Robert P. Jankov

Antioxidants as therapy in the newborn: some words of caution.

Reactive oxygen and nitrogen species are considered to play a major role in the pathogenesis of a wide range of human disorders. This may be a particularly important pathogenetic mechanism in the newborn nursery. The phrase “oxygen radical disease of prematurity” has been coined to collectively describe a wide range of neonatal disorders based on the belief that premature newborns are deficient in antioxidant defenses at a time when they are subjected to acute and chronic oxidant stresses. This belief has led to a number of clinical trials of antioxidant therapies being undertaken in neonatal patients. The realization that reactive oxygen species play a critical role in neonatal illnesses has only recently been paralleled by an increased understanding of their physiologic roles. A major concern is that effective scavenging of reactive oxygen species, to attenuate their toxic effects, will also inhibit essential cellular functions such as growth in potential target organs such as lung, brain, intestine, and retina.

Changes in expression of platelet-derived growth factor and its receptors in the lungs of newborn rats exposed to air or 60 % O2

PDGF-related gene expression has been well characterized during fetal rat lung development and adult rat lung injury, but not during normal postnatal lung growth or injury. Lung expression of the mRNA for PDGF-A, -B, -αR, and -βR and immunoreactive PDGF-AA, -BB, -αR, and -βR were assessed in rat pups raised in air or 60% O2 for up to 14 d after birth. Expression of mRNA and immunoreactive ligand did not correlate for pups raised in air. Immunoreactive PDGF-αR and -βR, but not PDGF-AA and -BB, were evident throughout the lung at birth. Both PDGF-AA and -BB were evident in airway epithelium, PDGF-BB in alveolar epithelial cells and PDGF-AA was widely distributed in parenchymal tissue at 4 d. PDGF-αR was localized to airway epithelium, and PDGF-βR to subendothelial perivascular regions and to airway and alveolar epithelium at 4 d. Immunoreactive PDGF ligands all declined after 4 d. Intraperitoneal injection of neutralizing antibodies or truncated soluble receptors to PDGF-BB reduced lung DNA synthesis in air. Exposure to 60% O2 significantly increased mRNA for PDGF-B, -βR, and -αR, but not PDGF-A, relative to air-exposed lung at various time points after birth. PDGF-A, -B, and -αR immunoreactivities in these lungs were reduced and delayed, consistent with a global inhibition of lung growth. Pups exposed to 60% O2 had a similar distribution of PDGF-βR to that seen in air, except that at 14 d PDGF-βR was distributed throughout the lung parenchyma. We conclude that PDGF ligands and receptors are important for normal postnatal lung growth and that their expression is delayed by O2 exposure.

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.

Rescue treatment with a Rho-kinase inhibitor normalizes right ventricular function and reverses remodeling in juvenile rats with chronic pulmonary hypertension

Chronic pulmonary hypertension in infancy and childhood is characterized by a fixed and progressive increase in pulmonary arterial pressure and resistance, pulmonary arterial remodeling, and right ventricular hypertrophy and systolic dysfunction. These abnormalities are replicated in neonatal rats chronically exposed to hypoxia from birth in which increased activity of Rho-kinase (ROCK) is critical to injury, as evidenced by preventive effects of ROCK inhibitors. Our objective in the present study was to examine the reversing effects of a late or rescue approach to treatment with a ROCK inhibitor on the pulmonary and cardiac manifestations of established chronic hypoxic pulmonary hypertension. Rat pups were exposed to air or hypoxia (13% O(2)) from postnatal day 1 and were treated with Y-27632 (15 mg/kg) or saline vehicle by twice daily subcutaneous injection commencing on day 14, for up to 7 days. Treatment with Y-27632 significantly attenuated right ventricular hypertrophy, reversed arterial wall remodeling, and completely normalized right ventricular systolic function in hypoxia-exposed animals. Reversal of arterial wall remodeling was accompanied by increased apoptosis and attenuated content of endothelin (ET)-1 and ET(A) receptors. Treatment of primary cultured juvenile rat pulmonary artery smooth muscle cells with Y-27632 attenuated serum-stimulated ROCK activity and proliferation and increased apoptosis. Smooth muscle apoptosis was also induced by short interfering RNA-mediated knockdown of ROCK-II, but not of ROCK-I. We conclude that sustained rescue treatment with a ROCK inhibitor reversed both the hemodynamic and structural abnormalities of chronic hypoxic pulmonary hypertension in juvenile rats and normalized right ventricular systolic function. Attenuated expression and activity of ET-1 and its A-type receptor on pulmonary arterial smooth muscle was a likely contributor to the stimulatory effects of ROCK inhibition on apoptosis. In addition, our data suggest that ROCK-II may be dominant in enhancing survival of pulmonary arterial smooth muscle.

