Asbestos inhalation induces reactive nitrogen species and nitrotyrosine formation in the lungs and pleura of the rat.

To determine whether asbestos inhalation induces the formation of reactive nitrogen species, three groups of rats were exposed intermittently over 2 wk to either filtered room air (sham-exposed) or to chrysotile or crocidolite asbestos fibers. The rats were killed at 1 or 6 wk after exposure. At 1 wk, significantly greater numbers of alveolar and pleural macrophages from asbestos-exposed rats than from sham-exposed rats demonstrated inducible nitric oxide synthase protein immunoreactivity. Alveolar macrophages from asbestos-exposed rats also generated significantly greater nitrite formation than did macrophages from sham-exposed rats. Strong immunoreactivity for nitrotyrosine, a marker of peroxynitrite formation, was evident in lungs from chrysotile- and crocidolite-exposed rats at 1 and 6 wk. Staining was most evident at alveolar duct bifurcations and within bronchiolar epithelium, alveolar macrophages, and the visceral and parietal pleural mesothelium. Lungs from sham-exposed rats demonstrated minimal immunoreactivity for nitrotyrosine. Significantly greater quantities of nitrotyrosine were detected by ELISA in lung extracts from asbestos-exposed rats than from sham-exposed rats. These findings suggest that asbestos inhalation can induce inducible nitric oxide synthase activation and peroxynitrite formation in vivo, and provide evidence of a possible alternative mechanism of asbestos-induced injury to that thought to be induced by Fenton reactions.     

Peroxynitrite is an important mediator in thermal injury-induced lung damage

OBJECTIVE: Intestinal ischemia and reperfusion injury was known to cause postinjury multiple organ failure by neutrophil and unclear nonneutrophil factors. Peroxynitrite formed by the rapid reaction between superoxide and nitric oxide, is a toxic substance that contributes to tissue injury in a number of biological systems. In this study, the role of nitric oxide and neutrophils on lung damage after burn was investigated. DESIGN: Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Thermal injury models in the rat. INTERVENTIONS: In experiment 1, specific pathogen-free Sprague-Dawley rats underwent 35% total body surface area burn. At 4, 8, 16, and 24 hrs after burn, intestinal mucosa and lung tissue were harvested for myeloperoxidase (MPO) assay, blood was collected for measurement of peroxynitrite-mediated oxidation of dihydrorhodamine 123, and pulmonary microvascular dysfunction was quantified by measuring the extravasation of Evans blue dye. In experiment 2, polymorphonuclear granulocyte antibody (0.12 mL/100 g administered intraperitoneally 16 hrs before burn), S-methylisothiourea (7.5 mg/kg, intraperitoneally, immediately after burn), a specific inducible nitric oxide synthase inhibitor, and sterile saline (15 mL/kg, intraperitoneally, immediately after burn) were given to different groups of thermally injured animals individually. The plasma dihydrorhodamine 123 oxidation level, intestinal and lung MPO activity, lung permeability, and lung histology were evaluated at 8 hrs after burn. The cellular localization of nitrotyrosine, a marker for peroxynitrite reactivity, was also examined by immunostaining. In experiment 3, 3-morpholinosydnonimine (10 mM, intraperitoneally), a peroxynitrite donor, was given to nonburned rats to examine the peroxynitrite effect on lung inducible nitric oxide synthase expression. MEASUREMENTS AND MAIN RESULTS: The level of MPO activity in intestine and lung, blood dihydrorhodamine 123 oxidation, and lung permeability were increased up to 2-fold, 2.5-fold, 2-fold, and 2-fold of normal, respectively, at 8 hrs after burn. S-methylisothiourea injection significantly decreased (p <.05) 31% of the lung MPO activity, 41% of the blood peroxynitrite level, 54% of the lung permeability, and the lung peroxynitrite production in burned rats. Polymorphonuclear granulocyte antibody pretreatment significantly decreased 60% of the intestinal MPO, 92% of the blood peroxynitrite level, and 56% the lung MPO activity in burned rats, but the lung permeability was only slightly decreased by polymorphonuclear granulocyte antibody pretreatment. Furthermore, 3-morpholinosydnonimine increased the lung inducible nitric oxide synthase messenger RNA levels. CONCLUSIONS: Thermal injury induces blood dihydrorhodamine 123 oxidation, intestinal and lung neutrophil deposition, lung nitrotyrosine production, and lung damage. Both specific inhibition of inducible nitric oxide synthase and polymorphonuclear granulocyte antibody pretreatment decrease blood dihydrorhodamine 123 oxidation and intestinal and lung neutrophil deposition, but only inducible nitric oxide synthase inhibition with S-methylisothiourea reduces lung peroxynitrite production and thermal injury-induced lung damage. Nitric oxide and the ensuing peroxynitrite production in lung play a more important role than neutrophil in contributing to thermal injury-induced lung damage.     

