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The beneficial effects of hypercapnia,
and the detrimental effects of
peroxynitrite, in chronic neonatal lung injury
By
Azhar Masood, M.D.
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Physiology
University of Toronto
© Copyright by Azhar Masood 2011
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ABSTRACT OF THESIS
The beneficial effects of hypercapnia, and the detrimental effects
of peroxynitrite, in chronic neonatal lung injury
by
Azhar Masood, M.D.
Ph.D., 2011, Department of Physiology, University of Toronto
Bronchopulmonary dysplasia (BPD) is a chronic neonatal lung injury (CNLI)
affecting infants of < 32 weeks gestation, which has a significant associated morbidity
and mortality. The hallmarks of BPD as seen in the current era are arrested
alveologenesis and parenchymal thickening. Those most severely affected may develop
pulmonary hypertension which worsens the prognosis. No effective preventive therapy
exists. Generation of damaging reactive oxygen species is implicated in its development.
The more recently recognized reactive nitrogen species may also contribute to this
disease. Thus, there is considerable interest in preventive antioxidant therapies, but
results to date have not been promising. Newborn rats, exposed to 60% O2 for 14 days,
develop a parenchymal injury and pulmonary hypertension that resembles the
morphological features of human BPD. Previous studies have shown that following
exposure to 60% O2, a pulmonary influx of neutrophils is followed by that of
macrophages. Inhibiting the influx of neutrophils prevents the generation of reactive
oxygen species, while simultaneously enhancing postnatal lung growth. Other
interventions have shown that development of pulmonary hypertension is dependent upon
increases in both 8-isoprostane and its downstream regulator of vascular tone, endothelin-
1. Gentler ventilation strategies, incorporated to minimize induction of stretch-mediated
pro-inflammatory cytokines, have shown benefits of permissive hypercapnia in adult lung
injury. Multicentre clinical trials of permissive hypercapnia in neonates have not shown
benefit. Therapeutic hypercapnia has been demonstrated to have a protective effect of
PaCO2 in both acute studies of ventilator-induced and ischemia-reperfusion injuries in
animal models. In the studies reported herein, therapeutic hypercapnia was found to
completely protect against CNLI and attenuate 60% O2-induced macrophage-derived
protein nitration. The likely nitrating agent was macrophage-derived peroxynitrite. The
critical role of peroxynitrite, in the development of chronic neonatal lung injury in this
model, was confirmed using a peroxynitrite decomposition catalyst. This protected
against the impairments of alveolarization and of pulmonary vascularization induced by
60% O2. These results suggest a more significant role for reactive nitrogen species than
previously recognized. Finally, preliminary evidence is presented supporting a role for
neutrophil-derived elastase in initiating the macrophage influx in the lungs, required for
peroxynitrite generation, during 60% O2-mediated injury.
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ACKNOWLEDGMENTS
I would like to begin by thanking Dr. Keith Tanswell, my thesis supervisor, for
his boundless support and mentorship throughout the years, and for his inexhaustible and
meticulous efforts geared towards perfecting my work. I would also like to sincerely
thank my supervisory committee members, Dr. Brian Kavanagh, Dr. Jaques Belik, Dr.
Martin Post, Dr. Robert Jankov and Dr. Patrick McNamara for their constant guidance,
constructive criticism and fitting encouragement. I want to make a special note of the
endless support by my fellow colleagues Ms. Rosetta Belcastro, Ms. Judy Cabacungan,
Dr. Man Yi, Mr. Samuel Shek, Ms. Crystal Kantores, Ms. Julijana Ivanovska, Dr. Jun Li
and especially Ms. Anna Decaria for her help with administrative work. I would
particularly like to thank Dr. André Kroon, Dr. Ben Hur Johnson, Dr. Adrian Ziino and
Dr. Darakhshanda Shehnaz for their contributions to the many scientific and intellectual
discussions we had. I wish to thank all the animal care staff at Sunnybrook Health
Science Centre for their continual assistance. I would like to thank the Canadian Institutes
of Health Research, the Hospital for Sick Children and the University of Toronto for their
support.
Most importantly, I would like to express my deepest gratitude to my late father
Professor Dr. Masood Alam for his endless scholarly, emotional and financial support
throughout the duration of this project. I would like to thank my loving mother for her
immeasurable encouragement. Lastly, I would like to thank my lovely wife Navera, my
daughters Hooriya and Huda, and my son Muhammad for their bountiful love and
support that helped me stay focused all the way through. Without their encouragement,
and the Creator’s blessings, this achievement would not have been possible.
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TABLE OF CONTENTS
List of figures................................................................................................................ x List of tables.................................................................................................................. xiii List of abbreviations ..................................................................................................... xiv Publication of thesis work............................................................................................. xvi Chapter 1 Introduction................................................................................................................. 1 The mammalian lung .................................................................................................... 2 Development ................................................................................................................ 2 Embryonic stage................................................................................................ 3 Pseudoglandular stage ...................................................................................... 4 Canalicular stage .............................................................................................. 4 Saccular stage.................................................................................................... 5 Alveolar stage ................................................................................................... 5 Alveolarization.............................................................................................................. 6 Two phase model of alveolarization ................................................................. 6 Factors influencing parenchymal growth...................................................................... 7 Pulmonary vascular development ................................................................................ 9 Factors influencing vascular growth ............................................................................ 12 Bronchopulmonary dysplasia........................................................................................ 13 Old (classic) BPD ............................................................................................. 13 “New” CNLI ..................................................................................................... 14 Aetiology of CNLI............................................................................................ 17 Management strategies for prevention of CNLI .......................................................... 18 Current therapies............................................................................................... 18 Permissive hypercapnia ................................................................................................ 21 Therapeutic hypercapnia............................................................................................... 23 Clinical trials of hypercapnia ........................................................................................ 24
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Oxidative/nitrative stress in CNLI ................................................................................ 25 Animals models of CNLI.............................................................................................. 33 60% O2-model of CNLI ................................................................................................ 35 Pulmonary hypertension/vascular injury/remodeling ................................................... 36 Origins of pulmonary hypertension in the 60% O2-model of CNLI............................. 38 Chapter 2 Aims/Hypotheses ........................................................................................................ 40 Global aims ................................................................................................................... 41 Specific hypotheses....................................................................................................... 41 Chapter 3 General Materials and Experimental Procedures ................................................... 43 Institutional review/in vivo interventions...................................................................... 44 Immunohistochemistry ................................................................................................. 44 Western blot analyses/immunoprecipitation................................................................. 45 Enzyme-linked immunosorbent assay (ELISA) analyses............................................. 46 Morphometric analyses................................................................................................. 46 Vessel counts and medial wall thickness analyses ....................................................... 51 Data presentation .......................................................................................................... 52 Chapter 4 Therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats............................................................................................................ 53 Abstract ......................................................................................................................... 54 Aims/objectives............................................................................................................. 55
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Hypothesis..................................................................................................................... 55 Introduction................................................................................................................... 55 Clinical relevance.......................................................................................................... 57 Specific materials and experimental procedures........................................................... 58 In vivo interventions.......................................................................................... 58 Von Willebrand factor immunohistochemistry ................................................ 58 Enzyme-linked immunosorbent assay (ELISA) analysis ................................. 58
Blood gas analyses............................................................................................ 58 Assessment of respiratory and heart rates......................................................... 59 Results ........................................................................................................................... 59 Exposure to hypercapnia resulted in a significant attenuation of increased tissue fraction routinely observed in pups exposed to 60% O2................................... 59 Concomitant hypercapnia and 60% O2 resulted in increased secondary crest
formation and alveolar growth.......................................................................... 65 Effects of therapeutic hypercapnia on inflammatory cell influx in the lung
parenchyma. ...................................................................................................... 66 Hypercapnia prevented vascular smooth muscle cell hyperplasia in 60% O2-
exposed pups..................................................................................................... 69 Hypercapnia attenuated the marked increase in nitrotyrosine immunoreactivity
observed in 60% O2-exposed pups. .................................................................. 69 Hypercapnia prevented α-smooth muscle actin nitration otherwise observed in
60% O2-exposed pups. ...................................................................................... 72 Hypercapnia abrogated the peripheral pulmonary vessel pruning in 60% O2-
exposed pups..................................................................................................... 72 Discussion..................................................................................................................... 74 Chapter 5 A peroxynitrite decomposition catalyst prevents 60% O2-mediated rat chronic neonatal lung injury.................................................................................................... 82 Abstract ......................................................................................................................... 83 Aims/objectives............................................................................................................. 83
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Hypothesis..................................................................................................................... 84 Introduction................................................................................................................... 84 Clinical relevance.......................................................................................................... 85 Peroxynitrite decomposition catalysts ......................................................................... 85 Specific materials and experimental procedures........................................................... 86 In vivo interventions.......................................................................................... 86 Total (free and esterified) 8-isoprostane measurement..................................... 86 Enzyme-linked immunosorbent assay (ELISA) analysis ................................. 87 Western blot analyses ....................................................................................... 87 Results ........................................................................................................................... 88 Effects of FeTPPS on lung and body weights and post-fixation lung volumes
nitration. ............................................................................................................ 88 Effects of FeTPPS on tyrosine nitration. .......................................................... 88 Protective effects of FeTPPS on the pulmonary vascular bed........................... 91 Protective effects of FeTPPS on alveolar development.................................... 93 Effects of FeTPPS on phagocyte influx............................................................ 97 Effects of FeTPPS on total and nitrated α-smooth muscle actin. .....................104 Effects of FeTPPS on nitrated PDGFR-α .........................................................104
Discussion.....................................................................................................................104 Chapter 6 Future directions and preliminary findings ............................................................112 Future directions ..........................................................................................................113 Clinical relevance .........................................................................................................115 An elastase inhibitor prevents 60% O2-mediated macrophage influx in neonatal rats.115 Aims/objectives ............................................................................................................115
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Hypothesis.....................................................................................................................115 Elafin ...........................................................................................................................116 Specific materials and experimental procedures...........................................................116 In vivo interventions .....................................................................................................116 Preliminary results .......................................................................................................116 Elafin prevented the 60% O2-induced macrophage induction .....................................116 Elafin partially reversed the reduced lung elastin content in 60% O2-exposed pups ...117 Discussion ....................................................................................................................117 Chapter 7 Conclusions..................................................................................................................121 Summary of findings ....................................................................................................122 Relevance .....................................................................................................................123 Limitations of this study ...............................................................................................123 Current concepts of the pathways involved in 60%-O2-induced CNLI........................124 Implications for CNLI...................................................................................................126 Clinical implications .....................................................................................................126 References ...................................................................................................................128
ix
LIST OF FIGURES
Chapter 1
Fig. 1.1 Survival of extremely-low-birth-weight infants Data from the Canadian Neonatal Network ............................................ 16 Fig. 1.2A The membrane chain reaction of lipid peroxidation ................................ 22 Fig. 1.2B NO as a lipid peroxidation chain breaker ................................................ 22 Fig. 1.3 The Haber-Weiss reaction........................................................................ 28
Chapter 3 Fig. 3.1 Illustration of crossed hairline to measure mean linear intercept ............ 47 Fig. 3.2 Cartoon of the 130-point contiguous grid used in measuring tissue fraction and secondary crest volume density ........................................................ 49
Chapter 4
Fig. 4.1 Lung histology and morphometric analysis of neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2.............................. 63 Fig. 4.2 Measurement of mean linear intercept and variance in average mean linear intercept measurements between fields within each lung section in neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2 ................................................................................................. 64 Fig. 4.3 Measurement of secondary crest density and secondary crest-to-tissue ratio and estimates of alveolar surface area and total alveolar number in neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2........................................................................................................... 67 Fig. 4.4 Neutrophil and macrophage immunostaining and counts per unit area in neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2........................................................................................................... 68
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Fig. 4.5 Immunostaining for α-smooth muscle actin and medial wall thickness (%), an index of pulmonary hypertension in neonatal rats ..................... 70 Fig. 4.6 Nitrotyrosine formation, a marker for peroxynitrite-mediated reactions, in the lung tissue of neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2........................................................................ 71 Fig. 4.7 Western blot analyses of nitrotyrosine and nitrated α-smooth muscle actin contents in neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2........................................................................ 73 Fig. 4.8 Immunofluorescent staining for von Willebrand factor in the lungs of neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2 ................................................................................................. 75 Fig. 4.9 Hart’s elastin staining of lung tissue from neonatal rats exposed to air or 60% O2 for 14 days with or without 5.5% CO2 ....................................... 76 Fig. 4.10 Counts of lung peripheral vessels and concentrations of VEGF-A, Ang-1 and Flt-1 in lung homogenates ..................................................... 77
Chapter 5
Fig. 5.1 Effects of FeTPPS on 60% O2-induced nitrotyrosine formation ............. 90 Fig. 5.2 Effects of FeTPPS on the 60% O2-mediated reduction in peripheral vessel density ........................................................................................... 92 Fig. 5.3 Effects of FeTPPS on the 60% O2-induced changes in lung content of mediators of angiogenesis........................................................................ 94 Fig. 5.4 Effects of FeTPPS on markers and mediators of 60% O2-induced pulmonary hypertension........................................................................... 95 Fig. 5.5 Effects of FeTPPS on 60% O2-induced changes in lung structure .......... 96 Fig. 5.6 Low-power photomicrographs to illustrate: (1) the heterogeneity in alveolar/saccular diameters observed in O2-exposed group; (2) enhanced alveologenesis with reduced diameters in the FeTPPS-treated and O2- exposed group .......................................................................................... 98 Fig. 5.7 Effects of FeTPPS on the 60% O2-mediated changes in alveologenesis. 99 Fig. 5.8 Lack of effect of FeTPPS on the 60% O2-mediated phagocyte influx ....101
xi
Fig. 5.9 Exposure to 60% O2 resulted in an influx of neutrophils, as assessed by myeloperoxidase immunoreactivity....................................................102 Fig. 5.10 Exposure to 60% O2 resulted in an influx of macrophages, as assessed by CD-68 immunoreactivity, which was apparently not affected by treatment with FeTPPS ............................................................................103 Fig. 5.11 Effect of FeTPPS on the 60% O2-induced changes in total and nitrated lung α-smooth muscle actin content ........................................................105 Fig. 5.12 Effect of FeTPPS on the 60% O2-induced nitration of PDGFR-α...........106
Chapter 6
Fig. 6.1 Preliminary data to assess the effect of subcutaneous elafin once daily for 6 days on lung macrophage accumulation in 60% O2...............118 Fig. 6.2 Hart’s stain of day-6 lung tissue after subtraction of background to show elastin in pups exposed to air or to 60% O2 ± elafin 8 mg/kg/d.....119
Chapter 7 Fig. 7.1 Schematic illustration of concepts of the pathways involved in the 60% O2-induced rat model of CNLI ................................................................125
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LIST OF TABLES
Table 1 Blood gases from day-14 rat pups exposed to air ± 5.5% CO2................ 60 Table 2 Respiratory and heart rates of day-14 rat pups before and after anaesthesia following exposure to air ± 5.5% CO2.................................. 61 Table 3 Lung and body weights and postfixation displacement lung volumes in 14-day-old rat pups exposed to air or 60% O2 ± 5.5% CO2 .................... 62 Table 4 Lung and body weights and postfixation displacement lung volumes in 14-day-old rat pups exposed to air or 60% O2 ± FeTPPS........................ 89
xiii
LIST OF ABBREVIATIONS AM Alveolar macrophages AMP Adenosine monophosphate AKT Not an abbreviation Ang-1 Angiopoietin-1 B Spherical constant of an alveolus of 1.55 bFGF Basic fibroblast growth factor BPD Bronchopulmonary dysplasia BrdU Bromodeoxyuridine (5-bromo-2'-deoxyuridine) BW Body weight CD-68 Cluster of differentiation 68 CGA Corrected gestational age cGMP Cyclic guanosine monophosphate CINC-1 Cytokine-induced neutrophil chemoattractant CNLI Chronic neonatal lung injury CO2 Carbon dioxide D Distribution variable of the characteristic linear dimensions of the alveoli DNA Deoxyribonucleic acid ECM Extracellular matrix EGF Epidermal growth factor ELISA Enzyme-linked immunosorbent assay ET-1 Endothelin-1 IL-1 Interleukin-1 FeTPPS 5,10,15,20 Tetrakis(4-sulfonatophenyl)porphyrinato iron (III), chloride FeTMPS Iron (III) Meso-tetra(2,4,6-trimethyl-3,5-disulfonato)porphine chloride FeTMPyP Iron (III) Tetrakis(1-methyl-4-pyridyl)porphyrins pentachlorideporphyrin pentachloride FGF Fibroblast growth factor Flt-1 Fms-like tyrosine kinase-1 FIO2 Fraction of inspired oxygen GAPDH Glyceraldehyde 3-phosphate dehydrogenase GF Growth factor(s) GF-R Growth factor receptors H2O2 Hydrogen peroxide HGF Hepatocyte growth factor IGF Insulin-like growth factor kDA KiloDalton L• Lipid radical LH Membrane lipid Lm Mean linear intercept LOO• Lipid peroxyl radical LOOH Lipid hydroperoxide
xiv
LOONO Alkyl peroxynitrite LV Lung volume in millilitres LV Lung volume LW Lung weight LW/BW Lung weight/body weight MCP-1 Monocyte chemotactic protein-1 MIP-1α Macrophage inflammatory protein-1 alpha MIP-1β Macrophage inflammatory protein-1 beta NA Corrected alveolar count per unit area N2 Nitrogen NADPH Nicotinamide adenine dinucleotide phosphate NICU Neonatal intensive care unit NO• Nitric oxide radical NOS Nitric oxide synthase NT Estimated total alveolar number NV Alveoli per unit volume O2 Oxygen O2
•- Superoxide ONOO¯ Peroxynitrite anion ONOOH Peroxynitrous acid PBS Phosphate buffered saline PDGF Platelet-derived growth factor PHT Pulmonary hypertension PI-3kinase Phophoinositide 3-kinase PMNL Polymorphonuclear leukocytes PO2 Partial pressure of oxygen PreproET-1 Preproendothelin-1 RDS Respiratory distress syndrome RNS Reactive nitrogen species ROS Reactive oxygen species SaO2 Oxygen saturation sc Subcutaneous SOD Superoxide dismutase TGF Transforming growth factor TIE Tyrosine kinase with immunoglobin and EGF-like domains TIE-2 Tyrosine kinase with immunoglobin and EGF-like domains-2 VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor VEGFR-2 Vascular endothelial growth factor receptor-2 VGVAPG Valine-Glycine-Valine-Alanine-Proline-Glycine VVas Fractional volume occupied by alveoli
xv
PUBLICATION OF THESIS WORK
1. Azhar Masood, Man Yi, Mandy Lau, Rosetta Belcastro, Samuel Shek, Jingyi Pan,
Crystal Kantores, Patrick J. McNamara, Brian P. Kavanagh, Jaques Belik, Robert
P. Jankov and A. Keith Tanswell “Therapeutic effects of hypercapnia on chronic
lung injury and vascular remodeling in neonatal rats”1
American Journal of Physiology: Lung, Cellular and Molecular Physiology
297:920-930, 2009.
