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

doi:10.1016/j.freeradbiomed.2010.07.001

Free Radical Biology and Medicine

Volume 49, Issue 7, 15 October 2010, Pages 1182-1191

https://doi.org/10.1016/j.freeradbiomed.2010.07.001 Get rights and content

Abstract

Exposure of newborn rats to 60% O₂ for 14 days 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 30 μg/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.

Section snippets

Reagents

5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS) was from Calbiochem (San Diego, CA, USA). A Vectastain ABC system and a peroxidase substrate kit were from Vector Laboratories (Burlingame, CA, USA). A rabbit polyclonal antibody to myeloperoxidase for neutrophil detection was from Dako Canada (Mississauga, ON, Canada). A mouse anti-rat cluster of differentiation 68 (CD-68) antibody for macrophage detection was from Serotec (Oxford, UK). A mouse monoclonal antibody

Effect of FeTPPS on lung and body weights and postfixation lung volumes

Exposure to 60% O₂, but not treatment with FeTPPS, had a significant independent effect on both lung and body weights, and on postfixation lung volumes, and there was no significant interaction between 60% O₂ and FeTPPS. As shown in Table 1, analysis of group differences revealed small, but statistically significant, effects of exposure to 60% O₂ for 14 days on lung and body weights, and on postfixation lung volumes, all of which differences were attenuated by concurrent treatment with FeTPPS.

Discussion

Consistent with our previous observations [17], [18], [19], exposure to 60% O₂ resulted in markedly increased lung tissue immunoreactivity for nitrotyrosine. The attenuation of this increase in nitrotyrosine immunoreactivity by FeTPPS suggests that peroxynitrite was also the nitrating species observed in previous studies with this model. FeTPPS acts by isomerizing peroxynitrite to nitrate [36]. Based on the literature, this effect seems to be specific, and we and others have observed no

Acknowledgments

This work was supported by group [A.K. Tanswell (MGC-25029)] and operating [R.P. Jankov (MOP-74506 and -84290), A.K. Tanswell (MOP-15276)] grants from the Canadian Institutes of Health Research (CIHR) and an infrastructure grant from the Canada Foundation for Innovation New Opportunities Fund (R.P. Jankov). R.P. Jankov holds a CIHR Independent Investigator award.