Intravasal peroxynitrite generation causes dysfunction in the isolated perfused rat lung via endothelin

In septic shock excessive nitric oxide and superoxide are produced, thus generating peroxynitrite. This study investigates whether and how intravasal peroxynitrite causes lung dysfunction. To generate peroxynitrite, isolated and ventilated rat lungs were perfused blood-free in a pressure-constant, recirculating mode with hypoxanthine/xanthine oxidase plus sodium nitroprusside. Airway and vascular resistance, and release of thromboxane A2, prostacyclin, and endothelin-1 were assessed over 200 min. Peroxynitrite generation, as demonstrated by oxidation of the marker 2',7'-dichlorodihydrofluorescein diacetate, caused broncho- and vasoconstriction starting after 100 min. Both reactants alone, i.e., NO. or O2, had no effect. The thromboxane A2/prostaglandin H2 receptor antagonist BM13.177 did not affect peroxynitrite-induced broncho- and vasoconstriction. Combined endothelin(A/B) (ET(A/B)) receptor antagonism (BQ123 plus BQ788) prevented broncho- and vasoconstriction more effectively than the ET(A) receptor antagonist BQ123 alone. In tissue from lungs exposed to peroxynitrite, significantly increased amounts of endothelin-1 were detected. This study identifies endothelin-1 rather than prostanoids as a distal mediator induced by the reaction product of superoxide and nitric oxide, i.e., peroxynitrite. It is concluded that 1) endothelin-1 is a causal mediator of peroxynitrite-induced acute rat lung injury, and 2) peroxynitrite-induced broncho- and vasoconstriction are mediated by both ET(A) and ET(B) receptors.     

Activation of alveolar macrophages in acid-injured lung in rats: different effects of pentoxifylline on tumor necrosis factor-alpha and nitric oxide production

OBJECTIVE: To determine whether acid instillation augments tumor necrosis factor-alpha and nitric oxide production by alveolar macrophages in rats, and to study the effects of treatment with pentoxifylline before acid instillation on the production of these inflammatory mediators. DESIGN: Controlled laboratory investigation on tumor necrosis factor-alpha and nitric oxide production by alveolar macrophages of rats that had acid-induced lung injury. SETTING: University research laboratory. SUBJECT: Alveolar macrophages of rats. INTERVENTIONS: Alveolar macrophages were recovered by bronchoalveolar lavage at 4, 10, 16, 24, and 72 hrs after unilateral hydrochloric acid (pH, 1.0; volume, 0.1 mL) instillation into the lungs of rats. Alveolar macrophages then were cultured with or without lipopolysaccharide. One group of rats was pretreated with pentoxifylline before acid instillation. MEASUREMENTS AND MAIN RESULTS: Alveolar macrophages from both acid-instilled and contralateral lungs, which had recovered 24 hrs after acid instillation, produced significantly greater tumor necrosis factor-alpha and nitric oxide. Subsequent exposure to lipopolysaccharide, as a surrogate for bacterial infection, further promoted tumor necrosis factor-alpha and nitric oxide release. Alveolar macrophages from rats pretreated with pentoxifylline before acid instillation produced significantly less tumor necrosis factor-alpha and did not overproduce tumor necrosis factor-alpha when exposed to lipopolysaccharide. In contrast, pretreatment with pentoxifylline had no effect on nitric oxide production by alveolar macrophages. CONCLUSIONS: Acid instillation stimulates alveolar macrophages to produce tumor necrosis factor-alpha and nitric oxide. Pentoxifylline preserved innate production of tumor necrosis factor-alpha to lipopolysaccharide and did not inhibit the production of bactericidal nitric oxide. This may partly explain why pentoxifylline reduces acid aspiration-induced lung injury while maintaining the host's ability to combat bacterial infection after acid aspiration.     