doi:10.1152/ajplung.00139.2009
2. Azhar Masood, Rosetta Belcastro, Jun Li, Crystal Kantores, Robert P. Jankov,
and A. Keith Tanswell “A peroxynitrite decomposition catalyst prevents 60% O2-
mediated rat chronic neonatal lung injury”2
Free Radical Biology and Medicine
49:1182–1191, 2010.
doi:10.1016/j.freeradbiomed.2010.07.001
1 Data reproduced (Chapter 4) with permission from The American Physiological Society 2 Data reproduced (Chapter 5) with permission from Elsevier
xvi
1
Chapter 1
Introduction
1. Introduction
1.1. The mammalian lung
The lung is a complex organ. Its development, which involves the differentiation
and proliferation of over forty different cells types [1], as well as the formation of a
system of branched conduits, commences during the embryonic period and continues into
adult life, during which there are distinct stages of maturation and growth. Once fully
differentiated and developed, the mature lung is comprised of 23 generations of
conducting airways that have branched repeatedly to terminate at the alveoli, the
spherical outcroppings that permit gas exchange with the blood. Each successive
branching decreases the airway luminal diameter with a simultaneous increase in total
surface area. The honeycomb-like network of alveolar units and the pulmonary vascular
circulation are separated by a fine alveolo-capillary barrier ~ 0.2 µm in thickness [2],
permitting efficient gas exchange. In humans, lung development at birth is far from
complete. Only 20% of alveoli have been formed at term, but are sufficient to sustain life.
The remaining 80% continues to develop into adult life. Once fully developed, the adult
human lung consists of more than 300 million alveoli [1], yielding a gas-exchange
surface area of ~ 70 m2.
1.2 Development
Lung development spans over three chronological periods. An embryonic period,
comprising of organogenesis, a fetal period, which is further sub-classified into three
distinct morphological stages, and a post-natal period, which continues into adulthood.
Having originated from the ventral surface of the primitive foregut, the respiratory tree
2
develops by branching morphogenesis [3], to form the 23 generations of the human
bronchial tree, which are housed in three lobes on the right and two lobes on the left. The
accompanying vascularization, including both angiogenesis and vasculogenesis, followed
by alveolarization, are precisely orchestrated by a number of genes, transcription factors,
growth factors, extracellular matrix molecules, integrins, and mechanical forces.
Morphologically, lung development can be divided into five distinct stages:
embryonic, pseudoglandular, canalicular, saccular and alveolar.
1.2 i a Embryonic stage
The embryonic stage spans from the 4th to the 7th week of gestation in humans
(gestational days 5-11 in rats). During the embryonic stage of development, the lung
forms as an outgrowth of the ventral wall of the primitive foregut as the laryngotracheal
groove. The epithelial cells from the endoderm of the foregut invade the surrounding
mesenchyme to form the larynx and trachea proximally, while the distal part of the
groove bifurcates into the left and right bronchial buds. Arrested growth of the epithelial
cells at branch points, in unison with the accelerated growth lateral to them, allows the
formation of left and right lobar airways. Further branching results in the formation of
two buds on the left and three buds on the right in humans, and one on the left and four
on the right in rodents. Subsequent branching forms segmental bronchi which continue to
divide to form 18 major lobules [4]. Pulmonary vessels arise from the 6th aortic arch and
grow around the airway buds by vasculogenesis [5].
3
1.2 i b Pseudoglandular stage
During the 7th till the 16th week of gestation in humans (gestational days 12-18 in
rats), the fastest division of intra-segmental airways is observed. Histologically, the
resemblance to a gland gives rise to the name of this stage of lung development.
Continued branching of airways, as well as the vascular network, coincides with
differentiation of epithelial cells into adult structures of cartilage, submucosal glands,
bronchial smooth muscle and epithelial cells [6]. The accelerated growth achieves almost
70% of total airway generation by the 14th week, and towards the end of this stage, the
formation of conducting airways and terminal bronchioles is complete. Bronchial
mesenchyme is involved in further branching of the trachea, while tracheal mesenchyme
inhibits branching of the bronchial tree. This epithelial-mesenchymal interaction
determines the branching pattern of future airways [6]. Columnar and cuboidal epithelial
cells replace the early pseudo-stratified epithelium cells in proximal and distal parts of
the dividing airways respectively. The de novo development of vessels, vasculogenesis,
forms the pre-acinar arteries and veins, growing parallel to the bronchus, bronchiole and
terminal bronchioles, separated by the mesenchyme. In essence, all pre-acinar structures
i.e. pre-acinar airways, pulmonary arteries and veins are developed during the
pseudoglandular stage.
1.2 i c Canalicular stage
Extending from the 16th to the 24th week of gestation in humans (gestational days
18-19.6 in rats), the canalicular stage is marked by two important milestones in the
development of the lung: differentiation of type I and type II pneumocytes, and the
4
formation of alveolar capillary barrier [6,7]. This, together with the onset of surfactant
formation, forms the platform for subsequent postnatal gas exchange. Respiratory
bronchioles, alveolar ducts and primitive alveoli are formed in acini.
1.2 i d Saccular stage
The saccular stage extends from the 24th to the 36th week of human gestation
(gestational days 19.6-postanatal day 8 in rats), during which enlargement of the
peripheral airways and dilatation of acinar tubules ensues, forming saccules [6]. The
appearance of thick ridges (primary septa) on the smooth parenchymal airspaces with
simultaneous thinning of the airspace walls, ensures increased surface area for future gas
exchange. This is accompanied by further differentiation of some type II cells to type I
cells. The fusion of two different networks that were originally formed through
angiogenesis and vasculogenesis, forms one complete vascular network. Within the
primary septa, increased vascular growth leads to the formation of a double capillary
network, sandwiching a central connective tissue layer [8, 9].
1.2 i e Alveolar stage
The alveolar stage of lung development extends from the 35th week of gestation in
humans till at least the 3rd year of life (postnatal day 3-28 in rats), involving a dramatic
increase in gas-exchange surface area by “bulk alveolarization”. This is a period of
colossal increase in the number of alveoli, which starts just before term and continues
into the first 6 months of life in humans. Subsequent development of alveoli occurs at a
much slower rate [10]. Formation of ridges on the surface of primary septa further
5
divides the saccules into alveolar sacs and alveolar ducts. This involves the extension of
the double-capillary network from within the primary septa into the newly formed
secondary septa. These secondary septa demarcate the sites of future alveoli.
Subsequently, following the proliferation and migration of alveolar myofibroblasts and
the deposition of elastic fibres at the tips of the secondary septa, substantial increase in
the gas-exchange surface area is achieved by the thinning of the septa, coinciding with
the development of the single capillary network. During the microvascular maturation,
transformation from the double to a single capillary network within the septa is achieved
by apoptosis of the central tissue layer separating the two capillaries, eventually leading
to their fusion to form a single capillary network. The growth of fused capillaries
increases the vascular surface area.
1.3 Alveolarization
1.3 i Two phase model of alveolarization
The original belief that secondary septation may only occur in a double capillary-
networked septum, and would cease to occur after microvascular network maturation,
was based on the observation that secondary septa were formed by the folding up of one
of the two capillary layers of the primary septa. Two studies in rats supported it: one
demonstrated a linear increase in alveolar number in the first 3 weeks of life [11], the
other exhibited a maximal increase in alveolar density occurring between postnatal day 3
-8 [12]. Accordingly, a two phase model of alveolarization was proposed, supporting
alveolar formation through secondary septation during the first postnatal week in rats,
6
followed by a subsequent increase in the alveolar number through peripheral extension
(without septation), once vascular maturation had concluded.
Recently, evidence of new alveolar formation in young adulthood in a species
(rhesus monkeys) in which microvascular maturation is completed at birth, has brought
the original two phase model of alveolarization into question [13]. Another recent study
has demonstrated evidence of new ongoing septation in rats, following vascular
maturation well into early adulthood [14]. New alveolar formation had already been
reported in mature rodents following starvation and refeeding [15, 16], as well as during
compensatory lung growth post-pneumonectomy and lobectomy [17, 18]. Consequently,
another two phase model of alveolarization has been proposed, supporting an early
“classical” rapid alveolarization (postnatal day 4-21) and septation exceeding the growth
of the lung parenchyma, along with an overlapping “late” second phase alveolarization,
following the maturation of the microvasculature (after postnatal day 14) [14]. Septal
formation in both phases commences by upfolding of the underlying capillary layer,
resulting in a double-layered capillary network within the nascent secondary crest.
Conversely, in late alveolarization, folding up and duplication of the single-capillary-
networked septum (by angiogenesis), results in the formation of a double capillary
network to initiate septal formation [14].
1.4 Factors influencing parenchymal growth
The complex regulation of lung development is dependent on both biochemical
and mechanical factors. Growth factors are important regulators of cellular growth,
proliferation, differentiation and maturation. They are polypeptides, that may reach a
7
target cell in many ways: synthesized elsewhere in the body and reach the target cell via
the bloodstream in an endocrine fashion; synthesized in the immediate vicinity by one
cell type and reach the target cell by passive diffusion in a paracrine fashion; synthesized
and act on the originating cell in an autocrine fashion; direct transfer via gap junctions
between cells, or present as a membrane-bound pro-growth factor, to act on a receptor of
an adjacent cell in a juxtracrine fashion.
Upon binding to its membrane-bound receptor, the growth factor activates a
signal cascade that affects DNA synthesis and cell division. Various types of signal
cascades and pathways exist, depending on the growth factor receptor that is activated.
Generally they utilize one of the following four signal transduction pathways. Firstly, G
protein-coupled receptors activate phospholipase C, resulting in the production of the
second messengers diacylglycerol and inositol triphosphate, that act on protein kinase C
which, upon phosphorylation (activation), activates various proteins that control cellular
processes [301, 309]. The second pathway involves binding of growth factors to their
tyrosine kinase receptors, which in turn activate a protein kinase C [302]. In the third
pathway, intracellular cyclic AMP is increased by the growth factor, stimulating or
inhibiting DNA synthesis by effects on protein kinases [303]. Lastly, activation of
cytokine receptors by certain cytokines, leads to phophorylation of the Stat proteins [304,
305], which form a transcription complex that will eventually affect cellular processes.
Various growth factors play an important role in lung development and their
expressions at different stages of growth have been studied. Any deviation from their
normal expression affects growth (either directly or indirectly). Regulators of
alveologenesis include insulin-like growth factors (IGFs) [19-21], fibroblast growth
8
factors (FGFs) [22-24], platelet derived growth factors (PDGFs) [25, 26], hepatocyte
growth factor (HGF) [27] and vascular endothelial growth factors (VEGFs) [28, 29].
The role of hormones in lung development has been well recognized. Maternal
administration of glucocorticoids reduces the ratio of alveolar type II to type I cells [31,
32] and impairs fetal lung growth [30]. Androgens are understood to influence lung
development because of the variability in lung growth between different sexes [33]. Prior
to epithelial differentiation, female rat lungs have an elevated number of cuboidal
epithelial cells when compared to the males. They also exhibit an higher rate of lung
growth during gestation. Thyroid hormone stimulates the functional maturation of
surfactant-producing type II cells [34].
Physical factors are known to affect lung development: restriction of the
intrathoracic space leads to pulmonary hypoplasia [35]; severing of the phrenic nerve in
animal models to eliminate breathing movements results in stunted pulmonary growth
[36, 37]; diminished cyclic stretch of the fetal lung reduces lung growth [38]; tracheal
occlusion in fetal sheep to increase the intraluminal pressure results in enhanced lung
growth [39]; decreased production of amniotic fluid (oligohydramnios) is associated with
pulmonary hypoplasia [40, 41].
1.5 Pulmonary vascular development
The vascular system of the mature lung consists of a dual arterial supply. The pulmonary
system originating from the pulmonary arteries carry deoxygenated blood to perfuse the
intrapulmonary structures and ultimately regulate gas exchange. The bronchial system, arising
from the descending aorta or its branches, carries oxygenated blood to the peri-hilar region and
9
the walls of the bronchial tree, as well as to the vasa vasora of large pulmonary vessels. The
pulmonary veins drain all the intrapulmonary structures and carry oxygenated blood to the right
atrium, whereas the bronchial veins receive the venous return from the hilar structures, and pass
it to the superior vena cava through the azygos system. Hence the postnatal lung comprises both
a low-pressure pulmonary system arising from the pulmonary artery, and an high-pressure
bronchial system arising from the aorta.
The pulmonary trunk is derived from the truncus arteriosus around the 8th week of
gestation. Once the pulmonary arch arteries are derived from the sixth branchial arch arteries,
they fuse with the pulmonary trunk, the most proximal part of the pulmonary circulation. The
development of pulmonary arteries is closely related to the formation of the bronchial tree, and
use it as a template during the pseudoglandular stage. New pre-acinar vessels are formed by
vasculogenesis; the de-novo formation of vessels from precursor cells. Towards the end of the
16th week of gestation, all pre-acinar bronchi have been formed and are accompanied by their
respective pulmonary branches [42]. The pre-acinar vessels end several generations proximal to
the terminal bronchioles, and lead to the partially muscular acinar vessels that lie around the
respiratory bronchioles. Further growth and division leads to a loss of the muscle coat, resulting
in the formation of the non-muscular precapillary arterioles followed by capillaries, which wrap
around the smaller bronchioles and the primitive distal airways [42, 43]. During the canalicular
stage, early development of the pulmonary parenchyma is accompanied by an increase in the
number of lung capillaries by angiogenesis; the formation of new vessels from pre-existing
vessels [44]. This major developmental transition converts a loose network of capillaries into an
arranged formation around the airspaces [8], subsequently establishing close contact with the
overlying cuboidal epithelium. Thinning of the epithelium initiates the formation of the future
10
air-blood barrier. During the saccular stage, when the airways have formed into thin-walled
saccules, capillaries form a double layer within intersaccular septa; this double layer of
capillaries eventually fuses to form a single adult form during the period of alveolarization.
At birth, the newborn circulation undergoes dramatic changes. During fetal life, owing to
the high resistance offered by the lungs filled with fetal lung liquid and intrauterine hypoxic
pulmonary vasoconstriction [45], approximately 90% of the outflow from the right ventricle
bypasses the pulmonary circulation. This is made possible by the presence of the foramen ovale
and the ductus arteriosus, which direct the circulating blood from the right atrium to the left
atrium, and from the pulmonary artery to the aortic arch, respectively. With the first breaths of
the newborn, pulmonary vascular resistance falls secondary to the reversal of hypoxic pulmonary
vasoconstriction, the physical opening of the pulmonary capillaries, increased production of
nitric oxide and decreased production of vasoconstrictors such as thromboxane and leukotrienes
[46]. Decreased pulmonary vascular resistance results in an 8-10 fold increase in pulmonary
blood flow to match the systemic circulation. The resultant increased flow into the pulmonary
circulation increases the pulmonary venous return, as well as the pressure in the left atrium,
thence functionally closing the foramen ovale [47] and preventing any right-to-left shunting of
blood [48]. Subsequently, the ductus arteriosus constricts within the next 4-10 days. The
transition from the fetal to the postnatal pulmonary circulation includes abolition of the placental
circulation, closure of the fetal pulmonary systemic shunts, and transfer of the function of gas
exchange from the placenta to the lungs.
11
1.6 Factors influencing vascular growth
Vascular development in the lung is under a complex but tight control, involving
specific regulators of vessel formation. The formation of new capillaries by
vasculogenesis occurring at a similar distance from the epithelial buds suggests a role of
epithelial cells in vascular development [44, 195]. Among the different regulators of
vessel formation, the endothelial regulators are best characterized, and include the
ligand/receptor pairs of: VEGF/VEGFR and angiopoietin/tyrosine kinase with
immunoglobin and EGF-like domains (TIE) [195].
