (rhKGF). A marked increase in proliferation of bronchiolar and alveolar epithelial cells was observed after intratracheal administration of rhKGF (30 mg/kg) to adult FVB/N mice. Two days after rhKGF or saline treatment, 10(7) AP+ FFU of LAPSN, a recombinant amphotropic retrovirus that expresses human placental alkaline phosphatase (AP), was instilled intratracheally into the mice. Transduction efficiency, measured 2 days after infection, was increased approximately 70-fold by rhKGF pretreatment. However, even after KGF treatment the total numbers of AP-expressing cells were few. Transduction efficiency was similar using either LAPSN packaged by amphotropic host range packaging cells or LAPSN pseudotyped with 10A1 MuLV envelope protein (0.091 +/- 0.006 versus 0.094 +/- 0.028 transduction events/mm2, respectively). Amphotropic vectors use Pit-2 for cell entry, while 10A1 MuLV vectors can use Pit-1 or Pit-2 for cell entry. By in situ hybridization the retroviral receptor Pit-2 (Ram-1) mRNA was expressed only in the pulmonary vasculature, and Pit-1 (Glvr-1) mRNA was expressed at low levels throughout the lung. In vitro studies demonstrated that retrovirus was inactivated by pulmonary surfactant. Stimulating proliferation of the respiratory epithelium increased retroviral transduction in vivo, but the paucity of retroviral receptors and inactivation by surfactant are additional barriers to high-level retroviral gene transfer in the lung.     

角化细胞生长因子对内毒素诱导急性肺损伤的保护作用

目的 初步研究角化细胞生长因子(KGF)对脂多糖(LPS)诱导大鼠急性肺损伤(ALI)的保护作用及可能的机制.方法 将36只SD大鼠按随机数字表法分为3组,每组12只.模型组尾静脉注射LPS 5 mg/kg建立ALI动物模型.对照组和KGF组注射等量生理盐水.KGF组在注射LPS后气道给予KGF 5 mg/kg.8 h后处死各组大鼠观察肺组织病理改变,并测量肺血管通透性、肺上皮细胞通透性、肺湿/干重(W/D)比值以及Ⅱ型肺泡上皮细胞(ATⅡ)增殖、修复功能改变.结果 光镜下观察显示KGF可有效减轻LPS所致ALI的肺组织病理改变,表现为肺血管充血、水肿减轻,几乎无炎性细胞浸润.与模型组比较,KGF组肺血管通透性[(0.026±0.049)%比(0.087±0.027)%]和肺泡上皮通透性[(0.692±0.017)%比(0.931±0.029)%]及W/D比值(4.778±0.243比6.869±0.153)均明显降低(P<0.05或P<0.01),ATⅡ细胞增殖及修复功能则明显提高[ATⅡ数量(个):6.083±1.781比4.666±1.923,损伤面积(mm2):2.946±0.453比6.181±0.975,P<0.05和P<0.01].结论 KGF可减轻LPS所致ALI,其机制可能是通过增强ATⅡ细胞的增殖及修复能力从而起到有效的保护作用.     

Pseudomonas aeruginosa-induced lung and pleural injury in sheep. Differential protective effect of circulating versus alveolar immunoglobulin G antibody.

The overall objective of these studies was to determine whether IgG antibody to Pseudomonas aeruginosa would modify the acute lung and pleural injury that developed over 24 h after the instillation of 10(10) live P. aeruginosa into the distal airspaces of one lung in unanesthetized sheep. Using a quantitative experimental model to measure protein permeability across the alveolar epithelial, lung endothelial, and pleural mesothelial barriers, the effect of IgG antibody to P. aeruginosa was examined under four different experimental conditions. First, the effect of IgG antibody to P. aeruginosa in the circulation was examined by instilling 10(10) live P. aeruginosa in 5% ovine albumin in sheep that had been vaccinated. Under these conditions, the presence of circulating IgG antibody to P. aeruginosa reduced lung endothelial injury but did not modify the lung epithelial or pleural injury caused by intraalveolar P. aeruginosa. Therefore, the second experimental protocol determined the effect of instilling immune serum from a sheep that had been vaccinated so that IgG antibody to P. aeruginosa was present in both the circulation and in the airspaces along with instillation of live bacteria. Under these conditions, injury to the lung endothelium, alveolar epithelium, and pleural space was completely prevented. Therefore, the third protocol examined the protective effect of instillation of IgG antibody to P. aeruginosa in the airspaces concurrent with the live bacteria. Interestingly, intraalveolar IgG antibody to P. aeruginosa prevented all evidence of lung epithelial and pleural injury, and this effect was associated with a marked decrease in the number of viable bacteria in the lung after 24 h. Therefore, the fourth protocol examined the prophylactic effect of instillation of the specific IgG antibody to P. aeruginosa 24 h before instillation of the bacteria. With this prophylactic regimen, epithelial, endothelial, and pleural injury were prevented, and there was a significant decrease in the number of bacteria recovered from the lung. Thus, delivery of IgG antibody to P. aeruginosa the distal airspaces of the lung alone may provide a novel therapeutic approach to preventing acute pulmonary infection caused by P. aeruginosa.     

Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia

The pathogenesis of septic shock occurring after Pseudomonas aeruginosa pneumonia was studied in a rabbit model. The airspace instillation of the cytotoxic P. aeruginosa strain PA103 into the rabbit caused a consistent alveolar epithelial injury, progressive bacteremia, and septic shock. The lung instillation of a noncytotoxic, isogenic mutant strain (PA103DeltaUT), which is defective for production of type III secreted toxins, did not cause either systemic inflammatory response or septic shock, despite a potent inflammatory response in the lung. The intravenous injection of PA103 did not cause shock or an increase in TNF-alpha, despite the fact that the animals were bacteremic. The systemic administration of either anti-TNF-alpha serum or recombinant human IL-10 improved both septic shock and bacteremia in the animals that were instilled with PA103. Radiolabeled TNF-alpha instilled in the lung significantly leaked into the circulation only in the presence of alveolar epithelial injury. We conclude that injury to the alveolar epithelium allows the release of proinflammatory mediators into the circulation that are primarily responsible for septic shock. Our results demonstrate the importance of compartmentalization of inflammatory mediators in the lung, and the crucial role of bacterial cytotoxins in causing alveolar epithelial damage in the pathogenesis of acute septic shock in P. aeruginosa pneumonia.     

Fibronectin mediates adherence of rat alveolar type II epithelial cells via the fibroblastic cell-attachment domain.

The lung alveolar surface is composed of types I and II epithelial cells. Extremely attenuated type I cells cover 90% of the surface and are prone to necrosis during acute lung injury. After denudation of type I cells, the alveolar epithelium is restored by proliferation of type II cells. During reepithelialization in vivo the type II cells have been observed to reorganize on an extracellular matrix that contains fibronectin. We thus sought to determine whether type II cells would adhere to purified fibronectin. Adherence assays of primary rat type II cells were performed in protein-coated bacteriologic microtiter wells for 24 h at 37 degrees C. Concentrations of fibronectin from 1 to 300 micrograms/ml mediated type II cell adherence, 10 micrograms/ml gave maximal adherence, and 4 micrograms/ml gave 50% maximal adherence. Adherence progressively increased from 1 to 72 h. Adherence on fibronectin was at least 50% greater than adherence on laminin, types I and III collagen, or IV collagen. Little or no adherence was observed on bacteriologic plastic or albumin. Spreading on these various substrata paralleled adherence. Adherence to fibronectin, laminin, and fibrinogen was specifically blocked by their respective polyclonal antibodies. Monoclonal antibodies (MoAb) to the fibronectin cell-attachment domain blocked adherence to fibronectin, whereas MoAb to other domains did not. From the data reported here and the previously mentioned in vivo study we propose that fibronectin is an important functional component of the extracellular matrix that supports type II cells during alveolar reepithelialization.     

高氧所致小鼠急性肺损伤时一氧化氮合成酶的表达

目的探讨一氧化氮合成酶(INOS,eNOS)在高氧所致急性肺损伤发病过程中的作用.方法将54只小鼠置于密闭的氧气室暴露于＞95%的高氧,另18只小鼠呼吸正常空气作为对照组;分别于24、48 h和72 h取出小鼠,评价肺损伤程度同时进行支气管肺泡灌洗液细胞分类和计数;免疫组织化学测定肺组织iNOS和eNOS的表达及组织分布.结果高氧能引起急性肺损伤伴支气管肺汽灌洗液细胞总数、巨噬细胞、中性粒细胞数均明显增加;免疫组织化学研究显示iNOS和eNOS蛋白主要表达于气道上皮细胞、血管平滑肌细胞和巨噬细胞的胞浆中,它们在气道上皮细胞的表达在高氧环境下明显升高.结论在高氧环境下一氧化氮合成酶通过促进肺组织中一氧化氮合成从而在高氧所致的急性肺损伤过程中发挥重要作用.     