Recombinant tumor necrosis factor/cachectin and interleukin 1 pretreatment decreases lung oxidized glutathione accumulation, lung injury, and mortality in rats exposed to hyperoxia

Single, preexposure, parenteral injection with both recombinant tumor necrosis factor/cachectin (TNF/C) and interleukin-1 (IL-1) prolonged the survival of rats (144 +/- 9 h) in continuous hyperoxia (greater than 99% O2 at 1 atm) when compared with rats injected with boiled TNF/C and boiled IL-1 (61 +/- 2 h), TNF/C alone (61 +/- 2 h), IL-1 alone (62 +/- 2 h), or saline (64 +/- 3 h). After exposure to hyperoxia for 52 h, pleural effusion volume, pulmonary artery pressure, total pulmonary resistance, and lung morphologic damage were decreased in those rats given TNF/C and IL-1 as compared with saline-injected rats. In parallel, ratios of reduced (GSH) to oxidized (GSSG) glutathione were greater (P less than 0.05) in lungs of TNF/C + IL-1-injected rats (91 +/- 20) than of saline-injected rats (30 +/- 4) that had been exposed to hyperoxia for 52 h. No differences were found in superoxide dismutase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, or catalase activities in lungs of TNF/C + IL-1- or saline-treated, hyperoxia-exposed rats. Our results indicate that pretreatment with TNF/C and IL-1 favorably altered lung glutathione redox status, decreased lung injury, and enhanced survival of rats exposed to hyperoxia.     

Nitric oxide production and perivascular nitration in brain after carbon monoxide poisoning in the rat.

Nitric oxide is a short-lived free radical and physiological mediator which has the potential to cause cytotoxicity. Studies were conducted to investigate whether nitric oxide, and the potent oxidant peroxynitrite, were generated in brain during experimental carbon monoxide (CO) poisoning in the rat. Nitric oxide production was documented by electron paramagnetic resonance spectroscopy, and found to be increased by ninefold immediately after CO poisoning. Evidence that peroxynitrite was generated was sought by looking for nitrotyrosine in the brains of CO-poisoned rats. Nitrotyrosine was found deposited in vascular walls, and also diffusely throughout the parenchyma in inummocytochemical studies. The affinity and specificity of an anti-nitrotyrosine antibody was investigated and a solid phase immunoradiochemical assay was developed to quantity nitrotyrosine in brain homogenates. A 10-fold increase in nitrotyrosine was found in the brains of CO-poisoned rats. Platelets were involved with production of nitrotyrosine in the early phase of exposure to CO. However, nitrotyrosine formation and leukocyte sequestration were not decreased in thrombocytopenic rats poisoned with CO according to the standard model. When rats were pre-treated with the nitric oxide synthase inhibitor, L-nitroarginine methyl ester, formation of both nitric oxide and nitrotyrosine in response to CO poisoning were abolished, as well as leukocyte sequestration in the microvasculature, endothelial xanthine dehydrogenase conversion to xanthine oxidase, and brain lipid peroxidation. We conclude that perivascular reactions mediated by peroxynitrite are important in the cascade of events which lead to brain oxidative stress in CO poisoning.     

胰岛素样生长因子结合蛋白-2与足月及早产新生大鼠高氧肺损伤的关系

目的 探讨高浓度氧对足月及早产新生大鼠肺发育的影响及高氧肺损伤与胰岛素样生长因子结合蛋白-2(IGFBP-2)的关系.方法 将孕22 d自然出生(足月)及孕21 d行剖宫术提前娩出(早产)的新生大鼠在生后2 d随机分为足月空气组(I组)、足月高氧组(Ⅱ组)、早产空气组(Ⅲ组)、早产高氧组(Ⅳ组).Ⅱ、Ⅳ组持续暴露于氧含量为85%的氧箱中,I、Ⅲ组置于同室空气中.每日记录大鼠存活率,分别于出生后4、7、10、14和21 d活杀取肺组织标本,作病理学检查、辐射状肺泡计数(RAC).用蛋白质免疫印迹法(Western blotting)及逆转录-聚合酶链反应(RT-PCR)分别测定IGFBP-2肺表达浓度及mRNA表达.结果 高氧组自出生7 d后大鼠存活率较空气组显著下降(P均0.05).空气组肺组织无炎症病变;高氧组出生7 d和14 d时呈肺泡炎改变,21 d时肺泡炎改变明显加重,且肺泡生成受阻滞,肺泡结构囊泡化;Ⅱ、Ⅳ组各时间点RAC值较相应I、Ⅲ组显著减少(P均<0.01).高氧Ⅱ、Ⅳ组出生4 d和14 d IGFBP-2表达强度均较对应的I、Ⅲ组增强(P均<0.01);IGFBP-2 mRNA表达变化与其肽浓度变化相似.结论 长期高氧暴露可引起足月和早产新生大鼠亚急性肺损伤和肺发育阻滞,IGFBP-2异常表达;这可能是高氧导致亚急性肺损伤及肺发育阻滞的重要机制之一.     