Vascular endothelial growth factor (VEGF), a sub-family of growth factors, is
known to be involved both in vasculogenesis and angiogenesis. The presence of VEGF in
the epithelial cells of human fetuses has been demonstrated [49]. Its presence has also
been demonstrated in the developing epithelium and mesenchyme [50-52]. The VEGF
family comprises five members including VEGF-A, -B, -C, -D and placenta growth
factor (PlGF). Rat VEGF is the product of VEGF-A gene containing eight exons, giving
rise to homodimers or heterodimers with VEGF-B or PlGF. Multiple isoforms of VEGF-
A exist, that contain 120, 144, 164 and 188 amino acid residue forms. Rat VEGF-A164
shares 98% and 90% amino acid sequence identity with its mouse and human
homologues respectively [195]. VEGF-A is known to be essential for embryonic
vascular development [53]. Studies involving inactivation of VEGF alleles and knockouts
of VEGFR-1 and VEGFR-2 have resulted in lethal phenotypes involving deficient
organization of endothelial cells [54, 55]. VEGFR-2’s presence has been documented
very early during lung development in the mesenchyme [56]. The three VEGF-A
isoforms in the mouse, namely VEGF120, VEGF164 and VEGF188, have been demonstrated
12
to be present in alveolar type II cells, peaking in expression during the canalicular stage
when most vessel growth occurs [52]. Mice expressing only the VEGF120 isoform and
lacking VEGF164 and VEGF188 isoforms survived, but generated fewer air-blood barriers
(“oval or round spaces, visualized by electron microscopy, either empty or containing
blood cells, abutting the airspace lumen and bounded on one side by an endothelial cell”)
and had underdeveloped alveoli when compared to wild-type litter mates [57]. Inhibition
of VEGF receptors in adult rats led to pruning of the vascular tree accompanied by
enlarged air spaces, signifying the importance of VEGF in the maintenance of the normal
pulmonary vasculature and structure [29].
Angiopoietins vital role in normal vascular development is exhibited by the fact
that disruption of angiopoietin-1 (Ang-1) genes resulted in embryonic lethality [58],
while mice that over-expressed Ang-1 formed vessels that were resistant to leaking [59].
Angiopoietin receptor TIE-2’s role is vital in vascular network formation [60].
1.7 Bronchopulmonary dysplasia
1.7 i Old (classic) BPD
The advent of positive-pressure ventilation for newborn infants in the 1960’s
revolutionized the practice of neonatal intensive care [61]. Mechanical (positive-pressure)
ventilation introduced a momentous era marked by the increased survival of premature
infants suffering from neonatal respiratory distress. The combination of mechanical
ventilation and supplemental O2 became the standard of care for respiratory distress
syndrome (RDS). In 1967, Northway and colleagues reported a chronic lung disease with
characteristic clinical, radiographic and pathological manifestations in some premature
13
infants receiving the aforementioned treatment [62]. These infants were mostly not very
preterm (>31 weeks of gestation) and weighed more than 1499 g. This syndrome was
called bronchopulmonary dysplasia (BPD) [63, 64], which effectively described its
histological presentation: alveolar overdistension interspersed with regions of atelectasis
and pulmonary fibrosis, squamous metaplasia of the airways with excessive smooth
muscle hyperplasia, necrotizing bronchiolitis, vascular smooth muscle hyperplasia and
infiltration of inflammatory cells [65, 66]. Uncertainty persisted over whether positive-
pressure ventilation or oxygen (O2) toxicity was the offending agent responsible for BPD.
It was widely understood that the underdeveloped injured lungs of premature infants had
inadequate repair mechanisms, culminating in the development of the chronic lung
disease.
1.7 ii “New” CNLI.
Subsequent improvements in neonatal intensive care in the past four decades have
resulted in a considerably altered presentation of BPD. The improvements include:
development of gentler ventilator techniques to reduce volutrauma/barotrauma to the
premature lung, use of antenatal steroids to mature the lung surfactant system, use of
exogenous surfactant at birth, and use of improved parenteral nutrition. These
improvements have led to the survival of smaller and more immature infants. The “new”
BPD lacks the airway injury and the pulmonary fibrosis as originally described by
Northway. The hallmark of “new” BPD is the arrest of alveologenesis and capillary
development, with variable interstitial thickening [66]. The “new” BPD is seen primarily
in extremely premature infants that are born at 23-28 weeks of gestation and weigh less
14
than 1000 gm. These infants may suffer minor or no respiratory distress syndrome
(RDS), requiring little or no ventilatory support at birth and have been maintained at low
inspired O2 concentrations. Their birth coincides with the canalicular phase of lung
development. As a consequence, these preterm infants are born with extremely premature
lungs, exhibiting impaired alveolar and capillary growth, coupled with abnormal
reparative processes [66]. Histologically they have decreased, large/simplified alveoli,
probably saccules which have failed to undergo secondary septation, with accompanying
decreased dysmorphic capillaries and variable airway smooth muscle hyperplasia. Those
with moderate to severe “new” BPD may have accompanying histological changes of
pulmonary hypertension (PHT). “New BPD” is subsequently called chronic neonatal lung
injury (CNLI) to avoid confusion between “old” and “new” BPD.
The incidence of CNLI is inversely proportional to the newborn’s gestational age
(Fig. 1.1) and has been reported to be 85% in extremely low-birth-weight infants (500 –
699 g) [67], while for infants with birthweights >1500 g, it has been reported to be
around 5%. Even though an apparent reduction in severe CNLI within different
gestational age categories has been reported between the years 1994-2002 [68], the actual
prevalence of CNLI could be higher, because of increased survival of extremely
premature infants of 23-28 weeks’ gestation exhibiting a high incidence of CNLI.
CNLI continues to be the most prevalent chronic lung disease of infancy. Being
one of the most serious long-term sequelae of preterm birth, it affects
approximately14000 preterm infants each year in the USA [69]. Most notable are the
respiratory and neurosensory outcomes. Long term adverse consequences of CNLI
include pulmonary impairments [70, 71] and higher rates of rehospitalizations [72]. The
15
health care costs of CNLI in children were second only to asthma in 1993 [73]. Although
few infants with CNLI will die from respiratory failure in the current era, those with
persistent PHT are associated with an high mortality [74]. Recent evidence points to
Data from Canadian Neonatal Network(2006)
Gestational age at birth (weeks)22 24 26 28 30 32 34 36 38 40
%of
NIC
Uad
mis
sion
s
0
20
40
60
80
100
SurvivalBPD (O2 at 36 wk CGA)
Fig. 1.1. Survival of extremely-low-birth-weight infants according to gestation age and
their risk of developing CNLI, defined as an O2 requirement at 36 weeks’ corrected
gestational age. NICU = neonatal intensive care unit and CGA = corrected gestational
age.
failure of alveolar formation into late childhood [75] and emphysema in the late teens in a
large percentage of those who survived severe CNLI [70] . When compared to preterm
16
survivors without CNLI, the preterm survivors with CNLI have a higher prevalence of
neurosensory problems [69]. More frequent reporting of cerebral palsy [76], cognitive
dysfunction [77-79], attention deficit-hyperactivity disorder [80], language impairment
[81] and behavioural problems [79, 82] have been observed.
1.7 iii Aetiology of CNLI
The aetiology of BPD is generally believed to be multifactorial. After the original
description of BPD by Northway, a dramatic change in maternal care (use of antenatal
steroids for < 34 weeks of gestation), introduction of surfactant therapy, use of a various
“gentler” ventilator strategies, continuous monitoring of O2 saturation and better
nutritional support have increased the survival of very premature infants, however, the
incidence of CNLI at 36 weeks’ postconceptional age has not reduced significantly [83].
CNLI (detailed earlier) has become the most frequent chronic lung disease in infancy [66,
84]. Genetic predisposition of male infants to its development has been demonstrated
[85]. Chorioamnionitis, the single most important cause of preterm birth and occurring
frequently in preterm deliveries before 30 weeks’ gestation has also been implicated [86],
but other evidence [87] refutes this link. Volutrauma, coupled with prolonged mechanical
ventilation, has also been blamed [88]. Several mechanisms have been shown to inhibit
angiogenesis and hence potentially alveologenesis. Elevated levels of O2 in extrauterine
life is implicated in the inhibition of the VEGF/VEGFR signaling pathway in the
premature newborn, with the resultant inhibition of vascular growth and alveolarization
[89, 90]. In short, complex interactions between various contributing factors have been
17
ascribed to cause BPD, but the central role of oxidant stress has generally been accepted
[91].
Northway’s original hypothesis that O2 toxicity was contributing towards BPD
has been supported by subsequent research [92]. Reactive oxygen species cause damage
by apoptosis and oxidation of lipids, proteins and DNA [93], to which an immature lung
is already susceptible due to an immaturity of antioxidant defences [94, 95]. The STOP-
ROP clinical trial targeted O2-saturations of 96 and 99% in premature infants with a
resultant increase in the incidence of CNLI in those exposed to the higher O2 saturations
[96]. Likewise, another study, targeting for and comparing O2-saturation ranges of 91-
94% vs. 95-98%, resulted in increased CNLI in those exposed to the higher O2 saturations
[97].
1.8 Management strategies for prevention of CNLI
1.8 i Current therapies
Management of infants suffering from CNLI is geared towards achieving an
adequate gas exchange, while simultaneously preventing the progression of the disease
by reducing the factors that are believed to cause lung damage. Interestingly, and
unfortunately, the main therapies that are utilized to maintain an adequate gas exchange
are also thought to be implicated in the pathogenesis of the disease.
Mechanical ventilation is considered by many to be a double-edged sword,
contributing to the pathogenesis of CNLI in preterm infants. In current practice, minimal
ventilator settings that would allow for a patent airway while permitting ample gas
exchange are used. Additionally, attempts are made to limit the duration of mechanical
18
ventilation. This approach is undertaken to minimize the barotrauma/volutrauma that may
be associated with mechanical ventilation and might contribute to, or worsen, CNLI. In
order to maintain a patent airway, a higher positive end-expiratory pressure is maintained
during mechanical ventilation for infants that demonstrate severe airway obstruction,
especially those with bronchomalacia [98]. Despite the widely held belief in a role for
volutrauma/barotrauma in the aetiology of CNLI, and the convincing evidence that high
volumes and pressures are damaging under experimental conditions, there is a dearth of
evidence to support lung injury at the volumes and pressures currently used in clinical
practice.
Prolonged exposure to increased concentrations of inspired O2 is another
causative agent in the pathogenesis of CNLI. Infants with CNLI may respond with
increased airway resistance to episodes of hypoxemia. Management of CNLI includes
maintaining the FIO2 at a level that is sufficient to ensure adequate tissue oxygenation,
yet as low as possible to limit O2 toxicity. Since an SaO2 above 95% is associated with an
higher incidence of retinopathy of prematurity [97, 99], care is taken to maintain the SaO2
between 90-95% (PaO2 between 50-70 mm Hg) to minimize the detrimental effects of
hypo- and hyperoxemia [98].
Infants with CNLI have a tendency to accumulate excessive fluids in their lungs,
even when normal amounts of fluid intake are maintained [98]. Excess fluid predisposes
them to hypoxia and this, compounded with a poor lung function, results in prolonged
episodes of hypercapnia extending their time on the ventilator. Cautious fluid
management combined with a sparing use of diuretics is frequently used in the
management of CNLI.
19
Bronchodilators are sometimes implemented in cases of severe CNLI. Inhaled
bronchodilators are preferred, but their effect is short-lived while many of these drugs
have cardiovascular side-effects limiting their repeated use [98]. Methylxanthines have
been shown to reduce airway resistance, and caffeine has the additional benefit of
stimulating respiration while acting as a mild diuretic [98].
Inflammation may have a significant role in the pathogenesis of CNLI [100-102].
Use of steroids for their anti-inflammatory effects had been used to achieve rapid
improvement in lung function by reducing pulmonary and bronchial edema, possibly
increasing surfactant and antioxidant enzyme production and decreasing the responses of
mediators of inflammation [103, 104]. This allowed for earlier weaning off the ventilator.
However, prolonged use of steroids had its own complications such as an increased
incidence of cerebral palsy [105, 106]. Newer studies propose use of inhaled steroids to
minimize the use of systemic steroids and their potential side effects [107], but caution is
advised since enough evidence does not exist to support their use [108].
Adequate nutrition is a critical component of the management of CNLI.
Malnutrition not only affects somatic growth [109], it also delays the formation of new
alveoli. An aggressive nutritional management implementing the use of high caloric
formulas can contribute to increased muscle strength that, in turn, might help in
successful weaning off the ventilator. The neonate’s limited ability to tolerate protein and
fat, compounded by decreased clearance of carbohydrate-generated CO2 (because of their
CNLI), makes this target hard to achieve.
Pulmonary vasodilators may be utilized in an attempt to ensure proper
oxygenation in infants with CNLI, since pulmonary vascular resistance is extremely
20
sensitive to changes in alveolar PO2. Calcium channel blockers have been used to address
the increased pulmonary vascular resistance, but their side effects on the myocardium
limit their use [98]. Although inhaled nitric oxide has been shown to improve
oxygenation in infants with CNLI [110], lack of evidence supporting its role in improving
long-term outcomes in CNLI, makes it a controversial treatment, and it is rarely used
outside a research protocol. The assumption that nitric oxide only acts through an effect
on the pulmonary vascular bed may be incorrect, since, depending on its concentration
relative to superoxide, nitric oxide can act as either a prooxidant or as an antioxidant
[111]. Any protection observed [112] might well be through its effect as a chain-breaking
antioxidant (Fig. 1.2 B). The antioxidant properties of nitric oxide may offer a potentially
attractive intervention in the prevention of CNLI once sufficient reported evidence
validates its use clinically.
1.9 Permissive hypercapnia
Mechanical ventilation is indicated when a patient’s spontaneous ventilation is
inadequate to sustain adequate gas exchange, and less invasive interventions have been
ineffective. Traditionally, mechanical ventilation has been employed to maintain patent
airways allowing for an efficient gas exchange, and to regulate the levels of inspired and
expired gases to maintain the concentrations of the arterial blood gases within a
physiologically acceptable range. However, over-distension of the lungs could lead to
stretch-induced lung injury, while attempts at normalizing the arterial CO2 could lead to
unintentional overcorrection, hence causing a relative depletion of CO2. The resultant
hypocapnic alkalosis is known to contribute to brain injury and neurosensory deficits
21
Fig. 1.2.
A
lipidhydroperoxide
+lipid radical
lipid peroxylradical
lipidradical
+ hydroxylradical
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
L
LH
LH + OH L + O2 LOO LOOH
The Membrane Chain Reactionof Lipid Peroxidation
Membrane lipid (LH) is oxidized by the hydroxyl radical (OH•) to form a lipid
radical (L•) and subsequently the intermediate lipid peroxyl radical (LOO•), which, upon
accepting a proton from adjacent lipids, forms a lipid hydroperoxide (LOOH) and a lipid
radical, reinitiating the cycle and setting up a chain reaction.
B
+ NO
alkylperoxynitrate
lipid peroxylradical
lipidradical
+ hydroxylradical
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH
LH + OH L + O2 LOO LOONO
NO as a Lipid Peroxidation Chain Breaker
Nitric oxide (NO•) reacts with LOO• to form alkyl peroxynitrate (LOONO), hence
acting as a chain-breaking antioxidant, while terminating a potential chain reaction.
22
[113]. Ventilation strategies, when modified to minimize the tidal volumes to prevent
stretch-mediated lung injury, result in the retention of CO2, which inadvertently prevents
hypocapnic alkalosis. As a result, there has been a shift in clinical practice towards
allowing for a build up of arterial CO2 by employing low-tidal volume ventilation
strategies. The resulting hypercapnia is termed “permissive hypercapnia”, which has been
successfully utilized in adult intensive care units, improving survival in cases of acute
adult respiratory distress syndrome [114, 115] and acute adult lung injury [116].
There has been a drive in neonatology to minimize the over-distension and
putative volutrauma of the neonatal lung [117]. Although proof of efficacy of this
technique is lacking, results do hold promise [113]. The aim of this approach is to
minimize the induction of pro-inflammatory cytokines resulting from stretch-mediated
lung injury. Relaxation in the acceptable upper limits of arterial CO2, secondary to
gentler respiratory management, has been widely adopted in clinical practice.
1.10 Therapeutic hypercapnia
There is growing evidence to support the notion that the advantages of protective
ventilation strategies, and of permissive hypercapnia, are manifested due to the ensuing
hypercapnic acidosis. Hypercapnia has been shown to cause increases in arterial O2 levels
[118], most likely secondary to reduced intrapulmonary shunting and ventilation-
perfusion mismatching as well as improved cardiac output [119]. Although hypercapnic
acidosis decreases the contractility of cardiac muscle, the resultant increased preload,
heart rate and coronary blood flow, complimented by a decreased afterload (secondary to
systemic vasodilation), result in an overall increase in cardiac output [119, 120].
23
Hypercapnic acidosis can therefore improve tissue oxygenation [121]. As a result,
hypercapnic acidosis and its resulting benefits have led to the recognition of CO2 as a
therapeutic agent, rather than being considered a by-product of ventilation strategies.
Consequently, the deliberate induction of hypercapnia by increasing the inspired CO2,
with the goal of limiting lung injury, is termed “therapeutic hypercapnia”. It is worth
mentioning that hypercapnic acidosis increases cerebral blood flow, as well as
intracranial pressure [122] and, during the first three days of life, hypercapnia may be
associated with severe intraventricular haemorrhage in very low-birth-weight infants
[123].
1.11 Clinical trials of hypercapnia
A pilot trial of permissive hypercapnia randomly assigning 601-1250 g preterm
infants to a partial pressure of ~ 45 mm Hg of CO2, resulted in rapid weaning off the
ventilator [124]. A large multicentre trial, in adults with acute RDS, showed a
combination of small tidal volumes and permissive hypercapnia significantly reduced
mortality rates and ventilatory needs [125]. These studies, in addition to observations in
two retrospective studies, suggested that relative hypocapnia during early neonatal life is
associated with an increased risk for lung injury [126, 127]. This led to a trial [113]
involving minimal ventilation and the use of tapered low-dose dexamethasone to prevent
CNLI, while allowing for a target PaCO2 ~ 52 mm Hg in extremely low-birth-weight
infants (501-1000g). Although this trial reported no reduction in the incidence of death or
CNLI in the sample size studied, the lack of benefits of minimal ventilation (and its
associated hypercapnia) might be secondary to an early termination of the trial secondary
24
to an unanticipated increased rate of (steroid-therapy-related) gastrointestinal
perforations.