高氧对胎鼠肺泡Ⅱ型上皮细胞Notch受体的影响

目的 检测Notch 1在肺泡Ⅱ型上皮细胞(ACEⅡ)与肺成纤维细胞共培养的高氧损伤模型中表达的变化,初步探讨Notch信号在高氧肺损伤中的作用,为临床防治新生儿急、慢性肺损伤提供理论的依据.方法 Spragne Dawkey雌鼠12只,体质量200～220 g,雄鼠3只,220～250 g,由华中科技大学同济医学院实验动物中心提供.将共培养的AEC Ⅱ随机分为高氧组和对照组.高氧组按3L/nlin通入950 ml/L O2和250 ml/L CO2,10 min后,立即用封口胶密封培养板,置于37℃、50 ml/L CO2培养箱.于给氧后24、48、72、96 h,收获各组细胞,测氧仪检测培养瓶中的O2体积分数,将低于900mi/L O2的标本弃去.免疫组织化学法鉴定细胞,台盼蓝拒染实验计算细胞纯度,确认共培养造模成功.每24 h更换培养基并给氧.每一时间点同时设对照组,置于37℃、50 ml/L CO2培养箱中培养.NTT法检测AECⅡ增殖活力,绘制生长曲线;免疫组织化学法定位Notch1蛋白;荧光定量PCR检测noteh1 mRNA;流式细胞术双标记法检测AEC Ⅱ、AEC I细胞百分率.结果 免疫组织化学法显示高氧组Notch1活化人核受抑;高氧组各时间点Notch1 mRNA分别下降为对照组的0.43、0.29、0.11、0.03倍(置信限95%),通氧后AECⅡ百分率显著降低[24 h高氧组vs.对照组:(68.92±6.88)%vs.(90.35±4.01)%,P=0.006;48 h高氧组vs.对照组:(38.03±3.27)%、vs.(61.47±4.81)%,P=0.000;72 h高氧组vs.对照组:(20.13±4.45)%vs.(52.05±3.35)%,P=0.000;96 h高氧组vs.对照组:(8.17±1.99)%vs.(52.59±2.93)%,P:0.000].AEC I百分率除96 h外均增高[24 h高氧组vs.对照组:(0.1l±0.03)%vs.(0.01±0.01)%,P=0.006;48 h高氧组vs.对照组:(49.73±3.45)%vs.(16.13±2.13)%,P=0.000;72 h高氧组vs.对照组:(52.43±3.14)%vs.(5.98±0.95)%,P=0.000;96 h高氧组vs.对照组:(19.85±3.26)%vs.(29.03±3.16)%,P=0.0007].结论 高氧可抑制AECⅡNotch1受体的活化,引起AECⅡ增殖减弱,转分化异常.研究如何调控Notch信号通路将为修复肺泡上皮,恢复其正常生理功能打开新思路.     

Integrin α6β4 identifies an adult distal lung epithelial population with regenerative potential in mice

Laminins and their integrin receptors are implicated in epithelial cell differentiation and progenitor cell maintenance. We report here that a previously unrecognized subpopulation of mouse alveolar epithelial cells (AECs) expressing the laminin receptor α6β4, but little or no pro-surfactant C (pro-SPC), is endowed with regenerative potential. Ex vivo, this subpopulation expanded clonally as progenitors but also differentiated toward mature cell types. Integrin β4 itself was not required for AEC proliferation or differentiation. An in vivo embryonic lung organoid assay, which we believe to be novel, was used to show that purified β4+ adult AECs admixed with E14.5 lung single-cell suspensions and implanted under kidney capsules self-organized into distinct Clara cell 10-kDa secretory protein (CC10+) airway-like and SPC+ saccular structures within 6 days. Using a bleomycin model of lung injury and an SPC-driven inducible cre to fate-map AECs, we found the majority of type II AECs in fibrotic areas were not derived from preexisting type II AECs, demonstrating that SPC- progenitor cells replenished type II AECs during repair. Our findings support the idea that there is a stable AEC progenitor population in the adult lung, provide in vivo evidence of AEC progenitor cell differentiation after parenchymal injury, and identify a strong candidate progenitor cell for maintenance of type II AECs during lung repair.     

