Neutrophil elastase-induced elastin degradation mediates macrophage influx and lung injury in 60% O2-exposed neonatal rats.

Neutrophil (PMNL) influx precedes lung macrophage (LM) influx into the lung following exposure of newborn pups to 60% O2. We hypothesized that PMNL were responsible for the signals leading to LM influx. This was confirmed when inhibition of PMNL influx with a CXC chemokine receptor-2 antagonist, SB-265610, also prevented the 60% O2-dependent LM influx, LM-derived nitrotyrosine formation, and pruning of small arterioles. Exposure to 60% O2 was associated with increased lung contents of neutrophil elastase and α-elastin, a marker of denatured elastin, and a decrease in elastin fiber density. This led us to speculate that neutrophil elastase-induced elastin fragments were the chemokines that led to a LM influx into the 60% O2-exposed lung. Inhibition of neutrophil elastase with sivelestat or elafin attenuated the LM influx. Sivelestat also attenuated the 60% O2-induced decrease in elastin fiber density. Daily injections of pups with an antibody to α-elastin prevented the 60% O2-dependent LM influx, impaired alveologenesis, and impaired small vessel formation. This suggests that neutrophil elastase inhibitors may protect against neonatal lung injury not only by preventing structural elastin degradation, but also by blocking elastin fragment-induced LM influx, thus preventing tissue injury from LM-derived peroxynitrite formation.

Mechanical ventilation induces neutrophil extracellular trap formation.

BACKGROUND: Mechanical ventilation can injure the lung and induce a proinflammatory state; such ventilator-induced lung injury (VILI) is associated with neutrophil influx. Neutrophils release DNA and granular proteins as cytotoxic neutrophil extracellular traps (NETs). The authors hypothesized that NETs were produced in a VILI model and may contribute to injury. METHODS: In a two-hit lipopolysaccharide/VILI mouse model with and without intratracheal deoxyribonuclease (DNase) treatment or blockade of known inducers of NET formation (NETosis), the authors assessed compliance, bronchoalveolar lavage fluid protein, markers of NETs (citrullinated histone-3 and DNA), and markers of inflammation. RESULTS: Although lipopolysaccharide recruited neutrophils to airways, the addition of high tidal mechanical ventilation was required for significant induction of NETs markers (e.g., bronchoalveolar lavage fluid DNA: 0.4 ± 0.07 µg/ml [mean ± SEM], P < 0.05 vs. all others, n = 10 per group). High tidal volume mechanical ventilation increased airway high-mobility group box 1 protein (0.91 ± 0.138 vs. 0.60 ± 0.095) and interleukin-1ß in lipopolysaccharide-treated mice (22.4 ± 0.87 vs. 17.0 ± 0.50 pg/ml, P < 0.001) and tended to increase monocyte chemoattractant protein-1 and interleukin-6. Intratracheal DNase treatment reduced NET markers (bronchoalveolar lavage fluid DNA: 0.23 ± 0.038 vs. 0.88 ± 0.135 µg/ml, P < 0.001; citrullinated histone-3: 443 ± 170 vs. 1,824 ± 403, P < 0.01, n = 8 to 10) and attenuated the loss of static compliance (0.9 ± 0.14 vs. 1.58 ± 0.17 ml/mmHg, P < 0.01, n = 19 to 20) without significantly impacting other measures of injury. Blockade of high-mobility group box 1 (with glycyrrhizin) or interleukin-1ß (with anakinra) did not prevent NETosis or protect against injury. CONCLUSIONS: NETosis was induced in VILI, and DNase treatment eliminated NETs. In contrast to experimental transfusion-related acute lung injury, NETs do not play a major pathogenic role in the current model of VILI.

Chronic lung injury in the neonatal rat: up-regulation of TGFß1 and nitration of IGF-R1 by peroxynitrite as likely contributors to impaired alveologenesis.