Extremely premature infants were randomized at birth to higher PaCO2 targets
(55-65 mm Hg) during the first week of life [128] to aim for lower, less traumatic,
ventilator settings, based on an earlier controlled trial’s findings of a reduced duration of
assisted ventilation and reintubation following the use of permissive hypercapnia (PaCO2
45-55 mm Hg) [124]. The trial was stopped early after enrolling 31 % of the projected
sample size secondary to the development of worsening neurodevelopmental outcomes
associated with non-significant clinical results. Why were these trials unsuccessful?
Several possible reasons exist. First, the targeted PaCO2 was not achieved in most
patients. This was most likely secondary to an increased respiratory drive, rendering
difficulties in achieving the desired PaCO2 in non-paralyzed preterm infants with
moderate lung disease. Additionally, administration of bicarbonate in combined acidosis
to avoid side-effects of low pH might have also prevented the benefits of hypercapnia
[124]. Second, extreme immaturity in this patient population (median gestational age <
25 weeks) might have rendered them resistant to therapy, since even with the most gentle
strategies such infants may still develop CNLI. In short, most likely, this trial did not
show benefit either because the level of PaCO2 was inadequate, or the newborns were
resistant to therapy, or a combination of both.
1.12 Oxidative/nitrative stress in CNLI
It has been known for some time that an increased FIO2 could contribute to the
development of BPD/CNLI by stimulating free radical generation. Free radicals, the
25
highly reactive molecules capable of independent existence, were implicated as mediators
of O2 toxicity as early as 1954 [129]. They contain one or more unpaired electrons and
are generally highly reactive. Although O2 is also a free radical because of its two
unpaired electrons, it is not a strong oxidant and is only toxic through the formation of
various reactive oxygen species (ROS). ROS comprise a large variety of free oxygen
radicals such as superoxide anion and hydroxyl radical, in addition to the derivatives of
O2 that do not contain unpaired electrons, e.g. hydrogen peroxide, hypochlorous acid and
ozone. ROS, including superoxide anion, singlet O2, hydroxyl radical and peroxides, are
produced constantly by the cellular metabolism of molecular O2 in the mitochondria. On-
going production also involves microsomes and enzymes, such as xanthine oxidase,
cyclooxygenase, lipoxygenase, monoamine oxidase and endothelial nitric oxide synthase
[299]. The Haber-Weiss reaction generates hydroxyl radicals from hydrogen peroxide
(H2O2) and superoxide ion, using iron as a catalyst. The first step of the catalytic reaction
involves reduction of ferric iron to ferrous iron and during the second step (the Fenton
reaction), ferrous iron is oxidized back to the ferric state. The net reaction consumes a
superoxide radical and H2O2 to yield a hydroxide ion, O2 and hydroxyl radical (Fig. 1.3).
Approximately 97% percent of molecular O2 undergoes a 4 electron-enzymatic reduction
in the mitochondria to form water. Only 3% undergoes a non-enzymatic reduction and,
with the addition of 1, 2 or 3 electrons, yields superoxide, hydrogen peroxide or the
hydroxyl radical respectively. The presence of antioxidant enzymes helps achieve the
elimination of these toxic radicals: superoxide dismutases (SODs): both manganese and
copper-zinc, act on the superoxide anion, while catalase and glutathione peroxidase act
on H2O2 in the peroxisomes and cytoplasm respectively [130]. It is noteworthy that free
26
radicals are also used by the body as second messengers in many systems (discussed
later). Additionally, glutathione peroxidase may also act on and detoxify the lipid
peroxides formed by the oxidizing of membrane lipids by the hydroxyl radical.
Peroxiredoxins, a family of peroxidases that reduce intracellular peroxides, with the
thioredoxin system as the electron donor, are important antioxidants.
Large amounts of ROS are produced during the activation of phagocytes, namely
neutrophils and macrophages, involved in the host’s defense mechanism. This happens
following the inhalation of micro-organisms or particles, or activation of the membrane-
bound NADPH oxidase complex by other mediators, which leads to the generation of
superoxide anion [131]. Individually, superoxide and hydrogen peroxide are not very
reactive with cellular components [132], but in the presence of divalent metal cations,
may produce the highly reactive hydroxyl radical. Phagocytic cells, in addition to the
release of oxygen radicals and nitric oxide (NO), also release mediators such as
lysozymes, peroxidases, hypochlorous acid and proteases to mediate their antimicrobial
functions.
NO, a free radical synthesized from the semi-essential amino acid L-arginine by
specific nitric oxide synthases [133], can interact with ROS to form reactive nitrogen
species (RNS). Both NO and superoxide, being free radicals, react rapidly with various
molecules, and also with their reactive downstream metabolites, to form products that are
more reactive than their precursors. The rapid iso-stoichiometric reaction between NO
and superoxide results in the formation of a very potent oxidant, peroxynitrite [134].
Peroxynitrite is capable of causing peroxidation of membrane lipids [135] and cellular
27
Fe3+ + •O2
_→ Fe2+ + O2
The second step is the Fenton reaction:
Fe2+ + H2O2→ Fe3+ + OH_
+ •OH
Net reaction:
•O2
_+ H2O2→ •OH + OH
_+ O2
The Haber-Weiss Reaction
Fig. 1.3. The Haber-Weiss Reaction.
components [136]. It can also cause nitration of proteins and membrane lipids, while also
leading to cellular toxicity after being protonated and subsequently undergoing
spontaneous decomposition to form the hydroxyl radical [137]. The reaction of
peroxynitrite formation has the fastest rate constant yet described, approaching the
diffusion control limit (4-7 X 109 ms-1), while peroxynitrite has a half life of 1 s at 37°C
and pH 7.4 [134, 306]. This renders it sufficient stability to diffuse several cell diameters
before decomposing. The rate of the reaction of superoxide with NO is almost ten times
faster than its reaction with SOD, but under normal conditions, owing to the 100- to
1000-fold higher concentration of SOD [138], the reaction of SOD with superoxide takes
precedence. Nevertheless, under pathological conditions, adequate endogenous
28
production of NO can compete with SOD, outperforming it and leading to an excess
production of peroxynitrite. Subsequent production of the hydroxyl radical can initiate
the lipid peroxidation chain reaction, where membrane lipids are oxidized to form a lipid
radical followed by a lipid peroxyl radical (LOO) which, after utilizing a proton from the
adjacent lipid, forms lipid hydroperoxide (LOOH) (Fig. 1.2 A). Glutathione peroxidase,
as mentioned previously, may detoxify these lipid hydroperoxide radicals. Depending on
its concentration relative to superoxide anion, NO can act as a pro-oxidant or antioxidant
[111].
An imbalance between the production of ROS and a biological system’s ability to
neutralize them, leading to a net increase of the ROS, is termed an ‘oxidative stress”. This
can arise by an increase in oxidants with or without a decrease in antioxidants or
antioxidant enzymes. In an analogous fashion, the condition in which an host system,
following the production of highly reactive nitrogen-containing molecules, is
overwhelmed by increased production of RNS and is unable to neutralize and/or
eliminate them, is labelled a “nitrosative stress”. ROS and RNS can damage DNA,
proteins, lipids and carbohydrates, leading to compromised cellular functions and
augmented inflammatory reactions.
The role of oxidative stress in the pathogenesis of human BPD/CNLI is supported
by evidence from animal studies, and by experimental and epidemiological evidence in
humans. In the late 1970s, higher than normal activities of SOD, catalase and glutathione
peroxidase were reported in the lungs of term newborn rats when compared to 2-day old
premature rat fetuses [139]. Within the next decade sufficient evidence existed detailing
the role of antioxidant enzymes and their maturation during the last 10-15% of
29
gestational age, with a 150-200% increase in the antioxidant enzyme activities [95, 140,
141]. This maturation of antioxidant enzymes occurred in parallel with the prenatal
maturation of the surfactant system of the lung [94], suggesting their joint role in
preparing fetuses to be born into an O2-rich environment. Preterm rats [142], as well as
rabbits [143], when exposed to hyperoxia, were unable to mount an efficient increase in
antioxidant enzymes in their lungs. Reduced (< 50%) activities of Cu/Zn SOD in cord
erythrocytes of preterm human infants (gestational ages 29-34 weeks) when compared to
term infants, further highlighted the compromised defenses due to prematuriy [144, 307].
Free radicals and hypoxanthine-xanthine oxidase were shown to damage lungs in animal
models [145, 146], with evidence of higher plasma hypoxanthine levels at birth in
premature infants who developed chronic lung disease [147]. Fetal mouse lung explants,
when grown under hyperoxic conditions, exhibited inhibited growth, which was
irreversible in exposures that maintained a >50% O2 concentration [148]. Furthermore,
newborn rats when exposed to 95% O2 for a week, displayed severe irreversible
inhibition of DNA synthesis that did not recover during a second week of 60% O2
exposure. In the same study, a continuous two-week exposure to 60% O2 showed a non-
homogeneous reduction in lung DNA synthesis [149]. Premature infants destined to
develop chronic lung disease demonstrated higher concentrations of markers of oxidative
stress in their exhaled breaths [150-152]. In a 140-day-gestation preterm baboon model of
chronic lung disease, induced by exposure to 100% O2, lung pathology resembling the
original “old” BPD was prevented when a catalytic (metalloporphyrin) antioxidant was
used [153]. Given that the baboon and human had essentially identical histopathologies,
30
these findings are consistent with an important role for oxidant stress in the development
of “old” BPD.
There is direct evidence for increased ROS generation in the lungs of preterm
infants [154]. Large multicentre trials demonstrate that targeting preterm infants to higher
concentrations of inspired O2 is associated with significantly higher respiratory morbidity
[155] (pneumonia and chronic lung disease) and an extended hospital stay [97],
compared to those managed with lower O2 saturations. Although ventilation strategies
devised to minimize volutrauma, and hence CNLI, have not demonstrated a decrease in
its incidence [113], a role for ventilator-induced lung injury has been suggested by its
effects with inappropriate ventilation [156] and the reported associated increase in
cytokines [157]. The close link between inflammation and oxidative stress is due to the
burst of oxygen radicals generated by activated phagocytes, as part of the host’s defense
mechanism [158, 307]. The involvement of inflammatory cells in the development of
CNLI is potentially of great importance [101]. Preterm infants who develop CNLI [159],
as well those who develop RDS and are treated using mechanical ventilation with O2,
demonstrate an enhanced inflammatory cell response [160]. As mentioned earlier,
neutrophils and macrophages release superoxide anion and H2O2, with resultant tissue
damage affecting lung morphogenesis. Newborn rats exposed to 60% O2 develop an
injury similar to human CNLI [149]. Blockage of the associated increase in lung
neutrophil content by a selective chemokine receptor antagonist reduced the production
of ROS, while improving alveologenesis [161]. Activated neutrophils and alveolar
epithelial type II cells are important inducers of the Fenton reaction [162], which may
contribute to pulmonary injury and subsequent CNLI. Whether it is deficient antioxidant
31
enzymes in extremely premature infants which predispose them to oxidant stress, or
volutrauma leading to an inflammatory response, it is likely that oxidant stress plays a
major role in the pathogenesis of CNLI.
RNS can have a detrimental effect on the premature lung [306]. Importantly, RNS
target tyrosine residues in proteins [163]. Various studies have shown that protein
function can change following nitration of their tyrosine residues [164]. Moreover, RNS
have been shown to alter lipid oxidation pathways [165], inhibit mitochondrial respiration
[166] and cause DNA damage [167]. Peroxynitrite has been shown to inactivate
surfactant [168], activate matrix metalloproteins [169], inhibit protein phoshorylation by
tyrosine kinases [170] (hence interfering with signal transduction) and augment the
production of interleukin-8 [171] (a potent neutrophil chemoattractant). Peroxynitrite
reacts with CO2 to produce the nitrosoperoxocarbonate adduct (ONOOCO2) which may
act as a more potent nitrating species than peroxynitrite [237]. The enhanced airway
inflammation may thus contribute to pulmonary damage. Insufficient direct studies
delineating the role of RNS on the pathogenesis of CNLI do not preclude one from
speculating the aforementioned mechanisms are instrumental in its development.
Moreover, the four-fold increase in the content of plasma 3-nitrotyrosine during the first
month of life in infants that develop CNLI, highlights the potential contribution of
peroxynitrite-mediated reactions in the development of this disease [172]. The degree of
nitration is also a predictor of the severity of CNLI [173].
The immature infant’s deficiency in endogenous pulmonary antioxidants, and the
putative role of ROS in the pathogenesis of CNLI, have generated considerable interest in
utilizing antioxidant therapies to prevent this disease. Nevertheless, the physiological role
32
of the endogenous ROS as intracellular second messengers is of utmost importance. In
contrast to the pathological conditions where abnormally high concentrations of ROS
lead to permanent and irreversible damage to signal transduction, physiological redox
regulation and signaling relies on the temporary shift of the intracellular redox state
towards more oxidizing conditions [174]. Many cells have been demonstrated to undergo
small oxidative bursts when stimulated by cytokines, growth factors and hormones [175].
ROS have been shown to play a role in cell growth [176]. Platelet-derived growth factors
(PDGFs) [177], basic fibroblast growth factor (bFGF or FGF-2) [178] and epidermal
growth factors (EGF) [179] make use of ROS as second messengers for cell proliferation.
As a consequence, antioxidant therapies have the potential of direly affecting the signals
for normal lung growth. Similarly, while therapies devised to restrict the formation of
RNS usually target nitric oxide synthases (NOS), non-specific NOS inhibition can lead to
undesirable outcomes [180] (such as hypertension). Additionally, limiting RNS formation
can lead to a compromised host defense against invading microorganisms. As a result,
therapies aimed to counter the deleterious effects of RNS and ROS may need to be
targeted at their downstream mediators.
1.13 Animal models of CNLI
CNLI has a multi-factorial aetiology. Our lack of insight into the mechanisms
responsible for CNLI has precluded the development of a single effective treatment to
counter this pathology. Perhaps because the disease is multifactorial, it may not be
possible to prevent it with only one therapy. Various treatment modalities are undertaken,
as mentioned earlier, which do have their respective limitations. In an attempt to prevent
33
BPD, many animal models have been used for research. Since BPD develops in a
developmentally immature host, provisions are taken to keep this in perspective when
developing an animal model of the disease. Other important objectives are similar clinical
and histological end-points at the conclusion of the intervention. All animal models of the
disease make an attempt at mimicking some or most of the characteristics of the disease,
and an ideal model does not exist at this point in time. Each model has its own
advantages and limitations, which will be discussed below.
The long-term lamb (147 days-term gestation) model has contributed significantly
towards the understanding of the pathogenesis of interstitial connective tissue elements
[181], lung fluid balance and pulmonary circulation [182], and the use of nitric oxide
[183, 184]. Although this model has contributed significantly towards the understanding
of various aspects of the disease, the ovine lung has the distinction of being fully
alveolarized at term [300]. Most studies on the lamb model of CNLI now incorporate
animals delivered at 84% gestation (124 days), to match the period of development
between the saccular and alveolar stages [300]. In another model, one week prior to
caesarean section delivery at 125 days of gestation, intra-amniotic administration of E.
Coli endotoxin is incorporated to mimic prenatal inflammation as a causative agent in the
development of BPD [185].
Similar to humans, alveolarization continues postnatally in the baboon. The 140-
days’ gestation baboon model of BPD (76% gestation), which aimed for the saccular to
early alveolar phase of lung development, reproduced the “old” BPD [186]. After
delivery at 140 days’ gestation, 100% O2 was administered for 10 days to create severe
airway and parenchymal injury, though severe arterial changes were absent [187, 300].
34
The 125-days’ gestation baboon model (67% of term) aims for the development of the
lung to be between the canalicular and saccular stages. This model was created to mimic
the “new” BPD of extremely immature infants; it included the use of exogenous
surfactant, and ventilating the survivors at low tidal volumes for two weeks [300]. The
histological findings mimic those observed in the human form of “new” BPD. Although
the baboon model is considered as close to ideal as one can get, the cost, availability and
ethical issues relating to the use of an higher animal species limit its use.
In rat and mouse lungs, the majority of post-natal alveolarization occurs over a
two-three week period following birth [300]. This short period of post-natal development
is well suited to creating an injury that would mimic BPD/CNLI. Exposure to 80-100%
O2 has been utilized to produce “old” as well as “new” [188] BPD in mice. Mechanical
ventilators have been used to generate a ventilator-induced lung injury in rats and mice
after birth. In our laboratory, exposure of neonatal rat pups to 60% O2 for 14 days has
generated a lung injury similar to that observed in the “new” BPD [149].
1.14 60% O2-model of CNLI
In this model, following exposure to 60% O2 for 14 days, an overall inhibition of
lung growth is observed in neonatal rat pups. Impaired alveolar septation and
heterogeneous areas of interstitial thickening are observed [189] along with PHT which,
together with the absence of pulmonary fibrosis, place this model very close to the
pathology observed in human BPD. PHT in this model is manifested by a significant
right ventricular hypertrophy and pulmonary vascular remodeling [190].
35
1.15 Pulmonary hypertension/vascular injury/remodeling
The human lung is the only organ in the body that receives the entire cardiac
output at all times. The pulmonary circulation is a high-flow, low-resistance, low-
pressure system responsible for carrying blood to the pulmonary microcirculation, where
the blood takes up O2 and unloads excess CO2. Owing to such a tremendous demand and
work load, the pulmonary circulation is vulnerable to injury, as a consequence of
developmental or acquired disorders affecting the lungs, the heart or the systemic
vasculature.
Premature birth, in addition to its adverse effects on the airway and distal
airspaces, also has a detrimental effect on the growth, structure and function of the
developing pulmonary circulation [74]. The endothelial cell’s susceptibility to oxidant
injury [191, 192], combined with smooth muscle cell proliferation and incorporation of
fibroblasts and myofibroblasts into mature smooth muscle [193, 194], lead to structural
changes that eventually lead to the narrowing of vessels, and their decreased compliance.
This is compounded by simultaneous abnormal vasoreactivity and decreased
angiogenesis, limiting the vascular surface area and leading to an increase in pulmonary
vascular resistance [195]. The resultant increase in the mean pulmonary artery pressure is
defined as pulmonary hypertension.