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.     

富氢生理盐水对新生SD大鼠高氧肺损伤白介素-8含量及病理的影响

目的观察富氢生理盐水对新生SD大鼠高氧肺损伤的预防作用。 方法将新生SD大鼠随机分为3组,空气对照组、高氧对照组、高氧＋氢水组。高氧对照组、高氧＋氢水组大鼠置于氧浓度为90%~95%的氧气箱中,高氧＋氢水组每日腹腔注射富氢生理盐水2次（10 ml/kg）,空气对照组、高氧对照组每日腹腔注射生理盐水2次（10 ml/kg）,高氧暴露后第1、3、7、10天分别测量各组白介素（IL）-8水平,并观察肺部病理切片。 结果与空气对照组对比,高氧对照组IL-8含量明显升高,且肺部病理切片出现肺出血、肺泡间隔变薄,并出现大量肺泡融合现象;与高氧对照组比较,高氧+氢水组IL-8含量明显降低,病理图示肺出血、肺泡融合明显减少。 结论富氢生理盐水可一定程度减轻高氧对新生SD大鼠的肺部炎症损伤。     

Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats.

Alveolar type II cell proliferation occurs after many forms of lung injury and is thought to play a critical role in alveolar epithelial repair. Keratinocyte growth factor/fibroblast growth factor 7 (KGF) has been shown to promote alveolar type II cell growth in primary culture and alveolar epithelial hyperplasia in vivo. In this study, we used immunohistochemical analysis to determine the intrapulmonary distribution and cellular localization of recombinant human KGF (rhKGF) instilled into the trachea of rats. 6 h after administration, immunoreactive KGF was observed within the lung parenchyma and along alveolar epithelial cell membranes. By 18-24 h, KGF was detected intracellularly in alveolar epithelial cells and intraalveolar macrophages. Immunoreactive KGF was not demonstrable 48 h after delivery or in lung sections from PBS-treated animals. Intratracheal instillation of 5 mg/kg rhKGF stimulated a marked, time-dependent increase in the alveolar type II cell specific labeling index to a maximum level of 33 +/- 3% 48 h after rhKGF administration compared with 1.3 +/- 0.3% after PBS instillation. In addition, this increase in type II cell proliferation in vivo was documented by flow cytometric analysis of isolated type II cells which revealed a nearly fivefold increase in the proportion of cells traversing through the S and G2/M phases of the cell cycle. To test the hypothesis that KGFs effects on type II cells in vivo might affect the response to lung injury, rats were treated with rhKGF and exposed to hyperoxia. Animals that received 1 or 5 mg/kg rhKGF exhibited dramatically reduced mortality (P < 0.001, for both doses). Survival for animals treated with 0.1 mg/kg rhKGF was not significantly different from either untreated rats or animals treated with heat-denatured rhKGF. The lungs of rhKGF-treated animals that survived hyperoxia exposure had minimal hemorrhage and no exudate within the intraalveolar space. These experiments established that intratracheal administration of rhKGF stimulated alveolar type II cell proliferation in vivo and reduced hyperoxia-induced lung injury in rats. Directed delivery of KGF to the lungs may provide a therapeutic strategy to preserve or restore the alveolar epithelium during exposure to hyperoxia or other injurious agents.     

Role of interferon-gamma in lung inflammation following cecal ligation and puncture in rats.

