Gene expression profile and histopathology of experimental bronchopulmonary dysplasia induced by prolonged oxidative stress

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Abstract

Oxidative stress is an important factor in the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of premature infants characterized by arrested alveolar and vascular development of the immature lung. We investigated differential gene expression with DNA microarray analysis in premature rat lungs exposed to prolonged hyperoxia during the saccular stage of development, which closely resembles the development of the lungs of premature infants receiving neonatal intensive care. Expression profiles were largely confirmed by real-time RT-PCR (27 genes) and in line with histopathology and fibrin deposition studied by Western blotting. Oxidative stress affected a complex orchestra of genes involved in inflammation, coagulation, fibrinolysis, extracellular matrix turnover, cell cycle, signal transduction, and alveolar enlargement and explains, at least in part, the pathological alterations that occur in lungs developing BPD. Exciting findings were the magnitude of fibrin deposition; the upregulation of chemokine-induced neutrophilic chemoattractant-1 (CINC-1), monocyte chemoattractant protein-1 (MCP-1), amphiregulin, plasminogen activator inhibitor-1 (PAI-1), secretory leukocyte proteinase inhibitor (SLPI), matrix metalloproteinase-12 (MMP12), p21, metallothionein, and heme oxygenase (HO); and the downregulation of fibroblast growth factor receptor-4 (FGFR4) and vascular endothelial growth factor (VEGF) receptor-2 (Flk-1). These findings are not only of fundamental importance in the understanding of the pathophysiology of BPD, but also essential for the development of new therapeutic strategies.

Introduction

Neonatal intensive care has been increasingly effective in reducing the mortality of very premature infants at the expense of an increasing number of survivors with bronchopulmonary dysplasia (BPD). BPD is a chronic lung disease that develops in newborn infants treated with oxygen and positive pressure ventilation for respiratory distress. Infants with BPD, defined clinically by a continuing need for oxygen supplementation at 36 weeks postmenstrual age, are at high risk for morbidity and mortality during the first years of life and many of them have respiratory problems throughout childhood and young adulthood [1]. BPD is particularly seen in infants born at less than 30 weeks of gestation and with a birth weight less than 1200 g [2], [3]. At birth the lungs of these infants are underdeveloped, surfactant-deficient, fluid-filled, and not supported by a stiff chest wall, which enhances their susceptibility to lung injury and inflammation [2]. Oxidative stress plays an important role in the development of BPD, which is characterized by decreased alveolarization and vascularization of the developing lung [2], [4].

Animal models of BPD are critical for characterizing the pathophysiology of BPD and testing of potential treatment options [4]. Exposure of premature baboons [5], neonatal mice [6], [7], and rats [8], [9], [10] to hyperoxia results in progressive lung disease, which strongly resembles BPD in premature infants. Although a recent NHLBI workshop proposed differential gene expression in uninjured and injured lungs as a research priority to learn how inflammation and injury are expressed by the developing lung [4], gene expression profiles of BPD in premature infants are still lacking. Therefore, we investigated histopathology and differential gene expression in experimental BPD using DNA microarray technology and real-time RT-PCR in a premature rat model with chronic lung injury, induced by prolonged exposure to hyperoxia, and demonstrate the significance of this model for studying BPD in premature infants.

Section snippets

Animals

Timed-pregnant Wistar rats were kept in a 12 h dark/light cycle and fed a standard chow diet (Special Diet Services, Witham, Essex, England) ad libitum. Animal care was in accordance with institutional guidelines of the Leiden University Medical Center. Spontaneous birth occurred 22 days after conception. After a gestation of 21 days, pregnant rats were killed by decapitation and pups were delivered by hysterectomy through a median abdominal incision. Immediately after birth, the premature rat

Survival, lung histology, morphometrical analysis, and fibrin deposition

At birth (day 1) body weight was 4.8 g (Fig. 1A). In both oxygen-exposed and control pups body weight increased during the first week to approximately 10 g. From then on, controls grew slightly faster than oxygen-exposed pups. Initially, survival of both groups was similar (95%, Fig. 1B). Lethal effects of hyperoxia were observed from day 8 onwards, resulting in a survival rate of 77% on day 10 and 15% on day 14. Therefore, we included only pups from birth until day 10 in our studies.

Rats are

Discussion

In this study, we focused on differential gene expression in neonatal rat lungs exposed to prolonged hyperoxia with clear pathological changes during the saccular stage of development, an experimental setting that closely resembles the developmental stage of very premature infants receiving neonatal intensive care. Rats were born at the saccular stage of lung development and alveolarization by secondary septation took place during the first 2 weeks of life. Prolonged exposure of premature rat

Acknowledgements

We thank Eveline M. Mank, Dr. Judith M. Boer, and Dr. Johan T. den Dunnen of the Leiden Genome Technology Center for expert technical assistance with the cDNA chip experiments. We gratefully acknowledge Dr. Joost C.M. Meijers for supplying us with the antibody 59D8. This study was supported in part by a grant from the Gisela Thier Fund.

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