Regardless of the factors inducing PHT, an early persistent pulmonary
vasoconstriction eventually leads to a later stage of vascular remodeling, which renders
the vessels unresponsive to vasodilator stimuli, and increases resistance to blood flow
[196]. Vascular remodeling includes structural modifications of the media of existing
vessels, in addition to the abnormal growth of new and/or inhibition of growth of the
36
existing vessels. Typically, when pulmonary vascular remodeling occurs in late
complicated BPD, thickening of the medial smooth muscle layer secondary to
hyperplasia, hypertrophy, or both, muscularization of the originally non-muscular
arteries, reduction in arterial to alveolar number, increased perivascular deposition of
extracellular matrix (ECM) proteins and right ventricular hypertrophy are seen [197].
Normal expansion of pulmonary vascular bed, which is required for the normal drop in
pulmonary pressures, is impaired in preterm infants that required intensive support soon
after birth [198, 308].
In the “old” (pre-surfactant) era, severe changes of pulmonary vascular
remodeling were witnessed in infants that succumbed to severe BPD [199], with the
severity of the disease directly related to the amount of supplemental O2 exposure [200].
Evidence suggests that in experimental PHT, structural remodeling occurs more rapidly
and severely in newborn animals than in adults, with an incomplete recovery from
vascular remodeling [201]. As a consequence, long-term inhibitory effects on lung
growth [10, 202] could lead to an enhanced predisposition to severe PHT in later life
[203]. Small pulmonary vessels from hyperoxia-exposed newborn rats with CNLI have
demonstrated a decreased tendency to relax and an exaggerated response to constrictor
stimuli [204].
In the surfactant era, a subset of infants requiring prolonged ventilation (and
possibly the need for systemic corticosteroids) still develop PHT [98]. Others, who have
recently recovered from acute RDS, develop late onset CNLI with persistent
echocardiographic evidence of a diminished fall in pulmonary artery pressure [205].
Higher estimated pulmonary artery pressures were reported in preterm infants who
37
developed RDS, when compared to those who did not develop RDS over the first 5 days
of life [206]. Infants who developed CNLI showed a sustained elevation of pulmonary
artery pressure during the first year of life [207]. In a recent study of premature infants
with CNLI and PHT lasting for two or more months after birth, approximately 40% of the
infants studied had severe PHT with right ventricular pressures equal to, or exceeding,
the systemic circulation. The estimated survival rates were 64% at 6 and 53% at 24
months respectively after the diagnosis of severe PHT. Multivariate analyses revealed
associations of severe PHT and low birth weights with reduced survival [208]. Using
cardiac catheterization, hyperoxia and inhaled NO have been shown to cause marked
pulmonary vasodilatation in older patients with CNLI, highlighting the contribution of
increased pulmonary vascular tone towards the pulmonary vascular disease of CNLI
[209]. Pathological examination of 15 infants with severe CNLI, who mostly died of non-
respiratory causes, revealed muscularized intra-acinar vessels with associated prominence
of the elastic lamina layer [210]. The aforementioned studies demonstrate that PHT
continues to be an important complication of CNLI, and may have an important impact
on long term survival.
1.16 Origins of pulmonary hypertension in the 60% O2-model of CNLI
As described earlier, following exposure to 60% O2 for 14 days, neonatal rats
develop an overall inhibition of lung cell growth. The associated dysregulated
parenchymal thickening and an absence of pulmonary fibrosis are features that this model
shares with human infants with CNLI, as well as the 125-days’ gestation baboon model.
An antioxidant, U74398G, specifically attenuated lipid peroxidation and hydroxyl radical
38
formation, with a resultant prevention of PHT. It did not offer protection against
parenchymal changes in the 60% O2-model of CNLI [211], leading to the conclusion that
PHT and parenchymal changes were regulated through different mechanisms. The PHT
was induced by lipid peroxidation and mediated by endothelin-1 [212], acting through 8-
isoprostane binding to the thromboxane A2 receptor [190]. Interestingly, when
macrophage influx in this injury model was prevented by using gadolinium chloride, it
prevented the development of PHT [213], with an associated marked reduction in
nitrotyrosine formation (assessed by immunohistochemistry and Western blot analysis).
Further studies revealed that in this model, at day 14, lung macrophages were a major
source of reactive oxygen species as well as the major contributor to peroxynitrite
formation [214]. These findings, in tandem with observations that peroxynitrite is a very
potent vasoconstrictor of small vessels from the neonatal lung [204], support a
pathological role for peroxynitrite in the pulmonary vascular bed. The mechanisms
involved most likely include consumption of the vasodilator nitric oxide, during
peroxynitrite formation, and peroxynitrite’s direct vasoconstrictive effect.
39
Chapter 2
Aims/Hypotheses
40
2.1 Global aims
1) Chapter 4: To study the effects of therapeutic hypercapnia on CNLI in an hyperoxia
model, in which rat pups exposed to 60% O2 for 14 days, develop inhibition of alveolar
formation and areas of diffuse interstitial thickening.
2) Chapter 5: To study the effects of FeTPPS, a peroxynitrite decomposition catalyst, on
60% O2 -induced CNLI and PHT.
3) Chapter 6: To study the effect of elafin, a neutrophil elastase inhibitor, on 60% O2 -
induced CNLI.
2.2 Specific hypotheses:
Chapter 4:
1) Therapeutic hypercapnia will prevent the increased tissue fraction routinely observed
in pups exposed to 60% O2.
2) Exposure to therapeutic hypercapnia will restore secondary crest formation in 60%-O2-
exposed pups.
3) The protective effects of therapeutic hypercapnia will result in prevention of
pulmonary hypertension by impeding the vascular smooth muscle hyperplasia in 60%-
O2-exposed pups.
4) The significant increase in RNS formation, as seen in pups exposed to 60% O2, will
be inhibited by therapeutic hypercapnia.
41
Chapter 5
1) Pruning of the peripheral vascular bed observed in 60%-O2-exposed pups will be
prevented by a peroxynitrite decomposition catalyst.
2) A peroxynitrite decomposition catalyst will prevent the pulmonary hypertension, as
well as the increase in its upstream regulators, in pups exposed to 60% O2.
3) The use of a peroxynitrite decomposition catalyst will prevent macrophage-derived
peroxynitrite-induced protein nitration.
Chapter 6:
1) Macrophage influx in 60%-O2-exposed pups is dependent on the macrophage
chemotactic properties of elastin fragments, formed by the action of neutrophil elastase.
2) A neutrophil elastase inhibitor will prevent elastin fragmentation caused by 60% O2
exposure, and will prevent the development of peroxynitrite-mediated CNLI and PHT.
42
Chapter 3
General Materials and Experimental Procedures
43
3.1 Institutional review/in vivo interventions
Animal experiments were conducted according to Canadian Council on Animal
Care guidelines. Approvals were obtained from the Animal Care Review Committees of
the Sunnybrook, and Hospital for Sick Children, Research Institutes. Rat pups (10–12 per
litter) were exposed to air or 60% O2 in paired chambers for up to 14 days as previously
described [26, 27, 149, 161, 213]. Pups delivered inside the chambers. Dams were
exchanged daily between chambers to avoid maternal O2 toxicity.
3.2 Immunohistochemistry
Lungs were initially flushed with PBS containing 1 U/ml heparin, while
undergoing manual inflations to clear the pulmonary circulation of blood, and were then
perfused with 4% (wt/vol) freshly dissolved paraformaldehyde before fixing over 12 h
suspended in 4% (wt/vol) freshly dissolved paraformaldehyde. Throughout the fixation
process, a constant airway pressure of 20 cm H2O was maintained with air, via a tracheal
catheter, to prevent lung recoil. For vessel counts, and measurement of vessel medial wall
thickness, a vascular perfusion pressure of 100 cm H2O was maintained after ligation of
the pulmonary veins. The lungs were briefly washed three times in PBS, followed by a
graded ethanol series, and then embedded in paraffin at 60°C. Randomly oriented lung
blocks were cut into 5-µm sections for staining with hematoxylin and eosin, or for
immunohistochemistry. Immunostaining was performed using an avidin-biotin-
peroxidase complex method [215]. Slides were incubated overnight at 4°C with the
primary antibody. After a 1-h incubation with biotin-conjugated secondary antibody, the
labelled Vectastain ABC system (Vector Laboratories, Burlingame, CA) was used with a
44
substrate of 3,3-diaminobenzidine (DAB peroxidase substrate kit; Vector Laboratories).
Slides were mounted in Permount mounting medium. Immunostaining for
myeloperoxidase was performed with 1:2,000 primary rabbit polyclonal antibody (Dako
Canada, Mississauga, Ontario, Canada) to identify neutrophils [161], with 1:300 mouse
anti-rat CD-68 antibody (Serotec, Oxford, United Kingdom) to identify macrophages
[216], with 1:800 mouse monoclonal antibody for α-smooth muscle actin (Neomarkers,
Fremont, CA) and with 1:100 rabbit polyclonal antibody (Upstate Biotechnology, Lake
Placid, NY) to identify nitrotyrosine residues [213]. Secondary species-specific antibody
concentrations were 1:200. Counterstaining was performed with Nuclear Red for
myeloperoxidase and with Carazzi hematoxylin for nitrotyrosine, CD-68, and α-smooth
muscle actin. Peptides against which antibodies to myeloperoxidase, CD-68, and α-
smooth muscle actin were raised were not commercially available. Specificity of
antibodies for their target proteins was therefore assessed by Western blot. Appropriately
sized single protein bands only, at 42 and 98 kDa, were observed for α-smooth muscle
actin and rat CD-68, respectively. For myeloperoxidase, only the expected four bands at
10, 15, 55, and 60 kDa were observed.
3.3 Western blot analyses/immunoprecipitation
Western blots of lysates and immunoprecipitated lysates from perfused lung tissue
were performed [217]. Protein content was measured as described by Bradford [218].
Membranes were incubated overnight at 4°C with 1:2,500 rabbit polyclonal antibody to
nitrotyrosine (Upstate Biotechnology) or 1:500 rabbit polyclonal antibody to
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa
45
Cruz, CA) for normalization in blocking solution. After a thorough wash, the membranes
were incubated with secondary horseradish peroxidase-conjugated anti-rabbit antibody
(Cell Signaling Technology, Beverly, MA), diluted 1:3,000, for 90 min. Protein bands on
blots were quantified by enhanced chemiluminescence detection, with integrated band
densities being calculated after subtraction of background values, as previously described
[213]. For studies of α-smooth muscle actin nitration, α-smooth muscle actin was
immunoprecipitated and then separated with protein A sepharose beads (Sigma, St.
Louis, MO) and centrifugation. Because GAPDH could not be used to normalize
immunoprecipitated bands, Ponceau S protein stain was used for normalization.
3.4 Enzyme-linked immunosorbent assay (ELISA) analyses
Angiopoietin-1 (Ang-1) and VEGF-A were quantified by ELISA using
commercially available kits (R&D Systems, Minneapolis, MN).
3.5 Morphometric analyses
Morphometric analyses of whole left lungs were performed as previously described
[161], with the measurer masked from group allocation for all counting procedures. Ten
random images were captured from non-overlapping fields from each section, with four
randomly oriented sections per animal and six animals from different litters per group.
Mean linear intercept (Lm), a measure of the average diameter of saccules/alveoli, was
calculated as described by Dunnill [219]. A crossed hairline of known length (see Fig.
3.1) was superimposed on images which were stained for α-smooth muscle actin.
46
Fig. 3.1. Illustration of crossed hairline used to measure mean linear intercept. In this
image, the cross intercepted the tissue (black arrows) and the vessel (red arrows) 4 times
each. Therefore, (4 × 1) + (4 × 0.5) = 6.
47
Intercept numbers were assessed as follows: 1 count for distal lung tissue and 0.5
for proximal airway/vessel intercept. Average values per animal were derived from
averages of each section, following which the length of the two traverses was divided by
the average number of intersections to calculate the average Lm, which was subsequently
analyzed statistically.
Sample calculation:
5.4 + 5.8 + 5.2 + 6.1 = 22.5/4 = 5.625 (average of intersections from 4 sections)
2 × 0.2 × 1000 = 400 μm (2 traverses × length of traverse which is 0.2 × 1000 μm)
400/5.625 = 71.11 μm (length of two traverses divided by average number of intersections)
Estimated alveolar surface area per unit lung volume was calculated as described
by Kawakami et al. [220] from the formula:
Svw = 2 × lw/LT, where lw = number of intersections with the crossed hairline
LT = hairline of known length (200 μm)
Therefore, Svw = 2 × 5.625 /200 = 0.056
The tissue fractions per image were calculated using a 130-point contiguous
counting grid superimposed on each (× 200) image and counting the proportion of grid
points that fell on tissue (Fig. 3.2). This same grid was used to determine secondary crest
volume density. Secondary crests were identified by immunoreactive α-smooth muscle
actin in their tips, and their density per unit area was derived from the total number of
attached secondary crests in an image of known area, irrespective of their length. Ratios
of secondary crests to tissue were derived using the superimposed grid and counting the
48
Fig. 3.2. Cartoon of the 130-point contiguous grid (superimposed on an image) used in
measuring tissue fraction and secondary crest volume density.
49
number of points overlying secondary crests or other tissue. The number of total crests
per field was counted manually without the use of the overlaying grid.
Estimated total alveolar numbers (NT) were calculated as described by Weibel and
Gomez [221]. To achieve statistical consistency and for counting purposes, any alveolus
falling partially within the field of view was counted as 1 if it entered the visual field at
the top or right hand borders of the image, but was not counted if it fell in the bottom or
left hand borders.
NT = NV × LV
where,
NV = alveoli per unit volume (e.g. ml)
LV = lung volume in ml
Variable NV is calculated by the following formula:
NV = NA 3/2 × D (B × VVas
½) where,
NA = Corrected alveolar count per unit area
= Alveolar count per unit area (e.g. cm2) divided by shrinkage factor of 1.32
B = Spherical constant of an alveolus of 1.55
VVas = Fractional volume occupied by alveoli (obtained by point counting)
= # of points within all alveoli ÷ 130 (# of points on grid)
D = Distribution variable of the characteristic linear dimensions of the alveoli=1
50
Post-fixation lung volumes (LV) were measured by water displacement. These
morphometric approaches are relatively crude compared with the elegant, precise, and
technologically advanced approaches applied by some investigators [14], and I readily
acknowledge that they provide only estimates of alveolar surface area and number.
However, they have the advantage of simplicity and are well-suited to identifying large
differences between groups. Some of my derived numbers could be subject to fixation
artifact, if lung inflation was inconsistent. This would not appear to be the case in my
studies based on the standard errors of 0.99–3.3% and 0.7-2.6% (in the first and second
study respectively) for postfixation lung volumes.
3.6 Vessel counts and medial wall thickness analyses
Tissue sections that had been paraffin-embedded were stained using Weigert’s
resorcin-fuchsin solution (Elastin Products, Owensville, MO) diluted in acidic 70%
(vol/vol) ethanol. Dewaxed and rehydrated sections were rinsed in distilled water and
placed in Weigert’s solution overnight. After a 10-min wash in lukewarm tap water,
slides were counterstained with 0.5% (wt/vol) tartrazine in 0.25% (vol/vol) acetic acid
and then rinsed in distilled water. Sections were then dehydrated, cleared in xylene, and
mounted using a Permount-xylene (1:1 vol/vol) mixture. Concentric rings with diameters
of 20 and 65 µm were superimposed on images, and only those vessels with both inner
and outer elastic lamina, to identify arterioles, and outer elastin band diameters within
this range were counted. The choice of 20- and 65-µm diameters was somewhat arbitrary,
with the intent of excluding capillaries, yet including the most distal arterioles. The
decision to only include vessels with both inner and outer elastin lamina was to identify
51
only fully muscularized vessels, which may be contributing to any observed PHT. The
number of peripheral vessels of 20–65 µm in diameter was counted in 10 random, non-
overlapping fields per lung with all fields being within 435 µm of the edge of the lung on
tissue cross-section. Fields containing no identifiable vessels within the selected range
were included in the analysis. Medial wall thickness of vessels of 20-65 µm outer
diameter was calculated from the formula “percent wall thickness = (2 × wall
thickness/external diameter) × 100” [189], with the following modification. Sixty to
eighty vessels per pup were photographed under an oil-immersion lens at a 1,000-fold
magnification. Vessels with an external diameter within 20–65 µm and having external
diameters measured at 2 perpendicular planes within 33% of each other were included in
the counts. Applying this exclusion eliminated ~20% of the total vessels that were cut too
obliquely and therefore had the potential to introduce bias.
3.7 Data presentation
All values are presented as means ± SEM. Data for multiple groups were
subjected to 1- or 2-way ANOVA, followed by the Tukey test, using the SigmaStat
(SPSS, Chicago, IL version 11.0) analysis program. A P value <0.05 was regarded as
statistically significant.
52
Chapter 4
Therapeutic effects of hypercapnia
on chronic lung injury and vascular remodeling in
neonatal rats
53
4. Therapeutic effects of hypercapnia on chronic lung injury and vascular
remodeling in neonatal rats
4.1 Abstract
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, were attenuated by concurrent
hypercapnia. I 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
54
model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a
phagocyte influx.
4.2 Aims/objectives
To study the effects of therapeutic hypercapnia on chronic neonatal lung injury in
a model of CNLI, in which rat pups exposed to 60% O2 for 14 days develop inhibition of
alveolar formation, areas of diffuse interstitial thickening and pulmonary hypertension.
4.3 Hypothesis
Therapeutic hypercapnia will protect against chronic neonatal lung injury in a
small animal model and in turn prevent the development of PHT.