Interferon-gamma (IFN-gamma) has been implicated in the mortality of animal models of endotoxemia. On the other hand, the specific role of IFN-gamma in the development of organ inflammation in a model of polymicrobial sepsis has not been elucidated. In this study, we hypothesized that IFN-gamma plays an important role in lung inflammation after cecal ligation and puncture (CLP). To verify this hypothesis, lung tissue was removed 5 h after CLP or from sham controls. The mRNA expression (by RT-PCR) of IFN-gamma was increased in lung homogenates of CLP rats compared to sham controls. Using immunohistochemistry, we show for the first time the increased presence of IFN-gamma staining cells in the lung following CLP. Only very small amounts of positive staining for IFN-gamma was observed in lungs of sham controls. The presence of IFN-gamma in the lung 5 h after CLP correlated with a twofold increases in lung superoxide generation and MPO activity (index of neutrophil sequestration). Plasma and lung nitrite levels (breakdown product of nitric oxide) were also significantly increased in CLP rats. IFN-gamma antibody (1.2 mg/kg, i.v.) administered immediately after CLP significantly decreased lung superoxide levels to levels similar to the sham controls without affecting MPO activity, or lung or plasma nitrite levels. These results provide evidence that IFN-gamma may contribute to lung inflammation 5 h following CLP via increased production of superoxide.     

Adenovirus-mediated transfer of an Na+/K+-ATPase beta1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats

Pulmonary edema is cleared via active Na(+) transport by alveolar epithelial Na(+)/K(+)-ATPases and Na(+) channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na(+)/K(+)-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na(+)/K(+)-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 x 10(9) PFU of recombinant adenoviruses containing rat alpha(1) and beta(1) Na(+)/K(+)-ATPase subunit cDNAs (adalpha(1) and adbeta(1), respectively) to rat lungs 7 days prior to exposure to 100% O(2) for 64 hr. As compared with controls and ad alpha(1), AFC in the adbeta(1) rats was increased by >300%. Permeability for large solutes was less in the ad beta(1) than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adbeta(1) group. Survival through 14 days of hyperoxia was 100% in the adbeta(1) group but was not different from hyperoxic controls in animals given adalpha(1). Our data show that overexpression of a beta(1) Na(+)/K(+)-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na(+)/K(+)-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.     

Effect of the oestrogen receptor antagonist fulvestrant on the cirrhotic rat lung

It has been postulated that cirrhosis‐related lung vasodilatation and the subsequent hepatopulmonary syndrome are partly explained by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In this study, we assessed whether the oestrogen receptor antagonist fulvestrant (F) improves cirrhosis‐related lung abnormalities. Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham, and sham+F. Histological, immunohistochemical, and Western blot analyses were performed on lung samples. In the lung, the endothelial NO synthase and the nitrotyrosine protein expressions were increased in CBDL as compared to sham rats. Both parameters were significantly reduced by fulvestrant in the CBDL rats. Surprisingly, the level of pVASP (an indirect marker of NO formation and action) was decreased in CBDL rats, and fulvestrant had no effect on this parameter. The level of the vascular endothelial growth factor, the diameter of small lung vessels, and the number of macrophages were increased in CBDL lungs in comparison with sham lungs, and these parameters were unaffected by fulvestrant treatment. In conclusion, fulvestrant may not be relevant to improve lung abnormalities in cirrhosis because NO may not be biologically active and because key events contributing to the lung abnormalities are not affected by fulvestrant.     

Opposite effects of pre- and postischemic treatments with nitric oxide donor on ischemia/reperfusion-induced renal injury

We have demonstrated previously that preischemic treatment with FK409 [(+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide], a spontaneous nitric oxide (NO) donor, markedly improves ischemia/reperfusion-induced renal injury. However, there is conflicting information (renoprotective or cytotoxic) as to the contribution of NO to ischemic acute renal failure (ARF). In the present study, we investigated the effect of postischemic treatment with FK409 (1, 3, and 10 mg/kg i.v.) at 6 h after reperfusion on ischemic ARF, in comparison with the preischemic treatment effect. Ischemic ARF was induced by clamping of the left renal artery and vein for 45 min, followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function in ARF rats markedly decreased at 24 h after reperfusion. Histopathological examination of the kidney of ARF rats revealed severe renal damage. In contrast to the renoprotective effect by preischemic treatment, postischemic treatment with FK409 aggravated the ischemia/reperfusion-induced renal dysfunction and histological damage. Immunohistochemical analysis of renal sections obtained from ARF rats revealed positive staining for nitrotyrosine, a biomarker of peroxynitrite formation, in injured tubular cells, and more intense staining was observed in renal tissues from the animals that received postischemic treatment with FK409. On the other hand, the formation of nitrotyrosine, neutrophil infiltration into renal tissues, and renal superoxide production, all of which were enhanced in ARF rats, were efficiently attenuated by the preischemic treatment with FK409. These results demonstrate that, although preischemic treatment with an NO donor is renoprotective, postischemic treatment with the same agent aggravates the ischemia/reperfusion-induced renal injury, probably through peroxynitrite overproduction.     