Adenovirus-mediated fibroblast growth factor 1 expression in the lung induces epithelial cell proliferation: consequences to hyperoxic lung injury in rats

High concentrations of oxygen can induce pulmonary toxicity and cause injury to alveolar epithelial and endothelial cells. The present study was performed to determine whether the potent epithelial and endothelial fibroblast growth factor 1 (FGF-1) protected against hyperoxia-induced lung injury. Recombinant adenovirus carrying the gene encoding human secreted FGF-1 (Ad. FGF1) increased the proliferation of lung epithelial cells in vitro. Ad.FGF1 or control vector with an empty expression cassette (Ad.V152) was administered intratracheally to Wistar rats. With Ad.FGF1 (10(9), 5 x 10(9), 10(10), or 5 x 10(10) viral particles [VP]), FGF-1 protein was found in bronchoalveolar lavage fluid 4 days postinfection at levels proportional to the viral dose and was detected in plasma after doses of 10(10) VP or more were administered. Histological examination of the lungs showed intense proliferation and apoptosis of alveolar and bronchial epithelial cells, with few inflammatory cells. The alveolar architecture returned to normal within 17 days. Rats pretreated with Ad.FGF1 (10(9) or 5 x 10(9) VP) 2 days before exposure to hyperoxia (95% O2) survived, whereas rats pretreated with Ad.V152 died within 3 days. In conclusion, adenovirus-mediated FGF-1 overexpression in the lungs causes epithelial cell proliferation and has beneficial effects in hyperoxic lung injury.     

Extracellular superoxide dismutase in the airways of transgenic mice reduces inflammation and attenuates lung toxicity following hyperoxia

Extracellular superoxide dismutase (EC-SOD, or SOD3) is the major extracellular antioxidant enzyme in the lung. To study the biologic role of EC-SOD in hyperoxic-induced pulmonary disease, we created transgenic (Tg) mice that specifically target overexpression of human EC-SOD (hEC-SOD) to alveolar type II and nonciliated bronchial epithelial cells. Mice heterozygous for the hEC-SOD transgene showed threefold higher EC-SOD levels in the lung compared with wild-type (Wt) littermate controls. A significant amount of hEC-SOD was present in the epithelial lining fluid layer. Both Tg and Wt mice were exposed to normobaric hyperoxia (>99% oxygen) for 48, 72, and 84 hours. Mice overexpressing hEC-SOD in the airways attenuated the hyperoxic lung injury response, showed decreased morphologic evidence of lung damage, had reduced numbers of recruited inflammatory cells, and had a reduced lung wet/dry ratio. To evaluate whether reduced numbers of neutrophil infiltration were directly responsible for the tolerance to oxygen toxicity observed in the Tg mice, we made Wt and Tg mice neutropenic using anti-neutrophil antibodies and subsequently exposed them to 72 hours of hyperoxia. Both Wt and Tg neutrophil-depleted (ND) mice have less severe lung injury compared with non-ND animals, thus providing direct evidence that neutrophils recruited to the lung during hyperoxia play a distinct role in the resultant acute lung injury. We conclude that oxidative and inflammatory processes in the extracellular lung compartment contribute to hyperoxic-induced lung damage and that overexpression of hEC-SOD mediates a protective response to hyperoxia, at least in part, by attenuating the neutrophil inflammatory response.     

Fas/Fas ligand pathway is involved in the resolution of type II pneumocyte hyperplasia after acute lung injury: evidence from a rat model