4.4 Introduction
CNLI affects extremely premature infants (< 32-week gestation) who have
required mechanical ventilation and O2 therapy following birth [220, 222]. Various
management strategies routinely utilized to reduce the likelihood of infants developing
CNLI have had no beneficial effects on the ultimate outcome [98]. A very promising
approach to the prevention of CNLI, developed from adult intensive care practices, has
been that of permissive hypercapnia, which has improved survival in patients suffering
from acute respiratory distress syndrome [114, 115, 223] and acute lung injury [116]. The
initial rationale underlying the use of permissive hypercapnia had been that ventilation
with reduced tidal volumes should protect against stretch-mediated lung injury [224]. An
alternative, or complementary, explanation for the beneficial effects of ventilation with
55
reduced tidal volumes is a direct protective effect of hypercapnia [225, 226]. Permissive
hypercapnia is now a widely used strategy in ventilation of the newborn, with a target
arterial partial pressure of CO2 (PaCO2) of 45–55 mm Hg [227], which appears to have
no identifiable short-term adverse consequences [124].
Multicentre trials of permissive hypercapnia conducted on premature human
infants, using a target PaCO2 of ~52 or 55–65 mm Hg to reduce minute ventilation below
that of control infants, have failed to demonstrate an altered incidence of death or CNLI
[113, 128]. Logical explanations for this lack of benefit include: inadequate sample sizes,
in that both of these trials had to be terminated before full patient recruitment; the
neonatal lung is resistant to the benefits of hypercapnia; and/or the target PaCO2 in these
clinical trials was less than that required to obtain benefit.
So-called therapeutic hypercapnia, in which the CO2 concentration in the inspired
gas has been increased, has been used to demonstrate a protective effect of an increased
PaCO2 in both acute studies of ventilator-induced [228, 229] and ischemia reperfusion-
induced [230] lung injuries in animal models. I propose to apply this same approach to an
established rat model of CNLI induced by exposure to 60% O2 for 14 days [149] to
determine whether either the neonatal lung is unresponsive to hypercapnia or whether a
greater degree of hypercapnia than that applied to human infants to date would offer
protection. It must, however, be acknowledged that there may be critical differences
between passive and therapeutic hypercapnia in their effects on the lung, based on
potentially opposite effects on minute ventilation, and therefore the degree of strain to
which the lung is subjected. Neonatal rats exposed to 60% O2 for 14 days have a lung
histology similar to that observed in human infants with CNLI [149] in that they have an
56
heterogeneous injury with impaired alveolar development and thickening of the lung
parenchyma [161]. The parenchymal lung injury is heterogeneous, with areas of
interstitial thickening and active DNA synthesis mixed with areas of arrested
alveolarization and growth [149]. This parenchymal thickening appears to be due to an
increase in cell number, in that there is no effect of 60% O2 on lung wet-to-dry weight
ratios [149], and that 60% O2 induces no qualitative difference in collagen deposition, as
assessed by Sirius red staining [231]. The cells represented in the thickened parenchyma
appear to be largely of epithelial origin [27]. Based on the protective effects observed
with adult human patients with acute respiratory distress when subjected to a target
PaCO2 of 67 mm Hg [232], I studied the effect of adding 5.5% carbon dioxide to the
inspired gas of neonatal rat pups, which I estimated, from the alveolar gas equation,
should achieve a PaCO2 of ~70 mm Hg.
4.5 Clinical relevance
Subsequent to the beneficial effects of permissive hypercapnia observed in adult
acute respiratory syndrome and acute lung injury, and the lack of benefit observed in
studies in neonates suffering from BPD, a preventive approach utilizing therapeutic
hypercapnia in neonatal rats is warranted. The objective is to determine if the newborn
are resistant to the beneficial effects of hypercapnia, or simply require a greater degree of
hypercapnia. Should this therapy render protection to the rat model of CNLI, within
clinically acceptable parameters of hypercapnia, it will open potential new avenues in the
treatment of this long-standing complex disease, provided any beneficial effects on the
lung are not accompanied by adverse effects on other organs such as the brain.
57
4.6 Specific materials and experimental procedures
4.6 i In vivo interventions
In this study, half the litters in air or 60% O2 had 5.5% CO2 included in their
inspired gas.
4.6 ii Von Willebrand factor immunohistochemistry
A rabbit polyclonal antibody raised against von Willebrand factor (Thermo Fisher
Scientific, Fremont, CA) was used (dilution 1:60) to identify endothelial cells. A
fluorescent secondary antibody was used for von Willebrand (1:200) factor
immunolocalization (Alexa Fluor 488; A-11008; Invitrogen, Carlsbad, CA).
4.6 iii Enzyme-linked immunosorbent assay (ELISA) analysis
VEGF-A receptor fms-like tyrosine kinase-1 (Flt-1) concentrations in lung
homogenates were quantified by ELISA using commercially available kit (R&D
Systems, Minneapolis, MN)
4.6 iv Blood gas analyses
An initial study was conducted to confirm that the PaCO2 predicted from the
alveolar gas equation had been achieved. Pups that had been exposed to air or concurrent
air and 5.5% CO2 were anaesthetized in their sealed exposure chambers on day 14 of life
using intraperitoneal ketamine:xylazine (80:20 mg/kg) for carotid artery puncture.
Analyses were performed using CG4+ cartridges with an i-STAT portable clinical
analyzer (Abbott Diagnostics, Abbott Park, IL).
58
4.6 v Assessment of respiratory and heart rates
Effects of anaesthesia on heart and respiratory rates were assessed using a tail
sensor pulse oximeter (MouseOx; STARR Life Sciences, Oakmont, PA).
4.7 Results
The initial assessment of terminal blood gases confirmed that the predicted
increase in PaCO2 following exposure to 5.5% CO2 had been achieved (Table 1). The
assessment of respiratory and heart rates before and after anaesthesia (Table 2) did not
indicate any obvious effect of anaesthesia on either parameter. Visual assessment of
respiratory rates correlated with the values determined by oximeter. Exposure to neither
60% O2 nor 5.5% CO2 had significant effects on lung or body weights, the lung-to-body
weight ratios or postfixation lung volumes (Table 3).
4.7 i Exposure to hypercapnia resulted in significant attenuation of increased tissue
fraction routinely observed in pups exposed to 60% O2
As shown in Fig. 4.1, exposure to 60% O2 caused marked interstitial thickening of
the lung that appeared, microscopically, to be attenuated by exposure to concomitant
hypercapnia. Neonatal rats exposed to both air and 5.5% CO2 had occasional areas that
appeared to have an increase in interstitial thickening compared with those exposed to air
alone. Calculation of the tissue fraction (proportion of field occupied by tissue)
confirmed a significant increase following exposure to 60% O2, which was attenuated in
pups exposed to the combination of 60% O2 and hypercapnia (Fig. 4.1B). The mild
patchy increase in interstitial thickening induced in air-exposed pups by hypercapnia was
59
Table 1
Blood gases from d-14 rat pups exposed to air or air + 5.5% CO2
Group
PaO2 (mm Hg)
PaCO2 (mm Hg)
pH
HCO3 (mEq/L)
Air
84 ± 3
46 ± 3
7.35 ± 0.02
25 ± 1
Air + 5.5% CO2
106 ± 1 *
70 ± 3 *
7.25 ± 0.03 *
31 ± 2 *
Data are presented as means ± SEM (n = 4 or 5 animals/group).
* p < 0.05 versus air control group by 1-way ANOVA. PaO2, arterial PO2; PaCO2, arterial PCO2; meq, milliequivalents.
60
Table 2.
Respiratory (RR) and heart rates (HR) of d-14 rat pups before and after anaesthesia
following exposure to air or air + 5.5% CO2
Group
RR before
RR after
HR before
HR after
Air
147 ± 4
146 ± 6
475 ± 17
400 ± 25
Air + 5.5% CO2
143 ± 1
142 ± 2
407 ± 6*
352 ± 16
Data are presented as means ± SEM (n = 4 or 6 animals/group). When assessed by 2-way
ANOVA, no overall statistically significant effect of anesthesia on respiratory (RR) or
heart rate (HR) was observed. There was an overall significant effect of CO2 on HR.
*Significantly different (P < 0.05) by 2-way ANOVA on pairwise testing from HR before
in air.
61
Table 3
Lung (LW) and body (BW) weights, and post-fixation displacement lung volumes (LV) in
14-d-old rat pups exposed to air or 60% O2 ± 5.5% CO2
Parameter
Air
(mean ± SEM)
Air + 5.5% CO2 (mean ± SEM)
60% O2
(mean ± SEM)
60% O2 + 5.5% CO2
(mean ± SEM)
LW (mg)
467 ± 4
477 ± 7
468 ± 1
488 ± 9
BW (gm)
30.1 ± 0.5
30.4 ± 0.1
31.4 ± 0.2
31.4 ± 0.3
LW/BW × 102
1.55 ± 0.03
1.57 ± 0.02
1.49 ± 0.01
1.56 ± 0.02
LV (μl)
1069 ± 16
1087 ± 22
1015 ± 10
1098 ± 36
Data were presented as means ± SEM (n=5 pups per group). Both O2 [body weight (BW)
and CO2 [lung weight (LW)] had significant effects (P < 0.05) by 2-way ANOVA. No
differences were detected for pairwise comparisons between individual groups. LV, lung
volume.
62
Day 14 Air
Day 14 Air + CO2
Day 14 60% O2
Day 14 60% O2 + CO2
A
Tis
suef
ract
ion
0.0
0.1
0.2
0.3
a a+c o o+c
*§
B
Fig. 4.1 (A) Lung histology of neonatal rats exposed to air or 60% O2 for 14 d, with or
without 5.5% CO2. Bar = 100 μm. (B) Morphometric analysis of the tissue fraction
(tissue:air ratio) in neonatal rats exposed to air (a) or 60% O2 (o) for 14 d, with or without
5.5% CO2 (a+c; o+c). Both O2 and CO2 had significant effects (P < 0.05) by 2-way
ANOVA. *Significantly different (P < 0.05) from the air group. §Significantly different
(P < 0.05) from the 60% O2 group. Values are means ± SEM for 6 average-sized pups
from different litters.
63
Mea
nlin
ear
inte
rcep
t(µm
)
0
25
50
75
100
a a+c o o+c
A *B
§
Var
ianc
eof
mea
nlin
ear
inte
rcep
t
0.0
0.5
1.0
1.5
2.0
2.5
a a+c o o+c
§
Fig. 4.2. Measurement of (A) mean linear intercept and (B) of variance in average mean
linear intercept measurements between fields within each lung section in neonatal rats
exposed to air (a) or 60% O2 (o) for 14 d, with or without 5.5% CO2 (a+c; o+c). Both O2
and CO2 had significant effects (P < 0.05) by 2-way ANOVA. *Significantly different (P
< 0.05) from the air group. §Significantly different (P < 0.05) from the 60% O2 group.
Values are means ± SEM for 6 average-sized pups from different litters.
64
not statistically significant. That exposure to 60% O2 resulted in heterogeneous changes
in alveolar diameter is well demonstrated in Fig. 4.1, yet there was no apparent effect on
mean linear intercept in Fig. 4.2A.
As discussed elsewhere [161], the process of averaging values for all fields in a
section masks heterogeneity occurring between individual fields. To assess this, I
analyzed the variance of values for mean linear intercepts between fields within each
section as a measure of heterogeneity [161], as shown in Fig. 4.2B. Exposure to 60% O2
induced significant heterogeneity. Concurrent exposure to hypercapnia both reduced
mean linear intercept values below that of air-exposed control animals (Fig. 4.2A) and
corrected the heterogeneity observed with 60% O2 (Fig. 4.2B).
4.7 ii Concomitant hypercapnia and 60% O2 resulted in increased secondary crest
formation and alveolar growth
Secondary crests growing in from the walls of precursor saccules initiate the
process of alveolar formation. A statistically significant increase in the number of
secondary crests per unit area was observed in the pups exposed to 60% O2 and 5.5%
CO2 for 14 days, relative to all other exposure groups (Fig. 4.3A). To exclude the
possibility that this increase was due to an inflation artifact, the number of secondary
crests was also expressed as a fraction of total tissue. Following this correction, it was
evident that the combination of 60% O2 and hypercapnia had indeed stimulated
secondary crest formation beyond that seen in the other exposure groups (Fig. 4.3B).
There was a significant reduction in the secondary crest-to-tissue ratio following
exposure to 60% O2. This could reflect the previously noted increase in tissue fraction
65
and also the fact that many of the secondary crests in 60% O2-exposed pups are stunted in
length, as previously described [161]. The enhanced secondary crest formation, following
exposure to a combination of 60% O2 and hypercapnia, resulted in a significant increase
in estimated gas exchange surface areas (Fig. 4.3C) and estimated total alveolar numbers
(Fig. 4.3D).
4.7 iii Effects of therapeutic hypercapnia on inflammatory cell influx in lung
parenchyma
This laboratory has previously reported enhanced secondary crest and alveolar
formation in 60% O2-exposed pups when neutrophil influx into their lungs was prevented
[161]. One possible explanation for the increased secondary crest formation observed in
animals exposed to both 60% O2 and hypercapnia was that the hypercapnia was
suppressing an inflammatory cell influx. Myeloperoxidase staining for neutrophils at day
14 revealed an anticipated [161] increase in number with 60% O2. The neutrophil influx
appeared to be reduced by concomitant hypercapnia (Fig. 4.4A). Direct cell counts
confirmed a significant but incomplete hypercapnia-dependent reduction in the neutrophil
influx induced by 60% O2 (Fig. 4.4B). CD-68 immunostaining for macrophages
confirmed the anticipated [213] influx of macrophages induced by exposure to 60% O2,
which also appeared to be attenuated by concomitant hypercapnia (Fig. 4.4C). A
complete inhibition of the 60% O2-mediated macrophage influx by concomitant
hypercapnia was confirmed by direct cell counts (Fig. 4.4D). Because lung tissue was
fixed during inflation with air, macrophages retained their position on alveolar walls
rather than floating into the lumen of the alveolus, as would have occurred during
66
DTo
tala
lveo
liin
whol
elun
g(x
10-6
)
0
1
2
3
4
a a+c o o+c
Seco
ndar
ycr
ests
/mm
2
0
25
50
75
100
125
150
175 A §
a a+c o o+c
Seco
ndar
ycr
ests
/tiss
uera
tio
0.00
0.02
0.04
0.06
0.08 B §
*
Alv
eola
rsu
rfac
ear
eape
r uni
tlu
ngvo
lum
e(c
m2 /c
m3 )
0.00
0.02
0.04
0.06
0.08 C § §
a a+c o o+c a a+c o o+c
Fig. 4.3. Measurement of secondary crest density (A), and secondary crest/tissue ratio
(B), and estimates of alveolar surface area (C) and total alveolar number (D) in neonatal
rats exposed to air (a) or 60% O2 (o) for 14 d, with or without 5.5% CO2 (a+c; o+c).
Both O2 (A, C, D) and CO2 (A-D) had significant effects (P < 0.05) by 2-way ANOVA.
*Significantly different (P < 0.05) from the air group. §Significantly different (P < 0.05)
from the 60% O2 group. Values are means ± SEM for 6 average-sized pups from different
litters.
67
Day 14 Air
Day 14 Air + CO2
Day 14 60% O2
Day 14 60% O2 + CO2
§
Mye
lope
roxi
dase
-pos
itive
cells
/mm
2
0
50
100
150
200
250
*BA
Day 14 Air
Day 14 Air + CO2
Day 14 60% O2
Day 14 60% O2 + CO2
C D
CD-6
8-po
sitiv
ecell
s/mm
2
0
20
40
60 *
a a+c o o+c
a a+c o o+c
§
*
Fig. 4.4. (A) Neutrophils were identified by myeloperoxidase immunoreactivity (black
stain; bar = 100 μm), in order to (B) calculate neutrophil counts per unit area, and (C)
macrophages were identified by CD-68 immunoreactivity (brown stain; bar = 100 μm) in
order to (D) calculate macrophage counts per unit area, in lung tissue of neonatal rats
exposed to air (a) or 60% O2 (o) for 14 d, with or without 5.5% CO2 (a+c; o+c). CO2 (B,
D), but not O2, had significant effects (P < 0.05) by 2-way ANOVA. *Significantly
different (P < 0.05) from the air group. §Significantly different (P < 0.05) from the 60%
O2 group. Values are means ± SEM for 4 average-sized pups from different litters.
68
inflation with fluid.
4.7 iv Hypercapnia prevented vascular smooth muscle hyperplasia in 60% O2-exposed
pups
Based on previous observations [213], I hypothesized that the observed inhibition
of a macrophage influx into the 60% O2-exposed lungs by concomitant hypercapnia
would also be associated with a reduction both in PHT, and of nitrotyrosine formation.
When assessed by immunohistochemistry for α-smooth muscle actin (Fig. 4.5A), the
normal spiral pattern of perivascular smooth muscle observed around vessels of 30- to
50-μm diameter in air-exposed pups appeared thickened and formed a complete ring
following exposure to 60% O2, consistent with the development of PHT-induced vascular
remodeling. This change appeared to be attenuated by concomitant hypercapnia. To
assess PHT-induced vascular remodeling, I measured vessel medial wall thickness. As
shown in Fig. 4.5B, exposure to 60% O2 significantly increased medial wall thickness,
which was prevented by concomitant hypercapnia. Hypercapnia alone had no effect on
medial wall thickness.
4.7 v Hypercapnia attenuated the marked increase in nitrotyrosine immunoreactivity
observed in 60% O2-exposed pups
As shown in Fig. 4.6, the marked increase in nitrotyrosine immunoreactivity
observed following exposure to 60% O2 was apparently attenuated by concomitant
hypercapnia. That hypercapnia completely attenuated the marked increase in
69
Day 14 Air
Day 14 Air + CO2
Day 14 60% O2
Day 14 60% O2 + CO2
BA
%M
edia
lwal
lthi
ckne
ssof
vess
els20
-65μ
mo.
d.