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.     

Effect of endogenous nitric oxide on hyperoxia and tumor necrosis factor-alpha-induced leukosequestration and proinflammatory cytokine release in rat airways

OBJECTIVE: To investigate the effects of endogenous nitric oxide on hyperoxia and tumor necrosis factor-alpha-induced leukosequestration and proinflammatory cytokine release in rat airways. DESIGN: Prospective, randomized, controlled animal study. SETTING: Experimental laboratory. SUBJECTS: Male Sprague-Dawley rats weighing 350-500 g. INTERVENTIONS: The rats were pretreated with N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg) or saline intravenously 4-6 mins before intratracheal administration of tumor necrosis factor-alpha, 95% oxygen, or both, when the vasopressor effect of L-NAME had reached a plateau. MEASUREMENTS AND MAIN RESULTS: Bronchoalveolar lavage fluid was recovered from the airway of rats after exposure to 95% oxygen and tumor necrosis factor-alpha for 6 hrs under ventilator support. Neutrophils in lavage fluid were isolated and examined for the inducible nitric oxide synthase expression by flow-cytometric assay. Tumor necrosis factor-alpha and interleukin-1 beta in lavage fluid were measured by enzyme-linked immunosorbent assay. The percentage of neutrophils in bronchoalveolar fluid was significantly higher in rats exposed to hyperoxia + tumor necrosis factor-alpha (29.7 +/- 12.5%) compared with rats with hyperoxia (16.3 +/- 1.2%), tumor necrosis factor-alpha (4.2 +/- 1.1%), or room air (5.0 +/- 1.8%) alone (p <.05). Rats exposed to hyperoxia + tumor necrosis factor-alpha had significantly higher concentrations of inducible nitric oxide synthase of neutrophils (350.1 +/- 75.7 mean fluorescence intensity), compared with rats with hyperoxia (64.9 +/- 1.6 mean fluorescence intensity), tumor necrosis factor-alpha (102.6 +/- 15.3 mean fluorescence intensity), or room air (111.2 +/- 25.8 mean fluorescence intensity) alone (p <.05). Rats exposed to hyperoxia + tumor necrosis factor-alpha significantly produced higher concentrations of tumor necrosis factor-alpha and interleukin-1 beta, compared with rats with tumor necrosis factor-alpha, hyperoxia, or room air alone. Hyperoxia + tumor necrosis factor-alpha also significantly increased growth-related oncogene/cytokine-induced neutrophil chemoattractant (GRO/CINC)-1 in bronchoalveolar fluid, compared with those receiving tumor necrosis factor-alpha alone, hyperoxia alone, or room air alone. L-NAME significantly enhanced the percentage of neutrophil recovery and the production of tumor necrosis factor-alpha, interleukin-1 beta, and GRO/CINC-1 in airways compared with the corresponding hyperoxia + tumor necrosis factor-alpha treatment alone. CONCLUSIONS: Endogenous nitric oxide may be an important endogenous inhibitor of hyperoxia + tumor necrosis factor-alpha-induced leukocyte recruitment and subsequently tumor necrosis factor-alpha, interleukin-1 beta, and GRO/CINC-1 release.     

Inhibition of MIP-1alpha-induced human neutrophil and monocyte chemotactic activity by reactive oxygen and nitrogen metabolites