Postnatal alveolarization is regulated by a number of growth factors, including insulin-like growth factor-I (IGF-I) acting through the insulin-like growth factor receptor-1 (IGF-R1). Exposure of the neonatal rat lung to 60% O2 for 14 days results in impairments of lung cell proliferation, secondary crest formation, and alveologenesis. This lung injury is mediated by peroxynitrite and is prevented by treatment with a peroxynitrite decomposition catalyst. We hypothesized that one of the mechanisms by which peroxynitrite induces lung injury in 60% O2 is through nitration and inactivation of critical growth factors or their receptors. Increased nitration of both IGF-I and IGF-R1 was evident in 60% O2-exposed lungs, which was reversible by concurrent treatment with a peroxynitrite decomposition catalyst. Increased nitration of the IGF-R1 was associated with its reduced activation, as assessed by IGF-R1 phosphotyrosine content. IGF-I displacement binding plots were conducted in vitro using rat fetal lung distal epithelial cells which respond to IGF-I by an increase in DNA synthesis. When IGF-I was nitrated to a degree similar to that observed in vivo there was minimal, if any, effect on IGF-I displacement binding. In contrast, nitrating cell IGF-R1 to a similar degree to that observed in vivo completely prevented specific binding of IGF-I to the IGF-R1, and attenuated an IGF-I-mediated increase in DNA synthesis. Additionally, we hypothesized that peroxynitrite also impairs alveologenesis by being an upstream regulator of the growth inhibitor, TGFß1. That 60% O2-induced impairment of alveologenesis was mediated in part by TGFß1 was confirmed by demonstrating an improvement in secondary crest formation when 60% O2-exposed pups received concurrent treatment with the TGFß1 activin receptor-like kinase, SB 431542. That the increased TGFß1 content in lungs of pups exposed to 60% O2 was regulated by peroxynitrite was confirmed by its attenuation by concurrent treatment with a peroxynitrite decomposition catalyst. We conclude that peroxynitrite contributes to the impaired alveologenesis observed following the exposure of neonatal rats to 60% O2 both by preventing binding of IGF-I to the IGF-R1, secondary to nitration of the IGF-R1, and by causing an up-regulation of the growth inhibitor, TGFß1.

Cyclooxygenase-2 inhibition partially protects against 60% O2 -mediated lung injury in neonatal rats.

RATIONALE: Use of the anti-inflammatory agent dexamethasone in premature infants with bronchopulmonary dysplasia has been curtailed, and no alternative anti-inflammatory agents are approved for this use. Our objective was to use a neonatal rat model of bronchopulmonary dysplasia to determine if an highly selective cyclooxygenase-2 inhibitor, 5,5-dimethyl-3-(3-fluorophenyl)4-(4-methylsulfonyl)phenyl-2(5H)-furanone (DFU; 10 µg/g body weight), could prevent inflammatory cell influx and protect against lung injury. METHODS: Neonatal rats exposed to air or 60% O2 for 14 days from birth either received daily i.p. injections of (i) vehicle or DFU or (ii) vehicle or an EP(1) receptor antagonist, SC-19220. RESULTS: DFU attenuated the lung macrophage and neutrophil influx, prevented interstitial thickening and prevented the loss of peripheral blood vessels induced by 60% O2 , but did not protect against the variance in alveolar diameter induced by 60% O2 . Exposure to 60% O2 caused both an increase in lung prostaglandin E2 content and a reduction in lung mesenchymal cell mass which was reversed by DFU. Prostaglandin E2 binding to the EP(1) receptor inhibited DNA synthesis in cultures of lung fibroblasts in a dose dependent fashion. Treatment with SC-19220 attenuated the reduction in lung mesenchymal mass observed following exposure of rat pups to 60% O2 . CONCLUSIONS: An highly selective cyclooxygenase-2 inhibitor is an effective anti-inflammatory substitute for dexamethasone for preventing phagocyte influx into the neonatal lung during 60% O2 -mediated lung injury, and can modify the severity of that injury.

The IGF-I/IGF-R1 pathway regulates postnatal lung growth and is a nonspecific regulator of alveologenesis in the neonatal rat.