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Chronic lung injury in the neonatal rat: Up-regulation of TGFβ1 and nitration of IGF-R1 by peroxynitrite as likely contributors to impaired alveologenesis
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Citation Excerpt :
Dams were exchanged daily between chambers to avoid maternal O2 toxicity. In some experiments, pups received daily subcutaneous (sc) injections (5 μl/g) of normal saline vehicle or 30 μg/g/day of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride (FeTPPS; Calbiochem, San Diego, CA, USA) in normal saline vehicle, as previously described [18]. Additionally, we investigated whether TGFß1 was a downstream regulator of peroxynitrite-induced effects by using a selective inhibitor of TGFß1 activin receptor-like kinase, SB 431542 (Sigma-Aldrich Canada).
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, TGFβ1. That 60% O₂-induced impairment of alveologenesis was mediated in part by TGFβ1 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 TGFβ1 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, TGFβ1.
Therapeutic hypercapnia prevents inhaled nitric oxide-induced right-ventricular systolic dysfunction in juvenile rats
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Chronic pulmonary hypertension in the neonate and infant frequently presents with right-ventricular (RV) failure. Current clinical management may include protracted treatment with inhaled nitric oxide (iNO), with the goal of reducing RV afterload. We have previously reported that prolonged exposure to iNO causes RV systolic dysfunction in the chronic hypoxia-exposed juvenile rat, which was prevented by a peroxynitrite decomposition catalyst. Given that inhalation of CO₂ (therapeutic hypercapnia) may limit oxidative stress and upregulated cytokine expression in the lung and other organs, we hypothesized that therapeutic hypercapnia would attenuate cytokine-mediated nitric oxide synthase (NOS) upregulation, thus limiting peroxynitrite generation. Sprague–Dawley rat pups were exposed to chronic hypoxia (13% O₂) from postnatal day 1 to 21, while receiving iNO (20 ppm) from day 14 to 21, with or without therapeutic hypercapnia (10% CO₂). Therapeutic hypercapnia completely normalized RV systolic function, RV hypertrophy, and remodeling of pulmonary resistance arteries in animals exposed to iNO. Inhaled nitric oxide-mediated increases in RV peroxynitrite, apoptosis, and contents of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, and NOS-2 were all attenuated by therapeutic hypercapnia. Inhibition of NOS-2 activity with 1400 W (1 mg/kg/day) prevented iNO-mediated upregulation of peroxynitrite and led to improved RV systolic function. Blockade of IL-1 receptor signaling with anakinra (500 mg/kg/day) decreased NOS-2 content and had similar effects compared to NOS-2 inhibition on iNO-mediated effects, whereas blockade of TNF-α signaling with etanercept (0.4 mg/kg on alternate days) had no effects on these parameters. We conclude that therapeutic hypercapnia prevents the adverse effects of sustained exposure to iNO on RV systolic function by limiting IL-1-mediated NOS-2 upregulation and consequent nitration. Therapeutic hypercapnia also acts synergistically with iNO in normalizing RV hypertrophy, vascular remodeling, and raised pulmonary vascular resistance secondary to chronic hypoxia.
Lipid hydroperoxide formation regulates postnatal rat lung cell apoptosis and alveologenesis
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Citation Excerpt :
Pups were killed on day 7 of life by inhalation of ethyl ether. Total (free and esterified) 8-isoprostane (8-iso-prostaglandin F2α) content was measured as previously described [12]. An enzyme-linked immunosorbent assay kit for 8-isoprostane was from Cayman Chemical Co. (Ann Arbor, MI, USA).
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 15 μg/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 187 μg/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.
Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle
2013, Free Radical Biology and Medicine
Peroxynitrite, the reaction product of nitric oxide and superoxide, contributes to the pathogenesis of chronic pulmonary hypertension in immature animals by stimulating proliferation of pulmonary arterial smooth muscle cells (PASMCs). Pulmonary vasoconstriction, secondary to hypoxia and other stimuli, leads to enhanced pulsatile stretch of cells in the vascular wall, particularly in smooth muscle, which we hypothesized would cause increased peroxynitrite generation. Our objectives in this study were to determine whether cyclic mechanical stretch, at supraphysiologic levels, would cause increased production of reactive oxygen species (ROS), nitric oxide, and peroxynitrite in vitro. Early passage neonatal rat PASMCs were seeded and grown to subconfluence on collagen-coated elastomer-bottom plates and subjected to cyclic mechanical stretch (10% (“physiologic”) or 20% (“supraphysiologic”) at 0.5 Hz) for up to 24 h. Compared to nonstretched controls and to cells subjected to 10% stretch, 20% stretch increased H₂O₂ (stable marker of ROS) and nitrate/nitrite (stable marker of nitric oxide) in conditioned medium. These effects were accompanied by increased peroxynitrite, as evidenced by increased in situ dihydroethidium fluorescence and immunoreactive nitrotyrosine and by increased expression of nitric oxide synthase (NOS)-1 and NADPH oxidase 4 (NOX4), but not NOS-2. Stretch-induced H₂O₂ release and increased dihydroethidium fluorescence were prevented by pretreatment with a superoxide scavenger, nonspecific inhibitors of NADPH oxidase or NOS, or a peroxynitrite decomposition catalyst. Short-interfering RNA-mediated knockdown of NOS-1 or NOX4 attenuated increased nitric oxide and H₂O₂ content, respectively, in stretched-cell-conditioned medium. Knockdown of NOS-1 also attenuated increased immunoreactive nitrotyrosine content and stretch-induced proliferation, whereas knockdown of NOS-2 had no effect. We conclude that increased peroxynitrite generation by neonatal rat PASMCs subjected to supraphysiologic levels of cyclic stretch is NOS-1-dependent and that increased ROS production is predominantly mediated by NADPH oxidase, specifically NOX4.
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Chronic pulmonary hypertension in infancy and childhood frequently culminates in right-ventricular (RV) failure and early death. Current management may include prolonged treatment with inhaled nitric oxide (iNO). Our objective was to examine the effects of iNO on established chronic hypoxic pulmonary hypertension in juvenile rats, a model of chronic neonatal pulmonary hypertension characterized by increased pulmonary vascular resistance, vascular remodeling (RV hypertrophy and arterial medial wall thickening), and significant RV dysfunction. Pups were exposed to air or hypoxia (13% O₂) from postnatal day 1 to 21 while receiving iNO (20 ppm) from day 14 to 21. In hypoxia-exposed animals, treatment with iNO decreased pulmonary vascular resistance, but did not augment RV output or reverse vascular remodeling. In addition, RV output was significantly reduced in air-exposed iNO-treated pups. Nitrotyrosine (a marker of peroxynitrite-mediated reactions), apoptosis, and expression of nitric oxide synthases 1 and 2 were increased in RV (but not left-ventricular) tissue from both air- and hypoxia-exposed pups treated with iNO. Concurrent treatment with a peroxynitrite decomposition catalyst (FeTPPS, 30 mg/kg/day, ip) prevented apoptosis and completely normalized RV output in iNO-exposed animals. Our results provide the first evidence that iNO may adversely impact the right ventricle through increased local generation of peroxynitrite.
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Original ContributionA peroxynitrite decomposition catalyst prevents 60% O2-mediated rat chronic neonatal lung injury