Peroxynitrite, formed by the reaction between nitric oxide (NO) and superoxide, has been implicated in the pathogenesis of numerous disease processes. Several studies have shown that peroxynitrite-induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may regulate cytokine function during inflammation. To test this hypothesis, the neutrophil and monocyte chemotactic responses of macrophage inflammatory protein-1alpha (MIP-1alpha) incubated with and without peroxynitrite were evaluated. Peroxynitrite attenuated neutrophil chemotactic activity (NCA) and monocyte chemotactic activity (MCA) by MIP-1alpha in a dose-dependent manner (P < .05). The inhibitory effects were not significant on NCA and MCA induced by leukotriene B4 or complement-activated serum incubated with peroxynitrite. The reducing agents deferoxamine, dithiothreitol, and exogenous L-tyrosine abrogated the NCA and MCA inhibition by peroxynitrite. Papa-NONOate, an NO donor, or a combination of xanthine and xanthine oxidase to generate superoxide, did not show an inhibitory effect on NCA and MCA induced by MIP-1alpha. In contrast, 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, elicited a concentration-dependent reduction in NCA and MCA induced by MIP-1alpha. Consistent with its capacity to reduce NCA and MCA, peroxynitrite treatment reduced MIP-1alpha binding to neutrophils and monocytes. Nitrotyrosine was detected in the MIP-1alpha incubated with peroxynitrite. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of MIP-1alpha binding to neutrophils and monocytes and a reduction in NCA and MCA. These data demonstrate that peroxynitrite modulates the inflammatory cell migration by MIP-1alpha, and they suggest that oxidants may play an important role in the regulation of MIP-1alpha-induced inflammatory cell chemotaxis.     

Azithromycin protects against hyperoxic lung injury in neonatal rats.

Bronchopulmonary dysplasia (BPD) is a pulmonary disorder that causes significant morbidity and mortality in premature infants. BPD is pathologically characterized by inflammation, fibrosis, and mucosal necrosis, which leads to emphysematous coalescence of alveoli. We tested the hypothesis that azithromycin, a macrolide antibiotic, would decrease the severity of lung injury in an animal model of BPD. Sixty-three rat pups were randomly divided equally into control, hyperoxia, and hyperoxia plus azithromycin groups. The hyperoxia groups were exposed to > 95% oxygen from days of life 4 to 14. On day 14, the animals were processed for lung histology and tissue analysis. Lung morphology was assessed by mean linear intercept, a measure of alveolar size, with larger values corresponding to lungs that are more emphysematous. The degree of lung inflammation was assessed by quantifying interleukin-6 (IL-6) from lung homogenate. Fifty pups survived to day 14 (control = 21, hyperoxia = 11, hyperoxia + azithromycin = 18). Mortality was increased in the hyperoxia group versus the control group (p < .0001). Treatment with azithromycin improved survival in animals subjected to hyperoxia (p < .05). Azithromycin significantly decreased lung damage as determined by the mean linear intercept in the hyperoxia groups (p < .001). Finally, azithromycin-treated pups had lower levels of IL-6 in lung homogenate from the hyperoxia groups (p < .05). Azithromycin treatment resulted in improved survival, less emphysematous change, and decreased IL-6 levels in an animal model of BPD.     

Prevention of Collagen Deposition Following Pulmonary Oxygen Toxicity in the Rat by Cis-4-Hydroxy-l-Proline

Exposure of rats to high oxygen tensions causes increased collagen content of lungs and alveolar enlargement in 3-6 wk. We tested whether cis-hydroxyproline, a proline analogue that inhibits collagen synthesis, could prevent the collagen accumulation and alveolar enlargement. Rats were exposed to hyperoxia for 60 h and then to room air and hyperoxia for alternate 24-h periods for 11.5 d. Treated oxygen-exposed rats received 200 mg/kg cis-hydroxyproline twice daily over the 14-d exposure period. Control rats breathed room air. Examination of lungs on day 14 showed collagen content of oxygen-exposed lungs to be 48% greater than control (P < 0.05). The collagen content of the treated oxygen-exposed lungs was −12% of control (NS). Total lung volume was 16% greater than control in oxygen-exposed rats (P < 0.05) and 8% greater than control in treated oxygen-exposed rats (NS). Morphometric studies showed alveolar size was greater than control in oxygen-exposed rats (188±11 [SE] vs. 143±6 μμl [P < 0.05]). Oxygen-exposed, treated rats had a mean alveolar volume of 150±7 μμl. Lung pressure-volume curves were significantly shifted to the left of control in the oxygen-exposed rats, whereas the curves of the oxygen-exposed, treated group were identical to control. These data suggest that cis-hydroxyproline prevented the accumulation of collagen in the lungs in pulmonary oxygen toxicity. In addition, there was apparent protection from airspace dilatation and decreased lung elasticity, suggesting that alveolar enlargement after oxygen toxicity is linked to the deposition in lung tissue of new connective tissue fibers.