Effects of Rho-Kinase Inhibition on Pulmonary Hypertension, Lung Growth, and Structure in Neonatal Rats Chronically Exposed to Hypoxia

Rho-kinase (ROCK) inhibitors prevent pulmonary hypertension (PHT) in adult rodents, but little is known about their effects on the neonatal lung. Our objective was to examine the effects of ROCK inhibition on chronic hypoxia (CH)-induced PHT and abnormal lung structure in the neonatal rat. Pups were exposed to air or CH from postnatal d 1-14 while receiving Y-27632 (5 or 10 mg · kg−1 · d−1), fasudil (20 mg · kg−1 · d−1), or saline intraperitoneally. Relative to air, CH-exposed pups had increased pulmonary vascular resistance, right ventricular hypertrophy, arterial medial wall thickening, and abnormal distal airway morphology characterized by septal thinning and decreased secondary septation. Treatment with 10 mg/kg Y-27632 or fasudil attenuated the structural and hemodynamic changes of PHT while having no effect on septal thinning or inhibited secondary septation. In addition, Y-27632 (10 mg/kg) and fasudil augmented CH-induced somatic growth restriction. Pulmonary arteries of CH-exposed pups had increased ROCK activity, up-regulated expression of PDGF-BB and increased smooth muscle DNA synthesis, all of which were attenuated by treatment with 10 mg/kg Y-27632. Systemically administered ROCK inhibitors prevented PHT in the CH-exposed neonatal rat but at the cost of inhibited somatic growth. Limiting effects on vascular remodeling likely resulted, in major part, from attenuated vascular PDGF-BB/β-receptor signaling.

Endothelin-1 Inhibits Apoptosis of Pulmonary Arterial Smooth Muscle in the Neonatal Rat

Vascular wall remodeling in pulmonary hypertension is contributed to by an aberration in the normal balance between proliferation and apoptosis of smooth muscle. We observed that endothelin (ET)-1 is a critical mediator of vascular remodeling in neonatal rats chronically exposed to 60% O2, but has no direct proliferative effects on cultured neonatal rat pulmonary artery smooth muscle cells (PASMCs). These findings led us to hypothesize that ET-1 may modulate remodeling by inhibiting apoptosis of smooth muscle. ET-1 (0.1 μM) was found to significantly attenuate both Paclitaxel- and serum deprivation-induced PASMC apoptosis, likely through stimulation of the ETA receptor (ETAR). ET-1 also prevented Paclitaxel-induced up-regulation of pro-apoptotic Bax and cleaved (activated) caspase-3. In rat pups exposed from birth to 60% O2 for 7 d, arterial wall expression of Bax was decreased and expression of both ETAR and anti-apoptotic Bcl-xL were increased. Furthermore, increased numbers of TUNEL-positive cells were evident in the walls of pulmonary arteries from 60% O2-exposed animals treated with a combined ET receptor antagonist, SB217242, relative to air-exposed and vehicle-treated groups. Together, these findings suggest that ET-1 mediates remodeling of neonatal rat pulmonary arteries by inhibiting smooth muscle apoptosis.

Hypercapnia and the neonate.

‘Permissive hypercapnia’ is a familiar term in neonatal intensive care, given the widespread adoption of low‐tidal‐volume ventilation strategies applied with the goal of decreasing respiratory morbidity. Recent evidence suggesting that hypercapnic acidosis may itself have protective effects on the lung and other organs has led to the coining of a new phrase, ‘therapeutic hypercapnia’, which also encompasses the use of supplemental inspired CO2.

Macrophages as a major source of oxygen radicals in the hyperoxic newborn rat lung.