OBJECTIVE: We used a rat model of acute lung injury to evaluate the role of apoptosis of type II pneumocytes in alveolar remodeling during the resolution phase. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Sprague-Dawley rats. INTERVENTIONS: Sprague-Dawley rats had Escherichia coli lipopolysaccharide instilled transtracheally to induce acute lung injury. Animals were killed on various days after lipopolysaccharide instillation. Lung specimens from all animals were examined for the presence of apoptosis in type II pneumocytes by an in situ apoptosis assay and for proliferative nuclear antigen, cytokeratin-18, Fas, and Fas ligand with an immunohistochemical stain. Fas and Fas ligand expression in both lung tissue and bronchoalveolar lavage fluid was examined by Western blot analysis. MEASUREMENTS AND MAIN RESULTS: Histologic examination revealed that the lungs of rats with acute lung injury showed infiltration of numerous inflammatory cells in the intra-alveolar and/or interstitial space and hyperplasia of type II pneumocytes. Type II pneumocyte proliferation, detected by proliferative nuclear antigen staining, developed maximally around day 3 after acute lung injury. In the in situ apoptosis assay, positive signals in type II pneumocytes were obvious and were distributed diffusely in the lung parenchyma from day 1 after acute lung injury, became maximal around day 7, then declined until day 21. DNA fragmentation analysis revealed that a DNA ladder pattern was detectable from day 3, persisted until day 10, and disappeared after day 14. The major cell types expressing Fas ligand are macrophages and neutrophils. Western blot analysis showed that Fas ligand, both membrane-bound form and soluble form, was present from day 1 to day 21 after acute lung injury, with highest level occurring during the first week of acute lung injury. Fas expression in type II pneumocytes reached its maximum on days 3-5 and then gradually declined until day 21. Fas and Fas ligand expression appeared to proceed type II pneumocyte apoptosis. After the acute stage, Fas and Fas ligand expression declined, and type II pneumocyte apoptosis also decreased. These findings correlate with histologic resolution of type II pneumocyte hyperplasia. CONCLUSIONS: Our results confirm that type II pneumocyte proliferation in response to acute lung injury is mainly a reparative phenomenon. During the resolution phase of acute lung injury, extensive apoptosis of type II pneumocytes is the main cellular mechanism that accounts for the disappearance of these cells, and Fas/Fas ligand is involved in the resolution of type II pneumocytes. Our model may provide a useful tool to assess the mechanisms of tissue remodeling after acute lung injury.     

Mapping alveolar binding sites in vivo using phage peptide libraries

Targeting lung tissue is nonselective due in part to the lack of specific cell-surface receptors identified on target lung cells. We used in vivo phage display to identify a panel of peptides that can bind selectively to lung epithelial cells with less binding to nonepithelial cells. By direct intratracheal instillation of phage libraries into the lung, we isolated and identified 143 individual phage clones. Three phage clones revealed enhanced binding to the lung in vitro and in vivo. These three identified peptides were synthesized and demonstrated selective binding to epithelial cells in lung tissue versus the control peptide. Further, the peptides specifically bound to freshly isolated type II alveolar epithelial cells compared with Hep2 cells. The results suggest that the airway phage display approach could be exploited for analyzing the molecular diversity in the lower respiratory tract.     

Dual function of pneumolysin in the early pathogenesis of murine pneumococcal pneumonia.

Streptococcus pneumoniae is one of the most common etiologic agents of community-acquired pneumonia, particularly bacteremic pneumonia. Pneumolysin, a multifunctional cytotoxin, is a putative virulence factor for S. pneumoniae; however, a direct role for pneumolysin in the early pathogenesis of pneumococcal pneumonia has not been confirmed in vivo. We compared the growth of a pneumolysin-deficient (PLY[-]) type 2 S. pneumoniae strain with its isogenic wild-type strain (PLY[+]) after direct endotracheal instillation of bacteria into murine lungs. Compared with PLY(-) bacteria, infection with PLY(+) bacteria produced greater injury to the alveolar-capillary barrier, as assayed by albumin concentrations in alveolar lavage, and substantially greater numbers of PLY(+) bacteria were recovered in alveolar lavages and lung homogenates at 3 and 6 h after infection. The presence of pneumolysin also contributed to the development of bacteremia, which was detected at 3 h after intratracheal instillation of PLY(+) bacteria. The direct effects of pneumolysin on lung injury and on the ability of pneumococci to evade local lung defenses was confirmed by addition of purified recombinant pneumolysin to inocula of PLY(-) pneumococci, which promoted growth of PLY(-) bacteria in the lung to levels comparable to those seen with the PLY(+) strain. We further demonstrated the contributions of both the cytolytic and the complement-activating properties of pneumolysin on enhanced bacterial growth in murine lungs using genetically modified pneumolysin congeners and genetically complement-deficient mice. Thus, pneumolysin facilitates intraalveolar replication of pneumococci, penetration of bacteria from alveoli into the interstitium of the lung, and dissemination of pneumococci into the bloodstream during experimental pneumonia. Moreover, both the cytotoxic and the complement-activating activities of pneumolysin may contribute independently to the acute pulmonary injury and the high rates of bacteremia which characterize pneumococcal pneumonia.     