0
5
10
15
20
25
a a+c o o+c
*
§
Fig. 4.5. (A) Immunostaining for α-smooth muscle actin (brown stain) in the lungs of
neonatal rats exposed to air or 60% O2 for 14 d, with or without 5.5% CO2. Bar = 30 μm.
(B) Medial wall thickness (%), an index of pulmonary hypertension, was measured in the
completely muscularized pulmonary vessels with a 20-65 μm outer elastic lamina
diameter (o.d.) in neonatal rats exposed to air (a) or 60% O2 (o) for 14 d, with or without
5.5% CO2 (a+c; o+c). Both O2 and CO2 had significant effects (P < 0.05) by 2-way
ANOVA. *Significantly different (P < 0.05) from the air group. §Significantly different
(P < 0.05) from the 60% O2 group. Values are means ± SEM for 4 average-sized pups
from different litters.
70
Day 14 Air Day 14 60% O2
Day 14 Air + CO2 Day 14 60% O2 + CO2
Negative Control
Positive Control
Fig. 4.6. Nitrotyrosine formation, a marker for peroxynitrite-mediated reactions, in the
lung tissue of neonatal rats exposed to air or 60% O2 for 14 d, with or without 5.5% CO2.
As a negative control, sections were immunostained after immunoadsorption of the
primary antibody. As a positive control, sections were immunostained after exposure to
peroxynitrite. Bar = 100 μm.
71
nitrotyrosine immunoreactivity observed following exposure to 60% O2 was confirmed
by measurement of nitrotyrosine content by Western blot (Fig. 4.7A).
4.7 iv Hypercapnia prevented α-smooth muscle actin nitration otherwise observed in
60% O2-exposed pups
Peroxynitrite is both a potent vasoconstrictor, and an inhibitor of relaxation, of
small pulmonary vessels [204]. One mechanism by which relaxation could be impaired is
through nitration of smooth muscle proteins. I, therefore, immunoprecipitated α-smooth
muscle actin to assess its degree of tyrosine nitration by Western blot. After
densitometry, it became apparent that exposure to 60% O2 significantly increased
tyrosine nitration of α-smooth muscle actin compared with controls, which was
attenuated by concomitant exposure to hypercapnia (Fig. 4.7B).
4.7 vii Hypercapnia abrogated the peripheral pulmonary vessel pruning in 60% O2-
exposed pups
When examining slides for changes in α-smooth muscle actin immunoreactivity,
as described above, I noted an apparent reduction in small vessels in the lung periphery
induced by 60% O2 was restored by concomitant hypercapnia. When endothelial cells
were identified, using an antibody to von Willebrand factor, there was a qualitatively
obvious reduction of vessels of all sizes, including capillaries (Fig. 4.8). I did not attempt
to perform capillary counts, which I find challenging due to the uncertainties involved in
labelling immunoreactive cell clusters with no apparent lumen. However, vessel
depletion included an apparent 60% O2-dependent reduction in the number of arterioles,
72
a a+c o o+c
66 kDa
37 kDa
Nitrotyrosine
GAPDHN
itrot
yros
ine
cont
ent
norm
aliz
edto
GA
PDH
0.0
0.5
1.0
1.5
a a+c o o+c
*
Nitr
ated
-sm
ooth
mus
cleac
tin
0.0
0.4
0.8
1.2
a a+c o o+c
*
Nitratedα -smooth
muscle actin42 kDa
a a+c o o+c
Ponceau
A B§
§
Fig. 4.7. Lung homogenates from neonatal rats exposed to air (a) or 60% O2 (o) for 14 d,
with or without 5.5% CO2 (a+c; o+c) were studied by Western blot, and densitometric
analysis, for (A) nitrotyrosine content and (B), after immunoprecipitation, for the content
of nitrated α-smooth muscle actin. Both O2 (A) and CO2 (A, B) had significant effects (P
< 0.05) by 2-way ANOVA. *Significantly different (P < 0.05) from the air group.
§Significantly different (P < 0.05) from the 60% O2 group. Values are means ± SEM for
4 average-sized pups from 4 different litters.
73
which was restored by concomitant hypercapnia. Hart’s elastin stain was used to better
demonstrate this finding (Fig. 4.9) and to allow direct counts of small vessels in the lung
periphery with both inner and outer elastic lamina, and an outer elastic lamina diameter
of 20–65 μm. As shown in Fig. 4.10A, 60% O2 caused a significant reduction in the
number of small vessels in the lung periphery, which was completely attenuated by
concomitant hypercapnia. The pattern of a reduction in vessel number in 60% O2, which
was restored by concomitant hypercapnia, was matched by parallel reductions in VEGF
(Fig. 4.10B) and Ang-1 (Fig. 4.10C) contents, which were also restored by concomitant
hypercapnia. Flt-1 content was unaffected by either 60% O2 or hypercapnia (Fig. 4.10D).
4.8 Discussion
Outside the neonatal period, hypercapnia is well-recognized to have anti-
inflammatory effects on the injured lung [233]. Intuitively, this would be a likely
significant contributor to any protective effect on lung injury. However, a recent study of
experimental pneumonia-induced lung injury suggested that the protective effect of
hypercapnia was neutrophil-independent [234]. The only laboratory study, of which I am
aware, that has addressed the question of whether hypercapnia is protective against injury
in the developing lung was a short term (6-h) study conducted in ventilated preterm
lambs [229]. The authors of that study found that a mean PaCO2 of 95 mm Hg was
protective against acute lung injury. Hypercapnia was also associated with a reduction in
indicators of inflammation, although only a reduction in airway inflammatory cells
achieved statistical significance. Most likely because the 60% O2-model is a chronic
74
Day 14 Air
Day 14 Air + CO2
Day 14 60% O2
Day 14 60% O2 + CO2
Fig. 4.8. Immunofluorescent staining for von Willebrand factor (white), a marker for
endothelial cells, in the lungs of neonatal rats exposed to air or 60% O2 for 14 days with
or without 5.5% CO2. Bar = 100 μm.
75
Day 14 Air
Day 14 Air + CO2
Day 14 60% O2
Day 14 60% O2 + CO2
Fig. 4.9. Hart’s elastin staining of lung tissue from neonatal rats exposed to air or 60% O2
for 14 days with or without 5.5% CO2 to identify vessels with elastic lamina. Bar = 200
μm.
76
0
2
4
6
8
a a+c o o+c
*
Peri
pher
alve
ssel
s(20
-65
µm)/m
m2
0
5
10
15
20
25
a a+c o o+ c
*
§
AA
ng-1
(pg/
µgpr
otei
n)
0.0
0.1
0.2
0.3
VE
GF-
A (p
g/m
g pr
otei
n)
a a+c o o+ c
Flt-1
(pg/
µgpr
otei
n)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
a a+c o o+c
B
C D
§§
*
Fig. 4.10. Neonatal rats exposed to air (a) or 60% O2 (o) for 14 d, with or without 5.5%
CO2 (a+c; o+c). (A) Counts of lung peripheral vessels with an outer elastic lamina
diameter (o.d.) of 20-65 μm expressed as vessel density per unit area. (B) VEGF-A
concentrations in lung homogenates. (C) Ang-1 concentrations in lung homogenates.
(D) Flt-1 concentrations in lung homogenates. Both O2 (A, B) and CO2 (A) had
significant effects (P < 0.05) by 2-way ANOVA. *Significantly different (P < 0.05) from
the air group. §Significantly different (P < 0.05) from the 60% O2 group. Values are
means ± SEM for 6 average-sized pups from different litters.
77
preparation, I was able to detect a significantly reduced influx of inflammatory cells into
the lung, including the lung parenchyma, in response to hypercapnia. A recent study
demonstrated antioxidant properties of hypercapnic acidosis, following endotoxin-
induced lung injury in mechanically ventilated adult rats [235]. A reduction of pulmonary
oxidative reactions
was evident within minutes of the onset of hypercapnic acidosis in acutely inflamed
lungs, independent of nitric oxide-dependent peroxynitrite production. One other study
examined gene expression in air-exposed neonatal mouse lungs and found hypercapnia to
downregulate the expression of a number of immune response genes, but no
corresponding protein measurements were made [236]. I am unaware of other studies
showing hypercapnia-mediated inhibition of pulmonary inflammation or protection
against lung injury in the neonate. My initial assessment of blood gases suggested that I
had achieved the target PaCO2. Subsequent assessment of the impact of anaesthesia on
respiratory and heart rates did not suggest any obvious impact. However, I acknowledge
that I cannot exclude some degree of anaesthesia effect with absolute certainty. I
observed hypercapnia to completely protect against 60% O2-mediated perivascular
smooth muscle hyperplasia and vascular pruning. This I attribute, at least in part, to the
previously described critical role of a macrophage influx in both protein nitration and
PHT in this model [213, 214]. I speculate that the two processes may be linked through
the potent vasoconstrictive properties of peroxynitrite [204], which could result in
sustained PHT with secondary smooth muscle cell hyperplasia and an eventual reduction
in vessel number. Alternatively, or additionally, the reduction in vessel number could be
due to a failure of arteriolar formation induced by 60% O2. Consistent with this latter
78
explanation was the reduced concentrations of VEGF and Ang-1 induced by 60% O2,
which was restored by concomitant hypercapnia. Some caution needs to be exerted with
respect to this interpretation of the VEGF/Ang-1 data, since only a single time point was
studied, and expression could have been quite different at earlier time points. It has
previously been documented [204] that peroxynitrite impairs relaxation of perivascular
smooth muscle. I speculate, based on my observations herein, that this could result, at
least in part, from protein modification of α-smooth muscle actin by nitration.
Hypercapnia reduced the protein nitration associated with exposure to 60% O2, which is
consistent with the previously observed findings in a different lung injury model [230],
but conflicts with expectations based on ex vivo analyses [237]. The effect of therapeutic
hypercapnia on preventing the neutrophil influx during lung injury, although statistically
significant, was incomplete. The partial limitation of a neutrophil influx was, however,
sufficient to unmask the stimulation of secondary crest formation by 60% O2, as
previously described [161], which resulted in increased alveolar formation. Inflammation
has long been recognized as an aggravating or exacerbating factor both in human BPD
[237] and in hyperoxic rat models of chronic neonatal lung injury [161, 213, 238, 239].
Based on one published study that used neonatal rat pups exposed to 95% O2 [239], I
might have expected the observed CO2-mediated inhibition of a macrophage influx to
have been accompanied by a similarly effective inhibition of neutrophil influx. However,
this was not the case, which is probably due to the very different inflammatory cell
responses observed in rat pups exposed to 60% O2 rather than 95% O2. In rat pups
exposed to 60% O2, the neutrophil influx precedes the macrophage influx [213],
suggesting that in the 60% O2-mediated neonatal lung injury it is not the macrophage
79
that initiates the neutrophil influx, as seen with pups in 95% O2. My study, although
clearly demonstrating the protective effects of therapeutic hypercapnia in this model,
leaves unanswered a number of important mechanistic questions. These include defining
the mediators of PHT that are downregulated by hypercapnia. Previous studies have
demonstrated O2-mediated upregulation of the vasoconstrictors endothelin-1, 8-
isoprostane [190, 240], and peroxynitrite [213]. In this study, I did not examine the effect
of hypercapnia on the expression of endothelin-1 or 8-isoprostane but did observe
reduced nitrotyrosine immunoreactivity, a marker for peroxynitrite-mediated reactions.
Determining whether peroxynitrite is a critical mediator of PHT will require intervention
studies using, for example, a peroxynitrite decomposition catalyst [241]. Additionally, I
did not determine whether hypercapnia affects apoptosis in vascular smooth muscle cells
or in lung parenchymal cells. It has been previously demonstrated that endothelin-1
inhibits apoptosis of neonatal rat pulmonary artery smooth muscle cells [212].
My primary objective in these studies was to determine whether the chronically
injured neonatal lung was capable of a beneficial response to therapeutic hypercapnia.
My observations that hypercapnia, in the presence of moderate hyperoxia, limited
pulmonary parenchymal injury, promoted alveolar growth, and prevented vascular
remodeling during neonatal lung injury clearly demonstrated that the neonatal lung does
have such a capacity. However, whether such a beneficial effect can be extrapolated to
the human clinical arena remains unclear. My calculated target PaCO2 was greater than
that accepted in normal clinical practice within neonatal intensive care units. Although
adult humans may tolerate extreme levels of hypercapnia without adverse effects [242,
243], the population of human infants most prone to develop BPD are at particular risk of
80
developing intracranial hemorrhage, retinopathy of prematurity and neurodevelopmental
impairment. Their risk of developing the latter morbidities may be increased by
hypercapnia [128, 244, 245], and any future studies designed to test the potential benefits
of a less conservative PaCO2 than previously used [113] would need to be undertaken
with an appropriate degree of caution.
81
Chapter 5
A peroxynitrite decomposition catalyst
prevents 60% O2-mediated rat chronic neonatal
lung injury
82
5. A peroxynitrite decomposition catalyst prevents 60% O2-mediated rat chronic
neonatal lung injury
5.1 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. I conclude
that peroxynitrite plays a critical role in the development of chronic neonatal lung injury.
5.2 Aims/objectives
To study the effects of FeTPPS, a peroxynitrite decomposition catalyst, on 60%
O2 -induced chronic neonatal lung injury and PHT.
83
5.3 Hypothesis
In the neonatal rat, peroxynitrite, the reaction product of nitric oxide and
superoxide, is an essential mediator of PHT that can be prevented by a peroxynitrite
decomposition catalyst.
5.4 Introduction
I have, as in my previous study, utilized a rat model of CNLI induced by exposure
to 60% O2 for 14 days, in which there is interstitial thickening with a marked
heterogeneity of alveolar diameters [149] and PHT [240]. The PHT can be prevented by
depletion of macrophages with gadolinium chloride, which also blocks the very marked
nitrotyrosine immunoreactivity, used as a marker of reactions mediated by peroxynitrite,
induced by 60% O2 [213]. Other interventions, such as induced hypercapnia [246] and
treatment with an interleukin-1 receptor antagonist [247], which led to a reduction in
macrophage influx after exposure to 60% O2, also reduced protein nitration and
prevented PHT and pruning of peripheral vessels. Treatment with a CXCR2-selective
antagonist depleted the 60% O2-exposed lung of neutrophils, protected against
impairment of alveologenesis [161], and reduced protein nitration [248], but the effects
on the pulmonary vascular bed or macrophage influx were not assessed. It has been
previously reported that peroxynitrite is a potent constrictor of small vessels from the
neonatal lung [204], in contrast to its reported relaxation of adult vessels [249, 250].
Small vessels from adult and neonatal lung are known to have quite distinct behaviors
[251, 252]. Together, these observations led me to hypothesize that peroxynitrite is a
critical mediator of the PHT observed in CNLI induced by exposure to 60% O2. To test
84
this hypothesis, neonatal rat pups were exposed to air or 60% O2 for 14 days and received
daily injections of a peroxynitrite decomposition catalyst, or vehicle, following which the
lungs were studied for changes in the peripheral pulmonary vascular bed, alveolar
development, and a number of their putative mediators.
5.5 Clinical relevance
PHT is a late sequel of severe CNLI that has long lasting implications.
Peroxynitrite could be a critical mediator of PHT. Should a peroxynitrite decomposition
catalyst effectively prevent PHT in neonatal rats, its future use (after appropriate clinical
trials) could become an adjunctive intervention to the current clinical practice of
neonatology designed to limit the development of PHT in moderate to severe CNLI.
5.6 Peroxynitrite decomposition catalysts
Unlike the formation of NO or the decomposition of superoxide, the formation as
well as the decomposition of peroxynitrite are enzyme-independent processes [253].
Peroxynitrite toxicity could be reduced by one of the following ways:
a) Utilizing a compound that would react with the ONOOH-derived radicals yielding
less reactive species (e.g. desferrioxamine doesn’t react directly with peroxynitrite
[254] but reacts with its derived hydroxyl radical [255].
b) Using a compound that would oxidize peroxynitrite, and the oxidized compound
be reduced back by endogenous reductants, hence establishing a catalysis of
peroxynitrite reduction (e.g. organoselenium compounds like ebselen that readily
react with peroxynitrite and use glutathione as a reductant [256])
85
c) Catalyze the isomerization of peroxynitrite to nitrate. (e.g. iron porphyrins
catalyze the isomerization of peroxynitrite to nitrate and have been used in cell
culture and animal models; FeTPPS, FeTMPS and FeTMPyP are examples
[257].)
I used FeTPPS, 5,10,15,20-Tetrakis(4-sulfonatophenyl)porphyrinato iron (III),
chloride in my experiments, which is a ferric porphyrin complex. It catalytically
isomerizes peroxynitrite to nitrate, both in vivo and in vitro. Hence, FeTPPS serves as a
selective peroxynitrite scavenger as well as a decomposition catalyst. It does not form a
complex with NO and exhibits minimal superoxide dismutase mimetic activity.
5.7 Specific materials and experimental procedures
5.7 i In vivo interventions
Rat pups (10-12/litter) were exposed to 60% O2 or air for up to 14 d in paired
chambers and received daily subcutaneous (s.c.) injections (5 μl/g) of saline vehicle or 30
μg/g/d of FeTPPS [258]. This dose was selected based on preliminary studies using a
range of doses available from literature. A dose of 20 μg/g/d did not completely attenuate
60% O2-induced lung tissue nitrotyrosine formation, as assessed by
immunohistochemistry, which was observed at 30 μg/g/d.