The lungs of newborn rats exposed to 60% O(2) for 14 d were found to have a greatly increased cyanide-insensitive O(2) consumption, reflecting increased reactive oxygen species (ROS) formation. Exposure of the lung to hyperoxia is known to increase the production of ROS by mitochondria. We hypothesized that macrophages may also be a major contributor to this increase. Newborn rat pups were exposed to either air or 60% O(2) for 14 d and received either intraperitoneal gadolinium chloride (GdCl(3)) to abrogate macrophage influx, or inert vehicle. Lung homogenates were equilibrated in either 21% or 100% O(2) and total and cyanide-insensitive O(2) consumption, as well as nitric oxide accumulation were measured polarographically. Citrate synthase, a marker of mitochondrial mass, and nitrotyrosine, a marker of peroxynitrite formation, were quantified by Western blot. In addition to increased macrophage numbers, the lungs of 60% O(2)-exposed animals had greatly increased cyanide-insensitive O(2) consumption (p <.05 compared to air controls) and immunoreactive nitrotyrosine (p <.05), which were all completely abrogated by treatment with GdCl(3). Exposure to 60% O(2) for 14 d had no effect on peroxynitrite-independent nitric oxide release or mitochondrial mass. We conclude that increased ROS in the lungs of newborn rats exposed to 60% O(2) for 14 d was likely to be caused, in significant part, by the presence of increased numbers of macrophages.

Growth factors, postnatal lung growth and bronchopulmonary dysplasia*

The term bronchopulmonary dysplasia (BPD) was coined by Northway et al.1 to reflect the involvement of all lung tissue elements in the pathology of a new disorder of postnatal lung growth described in ventilated infants. The described features included airway mucosal metaplasia, airway and vascular smooth muscle hyperplasia, saccular emphysema and atelectasis. The same group of investigators subsequently emphasised the additional feature of widespread interstitial fibrosis.2 An intriguing feature of BPD, as seen more recently, has been a reduced frequency of airway injury.3,4 This may have simply reflected a change in population characteristics, in that there has been a proportional increase in the representation of very low-birthweight infants amongst those who survive and go on to develop BPD.5 Alternatively, there may have been some unattributed change in management which has resulted in a decrease in the degree of airway injury. There are likely to be multiple factors that result in the histological changes seen with the development of BPD, but the three major candidates are pulmonary oxygen toxicity, volutrauma and cellular immaturity. Unlike injury to the adult lung, in which lung-cell proliferation

Rho-Kinase Inhibitor Prevents Bleomycin-Induced Injury in Neonatal Rats Independent of Effects on Lung Inflammation

Bleomycin-induced lung injury is characterized in the neonatal rat by inflammation dominated by neutrophils and macrophages, inhibited distal airway and vascular development, and pulmonary hypertension, similar to human infants with severe bronchopulmonary dysplasia. Rho-kinase (ROCK) is known to mediate lung injury in adult animals via stimulatory effects on inflammation. We therefore hypothesized that inhibition of ROCK may ameliorate bleomycin-induced lung injury in the neonatal rat. Pups received daily intraperitoneal bleomycin or saline from Postnatal Days 1 through 14 with or without Y-27632, a ROCK inhibitor. Treatment with Y-27632 prevented bleomycin-induced pulmonary hypertension, as evidenced by normalized pulmonary vascular resistance, decreased right-ventricular hypertrophy, and attenuated remodeling of pulmonary resistance arteries. Bleomycin-induced changes in distal lung architecture, including septal thinning, inhibited alveolarization, and decreased numbers of peripheral arteries and capillaries, were partially or completely normalized by Y-27632. Treatment with Y-27632 or a CXCR2 antagonist, SB265610, also abrogated tissue neutrophil influx, while having no effect on macrophages. However, treatment with SB265610 did not prevent bleomycin-induced lung injury. Lung content of angiostatic thrombospondin-1 (TSP1) was increased significantly in the lungs of bleomycin-exposed animals, and was completely attenuated by treatment with Y-27632. Thrombin-stimulated TSP1 production by primary cultured rat pulmonary artery endothelial cells was also attenuated by Y-27632. Taken together, our findings suggest a preventive effect of Y-27632 on bleomycin-mediated injury by a mechanism unrelated to inflammatory cells. Our data suggest that improvements in lung morphology may have been related to indirect stimulatory effects on angiogenesis via down-regulation of TSP1.