间质胶原酶在高氧所致急性肺损伤中的表达

目的 研究间质胶原酶(interstitial collagenase)在高氧所致急性肺损伤发病过程中的作用,探讨急性肺损伤的发病机理.方法 将72只C57BL/6小鼠随机分为正常对照组、高氧24 h组、高氧48 h组、高氧72 h组,每组18只;正常对照小鼠呼吸室内空气,高氧小鼠置于密闭的氧气室(氧浓度＞95%).评价肺损伤程度;逆转录-聚合酶链反应(RT-PCR)及免疫组织化学法测定肺组织间质胶原酶的表达及组织分布.统计分析采用单因素方差分析和q检验.结果 高氧能引起急性肺损伤;RT-PCR结果显示肺组织间质胶原酶mRNA表达量在暴露于高氧24 h后达(0.59±0.13)较对照组(0.07±0.01)明显增加(q≥3.15,P＜0.01),72 h达最高(0.68±0.12,q=3.78,P＜0.01);免疫组织化学研究显示间质胶原酶蛋白主要表达于气道上皮细胞、Ⅱ型肺泡上皮细胞和血管平滑肌细胞的胞浆中;间质胶原酶蛋白在气道上皮细胞的表达在高氧环境下24 h达(28.54±9.60)较对照组(13.48±4.32)明显升高(q≥2.62,P＜0.05),于48、72 h表达逐渐降低,分别为(20.32±5.68)和(15.24±4.65).结论 高氧能引起急性肺损伤伴间质胶原酶的表达增高,它们通过降解细胞外基质在高氧所致的急性肺损伤过程中发挥重要作用.     

Plasminogen activator inhibitor-1 in acute hyperoxic mouse lung injury.

Hyperoxia-induced lung disease is associated with prominent intraalveolar fibrin deposition. Fibrin turnover is tightly regulated by the concerted action of proteases and antiproteases, and inhibition of plasmin-mediated proteolysis could account for fibrin accumulation in lung alveoli. We show here that lungs of mice exposed to hyperoxia overproduce plasminogen activator inhibitor-1 (PAI-1), and that PAI-1 upregulation impairs fibrinolytic activity in the alveolar compartment. To explore whether increased PAI-1 production is a causal or only a correlative event for impaired intraalveolar fibrinolysis and the development of hyaline membrane disease, we studied mice genetically deficient in PAI-1. We found that these mice fail to develop intraalveolar fibrin deposits in response to hyperoxia and that they are more resistant to the lethal effects of hyperoxic stress. These observations provide clear and novel evidence for the pathogenic contribution of PAI-1 in the development of hyaline membrane disease. They identify PAI-1 as a major deleterious mediator of hyperoxic lung injury.     

Double impact of cigarette smoke and mechanical ventilation on the alveolar epithelial type II cell

Introduction

Ventilator-induced lung injury (VILI) impacts clinical outcomes in acute respiratory distress syndrome (ARDS), which is characterized by neutrophil-mediated inflammation and loss of alveolar barrier function. Recent epidemiological studies suggest that smoking may be a risk factor for the development of ARDS. Because alveolar type II cells are central to maintaining the alveolar epithelial barrier during oxidative stress, mediated in part by neutrophilic inflammation and mechanical ventilation, we hypothesized that exposure to cigarette smoke and mechanical strain have interactive effects leading to the activation of and damage to alveolar type II cells.

Methods

To determine if cigarette smoke increases susceptibility to VILI in vivo, a clinically relevant rat model was established. Rats were exposed to three research cigarettes per day for two weeks. After this period, some rats were mechanically ventilated for 4 hours. Bronchoalveolar lavage (BAL) and differential cell count was done and alveolar type II cells were isolated. Proteomic analysis was performed on the isolated alveolar type II cells to discover alterations in cellular pathways at the protein level that might contribute to injury. Effects on levels of proteins in pathways associated with innate immunity, oxidative stress and apoptosis were evaluated in alveolar type II cell lysates by enzyme-linked immunosorbent assay. Statistical comparisons were performed by t-tests, and the results were corrected for multiple comparisons using the false discovery rate.

Results

Tobacco smoke exposure increased airspace neutrophil influx in response to mechanical ventilation. The combined exposure to cigarette smoke and mechanical ventilation significantly increased BAL neutrophil count and protein content. Neutrophils were significantly higher after smoke exposure and ventilation than after ventilation alone. DNA fragments were significantly elevated in alveolar type II cells. Smoke exposure did not significantly alter other protein-level markers of cell activation, including Toll-like receptor 4; caspases 3, 8 and 9; and heat shock protein 70.

Conclusions

Cigarette smoke exposure may impact ventilator-associated alveolar epithelial injury by augmenting neutrophil influx. We found that cigarette smoke had less effect on other pathways previously associated with VILI, including innate immunity, oxidative stress and apoptosis.