IGF-I, IGF-II, and the IGF-I receptor are widely distributed throughout the neonatal rat lung on days 4, 7, 10, and 14 of life, with a similar abundance at each of these time points. Injection of 20 µg/g of a truncated soluble IGF-I receptor on days 2 and 5 of life, to decoy ligand away from the endogenous IGF-I receptor, reduced lung weight and lung-to-body weight ratio, reduced lung tissue fraction, and impaired alveolar formation, as assessed by secondary crest formation and mean linear intercepts on day 7 of life. Lung procollagen I content and elastin fiber density were also reduced. Injection of 100 µg/day of neutralizing anti-IGF-I, to prevent IGF-I from binding to the IGF-I receptor, on days 3, 4, and 5 of life reduced tissue fraction and elastin fiber density and impaired alveolar formation on day 6 of life. Both interventions reduced total lung cell and secondary crest cell DNA synthesis and small vessel counts per unit area, but these effects were lost after normalization to the reduced tissue fraction. These findings are consistent with a role for IGF-I binding to the IGF-I receptor in postnatal lung growth and on alveologenesis through a nonspecific positive effect on DNA synthesis. Injection of 100 µg/day of neutralizing anti-IGF-II, to prevent IGF-II from binding to the IGF-I receptor, on days 3, 4, and 5 of life had no effect on total lung cell DNA synthesis per unit area on day 6 of life, and a role for IGF-II in postnatal alveologenesis was not further pursued.

Lipid hydroperoxide formation regulates postnatal rat lung cell apoptosis and alveologenesis.

An acute increase in oxygen tension after birth imposes an oxidative stress upon the lung. We hypothesized that the resultant increase in reactive oxygen species, specifically lipid hydroperoxides, would trigger postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat. Neonatal rats were either untreated or treated daily with subcutaneous vehicle or diphenyl phenyl diamine, a scavenger of lipid hydroperoxides and inhibitor of lipid peroxidation, from day 1 to 6 of life. Alveolar formation and physiological lung cell apoptosis were assessed by morphometry, immunohistochemistry, and Western blot analyses on day 7 samples. Substitution experiments were conducted using the prototypic lipid hydroperoxide t-butylhydroperoxide. At a minimum effective dose of 15µg/g body wt, treatment with diphenyl phenyl diamine resulted in a significant increase in tissue fraction and mean linear intercept and significant reductions in small peripheral blood vessels, secondary crest formation, lung and secondary crest cell DNA synthesis, and estimated alveolar number. Decreased numbers of apoptotic type II pneumocytes and mesenchymal cells, and decreased contents of proapoptotic cleaved caspase-3 and -7 and cytoplasmic cytochrome c, and an increase in antiapoptotic Bcl-xL were found in lungs treated with diphenyl phenyl diamine. A prevention of selected changes induced by diphenyl phenyl diamine was observed with concurrent treatment with intraperitoneal t-butylhydroperoxide, at a minimally effective dose of 187µg/g body wt. We conclude that oxidative stress after birth induces lipid hydroperoxide formation, which, in turn, triggers postnatal alveologenesis and physiological lung cell apoptosis in the neonatal rat.

Therapeutic hypercapnia prevents bleomycin-induced pulmonary hypertension in neonatal rats by limiting macrophage-derived tumor necrosis factor-α.

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation, arrested lung growth, and pulmonary hypertension (PHT), as observed in human infants with severe bronchopulmonary dysplasia. Inhalation of CO(2) (therapeutic hypercapnia) has been described to limit cytokine production and to have anti-inflammatory effects on the injured lung; we therefore hypothesized that therapeutic hypercapnia would prevent bleomycin-induced lung injury. Spontaneously breathing rat pups were treated with bleomycin (1 mg/kg/d ip) or saline vehicle from postnatal days 1-14 while being continuously exposed to 5% CO(2) (Pa(CO(2)) elevated by 15-20 mmHg), 7% CO(2) (Pa(CO(2)) elevated by 35 mmHg), or normocapnia. Bleomycin-treated animals exposed to 7%, but not 5%, CO(2), had significantly attenuated lung tissue macrophage influx and PHT, as evidenced by normalized pulmonary vascular resistance and right ventricular systolic function, decreased right ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. The level of CO(2) neither prevented increased tissue neutrophil influx nor led to improvements in decreased lung weight, septal thinning, impaired alveolarization, or decreased numbers of peripheral arteries. Bleomycin led to increased expression and content of lung tumor necrosis factor (TNF)-α, which was found to colocalize with tissue macrophages and to be attenuated by exposure to 7% CO(2). Inhibition of TNF-α signaling with the soluble TNF-2 receptor etanercept (0.4 mg/kg ip from days 1-14 on alternate days) prevented bleomycin-induced PHT without decreasing tissue macrophages and, similar to CO(2), had no effect on arrested alveolar development. Our findings are consistent with a preventive effect of therapeutic hypercapnia with 7% CO(2) on bleomycin-induced PHT via attenuation of macrophage-derived TNF-α. Neither tissue macrophages nor TNF-α appeared to contribute to arrested lung development induced by bleomycin. That 7% CO(2) normalized pulmonary vascular resistance and right ventricular function without improving inhibited airway and vascular development suggests that vascular hypoplasia does not contribute significantly to functional changes of PHT in this model.