Abstract

Section snippets

Reagents

Effect of FeTPPS on lung and body weights and postfixation lung volumes

Discussion

Acknowledgments

Free Radic. Biol. Med.

Free Radic. Biol. Med.

Anal. Biochem.

J. Biol. Chem.

Free Radic. Biol. Med.

Cell

Biochim. Biophys. Acta

Arch. Biochem. Biophys.

J. Pediatr.

Pathology of chronic lung disease of early infancy

Acinar arterial changes with chronic lung disease of prematurity in the surfactant era

Pediatr. Pulmonol.

Expenditure for care of children with chronic illnesses enrolled in the Washington State Medicaid Program, fiscal year 1993

Pediatrics

Pulmonary hypertension in chronic lung disease of infancy: pathogenesis, pathophysiology, and treatment

Age and sex influence on pulmonary hypertension of chronic hypoxia and on recovery

Am. J. Physiol. Heart Circ. Physiol.

Three-week neonatal hypoxia reduces blood CGRP and causes persistent pulmonary hypertension in rats

Am. J. Physiol. Heart Circ. Physiol.

Ultrastructure of rat pulmonary arterioles after neonatal exposure to hypoxia and subsequent relief and treatment with monocrotaline

J. Pathol.

Progressive lung and cardiac changes associated with pulmonary hypertension in the fetal rat

Pediatr. Pulmonol.

The effect of chronic hypoxia on pulmonary arteries in young rats

Exp. Lung Res.

Normal postnatal development of the media of the rat hilar pulmonary artery and its remodelling by chronic hypoxia

Lab. Invest.

Influence of hypobaric hypoxia in infancy on the subsequent development of vasoconstrictive pulmonary vascular disease in the Wistar albino rat

J. Pathol.

Perinatal hypoxia increases hypoxic pulmonary vasoconstriction in adult rats recovering from chronic exposure to hypoxia

Am. Rev. Respir. Dis.

Chronic lung disease after premature birth

N Engl J. Med.

Emphysema in young adult survivors of moderate-to-severe bronchopulmonary dysplasia

Eur. Respir. J.

Changes in structure, mechanics and insulin-like growth factor-related gene expression in the lungs of newborn rats exposed to air or 60% O2

Pediatr. Res.

Endothelin-1 and O2-mediated pulmonary hypertension in neonatal rats: a role for products of lipid peroxidation

Pediatr. Res.

Gadolinium chloride inhibits pulmonary macrophage influx and prevents O2-induced pulmonary hypertension in the neonatal rat

Pediatr. Res.

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

Am. J. Physiol. Lung Cell. Mol. Physiol.

A critical role for interleukin-1 receptor in the lung injury induced in neonatal rats by 60% oxygen

Pediatr. Res.

Opposing effects of 60% oxygen and neutrophil influx on alveologenesis in the neonatal rat

Am. J. Respir. Crit. Care Med.

CXCR2 blockade reduces radical formation in hyperoxia-exposed newborn rat lung

Pediatr. Res.

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

Changes in structure, mechanics and insulin-like growth factor-related gene expression in the lungs of newborn rats exposed to air or 60% O₂

Endothelin-1 and O₂-mediated pulmonary hypertension in neonatal rats: a role for products of lipid peroxidation

Gadolinium chloride inhibits pulmonary macrophage influx and prevents O₂-induced pulmonary hypertension in the neonatal rat