Long-term failure of alveologenesis after an early short-term exposure to a PDGF-receptor antagonist.

Survivors of moderate-to-severe bronchopulmonary dysplasia have impaired alveologenesis lasting at least into early adult life. The mechanisms underlying this long-term effect are unknown. We hypothesized that short-term inhibition of growth factor-mediated early alveolar formation would result in a long-term impairment of subsequent alveologenesis. Neonatal rats were injected daily with the platelet-derived growth factor (PDGF) receptor antagonist, imatinib mesylate, from day 1-7 of life, to inhibit the early alveolar formation occurring by in-growth of secondary crests into precursor saccules. The pups were then allowed to recover for 7, 14, 21, or 58 days. In imatinib-treated pups, DNA synthesis in total lung cells, and specifically in cells of secondary crests, was reduced at day 8 of life, had rebounded on day 14 of life but was then again reduced by day 28 of life. At day 8 of life, imatinib-treated pups had impaired alveologenesis as reflected by a decrease in secondary crests, an increase in alveolar size, and an overall decrease in both estimated alveolar number and generations compared with age-matched controls. No meaningful recovery was observed, even after a 21- or 58-day recovery period. The lungs of imatinib-treated pups had increased fibulin-5 content and an abnormal deposition of elastin. We conclude that reduced signaling through the PDGF pathways, at an early stage of alveologenesis, can result in long-lasting changes in lung architecture. A likely mechanism is through impaired formation of the elastin scaffold required for alveolarization.

Inhibition of apoptosis by 60% oxygen: a novel pathway contributing to lung injury in neonatal rats.

During early postnatal alveolar formation, the lung tissue of rat pups undergoes a physiological remodeling involving apoptosis of distal lung cells. Exposure of neonatal rats to severe hyperoxia (≥95% O(2)) both arrests lung growth and results in increased lung cell apoptosis. In contrast, exposure to moderate hyperoxia (60% O(2)) for 14 days does not completely arrest lung cell proliferation and is associated with parenchymal thickening. On the basis of similarities in lung architecture observed following either exposure to 60% O(2), or pharmacological inhibition of physiological apoptosis, we hypothesized that exposure to 60% O(2) would result in an inhibition of physiological lung cell apoptosis. Consistent with this hypothesis, we observed that the parenchymal thickening induced by exposure to 60% O(2) was associated with decreased numbers of apoptotic cells, increased expressions of the antiapoptotic regulator Bcl-xL, and the putative antiapoptotic protein survivin, and decreased expressions of the proapoptotic cleaved caspases-3 and -7. In summary, exposure of the neonatal rat lung to moderate hyperoxia results in an inhibition of physiological apoptosis, which contributes to the parenchymal thickening observed in the resultant lung injury.

A peroxynitrite decomposition catalyst prevents 60% O2-mediated rat chronic neonatal lung injury.

Exposure of newborn rats to 60% O2 for 14days results in a chronic neonatal lung injury characterized by parenchymal thickening, impaired alveolarization, evidence of pulmonary hypertension, and pulmonary vascular pruning. The contribution of peroxynitrite to this injury was assessed by treating pups with a peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS), at 30microg/g/day. Body and lung weights and postfixation lung volumes were all slightly, but significantly, reduced by exposure to 60% O2 and this was attenuated by a concurrent FeTPPS intervention. The FeTPPS intervention had no impact on increased neutrophil or macrophage influx into the lung, but attenuated 60% O2-induced reductions in the lung contents of vascular endothelial-derived growth factor, its receptor-2, and angiopoietin and increases in 8-isoprostane and preproendothelin-1. The 60% O2-induced parenchymal thickening and impairment of alveologenesis, as well as vascular pruning and peripheral vessel medial wall thickening, were attenuated by FeTPPS, despite a persistent inflammatory cell influx. Pups exposed to 60% O2 and treated with FeTPPS had enhanced alveolar formation relative to control pups. We conclude that peroxynitrite plays a critical role in the development of chronic neonatal lung injury.

Therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats.

Permissive hypercapnia, achieved using low tidal volume ventilation, has been an effective protective strategy in patients with acute respiratory distress syndrome. To date, no such protective effect has been demonstrated for the chronic neonatal lung injury, bronchopulmonary dysplasia. The objective of our study was to determine whether evolving chronic neonatal lung injury, using a rat model, is resistant to the beneficial effects of hypercapnia or simply requires a less conservative approach to hypercapnia than that applied clinically to date. Neonatal rats inhaled air or 60% O2 for 14 days with or without 5.5% CO2. Lung parenchymal neutrophil and macrophage numbers were significantly increased by hyperoxia alone, which was associated with interstitial thickening and reduced secondary crest formation. The phagocyte influx, interstitial thickening, and impaired alveolar formation were significantly attenuated by concurrent hypercapnia. Hyperoxic pups that received 5.5% CO2 had a significant increase in alveolar number relative to air-exposed pups. Increased tyrosine nitration, a footprint for peroxynitrite-mediated reactions, arteriolar medial wall thickening, and both reduced small peripheral pulmonary vessel number and VEGF and angiopoietin-1 (Ang-1) expression, which were observed with hyperoxia, was attenuated by concurrent hypercapnia. We conclude that evolving chronic neonatal lung injury in a rat model is responsive to the beneficial effects of hypercapnia. Inhaled 5.5% CO2 provided a significant degree of protection against parenchymal and vascular injury in an animal model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a phagocyte influx.

A critical role for the IL-1 receptor in lung injury induced in neonatal rats by 60% O2.

IL-1 beta, a proinflammatory cytokine, may contribute to the development of the chronic neonatal lung injury, bronchopulmonary dysplasia. Chronic neonatal lung injury was induced in rats, by exposure to 60% O2 for 14 d from birth, to determine whether pulmonary IL-1 expression was up-regulated and, if so, whether a daily s.c. IL-1 receptor antagonist injections would be protective. Exposure to 60% O2 for 14 d caused pulmonary neutrophil and macrophage influx, increased tissue fraction and tyrosine nitration, reduced VEGF-A and angiopoietin-1 expression, and reduced small vessel (20-65 microm) and alveolar numbers. Lung IL-1 alpha and -1 beta contents were increased after a 4-d exposure to 60% O2. IL-1 receptor antagonist treatment attenuated the 60% O2-dependent neutrophil influx, the increased tissue fraction, and the reduced alveolar number. Treatment did not restore VEGF-A or angiopoietin-1 expression and only partially attenuated the reduced vessel number in 60% O2-exposed pups. It also caused a paradoxical increase in macrophage influx and a reduction in small vessels in air-exposed pups. We conclude that antagonism of IL-1-mediated effects can, in major part, protect against lung injury in a rat model of 60% O2-induced chronic neonatal lung injury.

A critical role for fibroblast growth factor-7 during early alveolar formation in the neonatal rat.

Mesenchymal cell-derived FGF-7 (fibroblast growth factor-7) induces proliferation in both epithelial and endothelial cells. We found FGF-7 to be expressed in the lungs of neonatal rats from birth to d 14 of age. A role for FGF-7 in early postnatal lung growth and alveolar formation, by an action on type II pneumocytes, has been excluded by the work of others. However, a role through an action of FGF-7 on other cell types has not been excluded. We used intraperitoneal injections of neutralizing antibodies on d 3, 4, and 5 of life to inhibit binding of FGF-7 to its receptors, and assessed alveolar formation on d 6 of life. This intervention inhibited DNA synthesis in, and number of, alveoli-forming secondary crests, resulting in a significantly reduced alveolar number. This failure of alveolar formation was associated with a reduction in the number of small blood vessels in the lung periphery. We conclude that FGF-7, most likely through its effect on the vascular bed, is required for normal early postnatal alveolar formation from secondary crests.