5.7 ii Total (free and esterified) 8-isoprostane measurement
Lung tissue was flushed of blood and flash frozen in liquid nitrogen to prevent
auto-oxidation and stored at -80°C till analysis. After thawing, 0.01% (w/v) butylated
86
hydroxytoluene was added to the homogenized tissue and a small aliquot was removed
for protein measurement. To the remaining sample, an equivalent volume of 15% (w/v)
KOH was added (to hydrolyse esterified 8-isoprostane) and the mixture was incubated at
40 °C for 1 hour. After neutralization with KH2PO4 (pH 7.2-7.4), proteins were
precipitated by the addition of 2-4 volumes of ice cold 100% ethanol containing 0.01%
(w/v) butylated hydroxytoluene. The sample was centrifuged at 1500 × g for 10 minutes
to remove precipitated proteins and the decanted supernatant was evaporated under a
stream of dry N2. After acidification to a pH of 4.0 (by the addition of 30% acetic acid)
the sample was purified using a commercially available affinity column according to the
supplier’s instructions. Samples and standards were then analyzed in duplicates for total
8-isoprostane content using a commercially available enzyme immunoassay kit (Cayman
Chemicals, Ann Arbor, MI, USA).
5.7 iii Enzyme-linked immunosorbent assay (ELISA) analysis
VEGF-R2 receptor concentrations in lung homogenates were quantified by
ELISA using commercially available kit (R&D Systems, Minneapolis, MN).
5.7 iv Western blot analyses
For preproendothelin-1 (Pierce Biotechnology, Rockford, IL, USA) and α-smooth
muscle actin, membranes were incubated overnight at 4 °C with 1:1500 and 1:4000
dilutions respectively. For platelet-derived growth factor receptor-α (PDGFR-α) antibody
(Santa Cruz, CA, USA), a dilution of 1:200 was used for incubation of membranes.
87
5.8 Results
5.8 i Effects of FeTPPS on lung and body weights and post-fixation lung volumes
As shown in Table 4, exposure to 60% O2 for 14 d resulted in a small, but
statistically significant, reduction in lung weights which was attenuated by concurrent
injections of FeTPPS. Neither 60% O2 nor FeTPPS had significant effects on body
weights, the lung-to-body weight ratios or postfixation lung volumes.
5.8 ii Effects of FeTPPS on tyrosine nitration
Tissue immunoreactivity for nitrotyrosine, including the tips of secondary septa,
appeared markedly increased in rat pups exposed to 60% O2 for 14 d (Fig. 5.1A ), which
appeared to be attenuated by concurrent treatment with FeTPPS. When nitrotyrosine
immunoreactivity was quantified by Western analysis (Fig. 5.1B), the major nitrated
protein band was present at 57 kDa, as previously observed [213], which was used for
densitometry. Analysis of group differences revealed a significant increase in
nitrotyrosine content following exposure to 60% O2, which was attenuated by concurrent
treatment with FeTPPS.
88
Table 4
Lung (LW) and body (BW) weights, and post-fixation displacement lung volumes (LV)in
14-day-old rat pups exposed to air or 60% O2 ± FeTPPS or vehicle
Parameter
Air + vehicle Air + FeTPPS 60% O2 + vehicle 60% O2 + FeTPPS
LW (mg)
478 ± 8 475 ± 3 445 ± 12 * 473 ± 3
BW (gm)
29.7 ± 0.4 29.0 ± 0.2 28.4 ± 0.6 28.6 ± 0.2
LW/BW × 102
1.61 ± 0.04 1.64 ± 0.01 1.57 ± 0.04 1.66 ± 0.02
LV (μl)
1188 ± 8 1155 ± 30 1100 ± 27 1120 ± 14
Data are presented as means ± SEM (n=4 pups from different litters per group).
* P < 0.05; significantly different by 2-way ANOVA from the matching air group.
89
a:v
a:f
o:v
o:f
A BNitrotyrosine 66 kDa
a:v a:f o:v o:f
GAPDH
a:v a:f o:v o:f
Nitr
otyr
osin
eco
nten
tno
rmal
ized
toG
APD
H
0.0
0.4
0.8
1.2
1.6
*
37 kDa
Fig. 5.1. Effect of FeTPPS on 60% O2-induced nitrotyrosine formation. Rat pups were
exposed for 14 days to air (a) and received daily s.c. injections of saline vehicle (a:v) or
FeTPPS (a:f) or were exposed to 60% O2 (o) and received daily s.c. injections of saline
vehicle (o:v) or FeTPPS (o:f). (A) Exposure to 60% O2 resulted in marked thickening of
the lung parenchyma and intense nitrotyrosine immunoreactivity, both of which appeared
to be attenuated by treatment with FeTPPS. Secondary crests displayed increased
nitrotyrosine immunoreactivity (inset). Scale bar, 100 μm. (B) Western blots confirmed
that the significant increase in nitrotyrosine immunoreactivity induced by 60% O2 was
attenuated by treatment with FeTPPS. Representative blots are shown. *Significantly
different (P < 0.05) by 2-way ANOVA from the a:v and o:f groups. Values are means ±
SEM for pooled homogenates from 4 litters.
90
5.8 iii Protective effects of FeTPPS on the pulmonary vascular bed
Vessels were identified by staining tissue for elastin (Fig. 5.2A). When vessels of
20-65 μm diameter that had both inner and outer elastin lamina were counted and
analyzed, it revealed a significant decrease in vessel density following exposure to 60%
O2, which was attenuated by concurrent treatment with FeTPPS (Fig. 5.2B ). The lung
contents of the regulators of vessel growth VEGF, VEGF-R2 and Ang-1 were measured.
Exposure to 60% O2 caused a reduction in the lung content of VEGF, which was not
significantly influenced by concurrent treatment with FeTPPS (Fig. 5.3A). Analysis of
group differences revealed a significant decrease in VEGF-R2 content following
exposure to 60% O2, which was attenuated by concurrent treatment with FeTPPS (Fig.
5.3B). Analysis of group differences (Fig. 5.3C) revealed a significant decrease in Ang-1
content following exposure to 60% O2, which was attenuated by concurrent treatment
with FeTPPS. Ang-1 values for the FeTPPS-treated and 60% O2-exposed group were
significantly greater than values for the air-exposed and vehicle-treated control group.
As a well-recognized index of PHT, I calculated percent medial wall thickness for
vessels with the characteristics described above. Analysis of group differences revealed a
significant increase in medial wall thickness following exposure to 60% O2, which was
attenuated by concurrent treatment with FeTPPS (Fig. 5.4A). Because PHT in this model
is thought to be mediated by 8-isoprostane acting through the thromboxane A2 receptor
[190] and by down-stream up-regulation of endothelin-1 [240], I also measured 8-
91
a:v
a:f
o:v
o:f
A B
a:v a:f o:v o:f
Peri
pher
alve
ssel
s/m
m2
0
5
10
15
20
*
Fig. 5.2. Effect of FeTPPS on the 60% O2-mediated reduction in peripheral vessel
density. Rat pups were exposed for 14 days to air (a) and received daily s.c. injections of
saline vehicle (a:v) or FeTPPS (a:f) or were exposed to 60% O2 (o) and received daily s.c.
injections of saline vehicle (o:v) or FeTPPS (o:f). (A) Exposure to 60% O2 resulted in an
apparent reduction in peripheral vessel density, as assessed by elastin staining, which was
restored by treatment with FeTPPS. Small vessels are identified by arrows. Scale bar, 200
μm. (B) Counting of vessels confirmed that 60% O2 reduced peripheral vessel numbers,
which was attenuated by treatment with FeTPPS. *Significantly different (P < 0.05) by 2-
way ANOVA from the a:v and o:f groups. Values are means ± SEM for 4 average-sized
pups from 4 different litters.
92
isoprostane and preproendothelin-1 contents. Analysis of group differences revealed
significant increases in 8-isoprostane (Fig. 5.4B) and preproendothelin-1 (Fig. 5.4C)
following exposure to 60% O2, which were attenuated (8-isoprostane) or partially
attenuated (preproendothelin-1) by concurrent treatment with FeTPPS. Preproendothelin-
1 values for the group exposed to 60% O2 and treated with FeTPPS remained
significantly greater than values for the air-exposed and vehicle-treated control group, but
were significantly less than values for the 60% O2-exposed and FeTPPS-treated group.
5.8 iv Protective effects of FeTPPS on alveolar development
Because I had observed an apparent effect of FeTPPS on the lung parenchymal
tissue thickening induced by 60% O2, (Fig. 5.1A), I quantified this thickening as the
tissue fraction. Analysis of values for the tissue fraction revealed a significant increase in
tissue fraction following exposure to 60% O2, which was attenuated by concurrent
treatment with FeTPPS (Fig. 5.5A). Analysis of values for the mean linear intercept, an
index of alveolar diameter, as previously reported, showed no significant change in mean
linear intercept following exposure to 60% O2 (Fig. 5.5B). However, 60% O2 is known to
induce a marked heterogeneity in mean linear intercepts within different areas on a lung
section [161, 246]. Mean linear intercept heterogeneity was also assessed in this study.
Analysis of group differences revealed a significant increase in mean linear intercept
heterogeneity following exposure to 60% O2, which was attenuated by concurrent
treatment with FeTPPS (Fig. 5.5C). Treatment of 60% O2-exposed pups with FeTPPS
resulted in mean linear intercept values less than those observed in air-exposed control
pups (Fig. 5.5B), consistent with enhanced alveolar formation. Low-power
93
A
a:v a:f o:v o:f
VE
GF-
A(%
con
trol
)
a:v a:f o:v o:f
VE
GF-
R2
(% c
ontr
ol) B
a:v a:f o:v o:f
Ang
-1(%
con
trol
)
0
*
#C†
0
100
150
50
0
100
150
50
*100
200
Fig. 5.3. Effect of FeTPPS on 60% O2-induced changes in lung content of mediators of
angiogenesis. Rat pups were exposed for 14 days to air (a) and received daily s.c.
injections of saline vehicle (a:v) or FeTPPS (a:f) or were exposed to 60% O2 (o) and
received daily s.c. injections of saline vehicle (o:v) or FeTPPS (o:f). Exposure to 60% O2
resulted in significant reductions in (A) VEGF-A, (B) VEGF-R2, and (C) Ang-1 lung
tissue content, which was attenuated by treatment with FeTPPS. †Groups are
significantly different (P < 0.05) by 2-way ANOVA. *Significantly different (P < 0.05)
from a:v and o:f group by 2-way ANOVA. #Significantly different (P < 0.05) from all
other groups by 1-way ANOVA. Values are means ± SEM for pooled homogenates from
4 litters.
94
A
a:v a:f o:v o:fTot
al8-
isopr
osta
ne (p
g/m
g pr
otei
n)
0
200
400
600
800 *
B PreproET-1 24 kDa
a:v a:f o:v o:f
GAPDH 37 kDa
a:v a:f o:v o:f
Prep
roen
doth
elin
-1co
nten
tno
rmal
ized
toG
APD
H
0.0
0.5
1.0
1.5 *
C
a:v a:f o:v o:f
%M
edia
lwal
lthi
ckne
ssof
vess
els2
0-65
μm o
.d.
0
5
10
15
20
25
*#
Fig. 5.4. Effects of FeTPPS on markers and mediators of 60% O2-induced pulmonary
hypertension. Rat pups were exposed for 14 days to air (a) and received daily s.c.
injections of saline vehicle (a:v) or FeTPPS (a:f) or were exposed to 60% O2 (o) and
received daily sc injections of saline vehicle (o:v) or FeTPPS (o:f). (A) Pulmonary artery
medial wall thickness, (B) lung 8-isoprostane content, and (C) preproendothelin-1
content were all increased by exposure to 60% O2. These increases were attenuated by
treatment with FeTPPS, though this attenuation was only partial for preproendothelin-1.
Representative blots are shown. *Significantly different (P < 0.05) from a:v and o:f
groups by 2-way ANOVA. #Significantly different (P < 0.05) from all other groups by 1-
way ANOVA. Values are means ± SEM for 4 average-sized pups from different litters or
for pooled homogenates from 4 litters. PreproET-1 = Preproendothelin-1.
95
#
B C
a:v a:f o:v o:f
Tiss
uefr
actio
n
0.0
0.1
0.2
0.3
0.4
A
a:v a:f o:v o:f
Mea
nlin
ear
inte
rcep
ts
0
20
40
60
80
100
a:v a:f o:v o:f
Mea
nlin
ear
inte
rcep
t var
ianc
e
0
1
2
3
4
**#
Fig. 5.5. Effect of FeTPPS on 60% O2-induced changes in lung structure. Rat pups were
exposed for 14 days to air (a) and received daily s.c. injections of saline vehicle (a:v) or
FeTPPS (a:f) or were exposed to 60% O2 (o) and received daily s.c. injections of saline
vehicle (o:v) or FeTPPS (o:f). (A) Lung parenchymal thickening after exposure to 60%
O2, and attenuation by treatment with FeTPPS, was confirmed by calculating the tissue
fraction. (B) Mean linear intercept was significantly reduced in the group receiving both
60% O2 and FeTPPS. (C) Exposure to 60% O2 resulted in a significant heterogeneity of
mean linear intercepts between fields in individual tissue sections, which was attenuated
by treatment with FeTPPS. *Significantly different (P < 0.05) from a:v and o:f groups by
2-way ANOVA. #Significantly different (P < 0.05) from all other groups by 1-way
ANOVA. Values are means ± SEM for 4 average-sized pups from 4 different litters.
96
photomicrographs of peripheral lung tissue were consistent with this interpretation (Fig.
5.6). These results suggested that FeTPPS was having an impact on not just pulmonary
vascular development, but also on postnatal alveolarization. Lung tissue was therefore
subjected to additional morphometric analyses. Analysis of values for both secondary
crest density and secondary crest/tissue ratio was performed. Analysis of group
differences revealed significant decreases in both secondary crest density (Fig. 5.7A) and
secondary crest/tissue ratio (Fig. 5.7B) following exposure to 60% O2, which were
attenuated by concurrent treatment with FeTPPS. The estimated alveolar surface area for
the group that had received FeTPPS and was exposed to 60% O2 was significantly greater
than all other groups (Fig. 5.7C). The estimated total alveolar number for the group that
had received FeTPPS and was exposed to 60% O2 was significantly greater than all other
groups (Fig. 5.7D).
5.8 v Effects of FeTPPS on phagocyte influx
Analysis of values for both neutrophil (Fig. 5.8A) and macrophage (Fig. 5.8B)
densities revealed a significant increase following exposure to 60% O2, which remained
unaffected in the group that had received FeTPPS and was exposed to 60% O2. See
colour images of myeloperoxidase (Fig. 5.9) and CD-68 (Fig. 5.10) immunostaining used
for counts.
97
a:v
a:f
o:v
o:f
Fig. 5.6. Effects of FeTPPS on alveolar diameter. Rat pups were exposed for 14 days to
air (a) and received daily s.c. injections of saline vehicle (a:v) or FeTPPS (a:f) or were
exposed to 60% O2 (o) and received daily s.c. injections of saline vehicle (o:v) or
FeTPPS (o:f). Low-power photomicrographs to illustrate: (1) the heterogeneity in
alveolar/saccular diameters observed in O2-exposed (o:v) group; (2) enhanced
alveologenesis with reduced diameters in the FeTPPS-treated and O2-
exposed (o:f) group. Scale bar = 200 μm
98
a:v a:f o:v o:f
Seco
ndar
ycr
ests
/mm
2
0
50
100
150
200
#
B
DC
A
a:v a:f o:v o:f
Seco
ndar
ycr
ests
/tiss
uera
tio
0.00
0.02
0.04
0.06
0.08
0.10
#
a:v a:f o:v o:f
Alv
eola
r su
rfac
e ar
ea(1
0-1 m2 /c
m3 )
0.00
0.02
0.04
0.06
0.08
*
a:v a:f o:v o:f
Tota
lalv
eoli
inw
hole
lung
(x10
-6)
0
1
2
3
4
5
*
Fig. 5.7. Effects of FeTPPS on 60% O2-mediated changes in alveologenesis. Rat pups
were exposed for 14 days to air (a) and received daily s.c. injections of saline vehicle
(a:v) or FeTPPS (a:f) or were exposed to 60% O2 (o) and received daily s.c. injections of
saline vehicle (o:v) or FeTPPS (o:f). Exposure to 60% O2 reduced both (A) secondary
crest density per unit area and (B) the secondary crest-to tissue ratio, which were restored
by treatment with FeTPPS. Concurrent exposure to 60% O2 and treatment with FeTPPS
resulted in an increase in both (C) alveolar surface area and (D) total alveolar number.
*Significantly different (P < 0.05) from a:v and o:f groups by 2-way ANOVA.
99
#Significantly different (P < 0.05) from all other groups by 1-way ANOVA. Values are
means ± SEM for 4 average-sized pups from 4 different litters.
100
A B
a:v a:f o:v o:f
Mye
lope
roxi
dase
-pos
itive
cells
/mm
2
0
40
80
120
160
a:v a:f o:v o:f
CD
-68-
posi
tive
cells
/mm
2
0
40
60
80
100
120
20
††
Fig. 5.8. Lack of effect of FeTPPS on 60% O2-mediated phagocyte influx. Rat pups were
exposed for 14 days to air (a) and received daily s.c. injections of saline vehicle (a:v) or
FeTPPS (a:f) or were exposed to 60% O2 (o) and received daily s.c. injections of saline
vehicle (o:v) or FeTPPS (o:f). (A) Counting neutrophil densities confirmed that 60% O2
increased neutrophil influx with or without treatment with FeTPPS. (B) Counting
macrophage densities confirmed that 60% O2 increased macrophage influx with or
without treatment with FeTPPS. †Groups are significantly different (P < 0.05) by 2-way
ANOVA. Values are means ± SEM for 4 average-sized pups from 4 different litters.
101
a:v
a:f
o:v
o:f
Fig. 5.9. Effects of FeTPPS on neutrophils. Rat pups were exposed for 14 days to air (a)
and received daily s.c. injections of saline vehicle (a:v) or FeTPPS (a:f) or were exposed
to 60% O2 (o) and received daily s.c. injections of saline vehicle (o:v) or FeTPPS (o:f).
Exposure to 60% O2 resulted in an influx of neutrophils, as assessed by myeloperoxidase
immunoreactivity (black stain) which was not apparently affected by treatment with
FeTPPS. Scale bar = 100 μm.
102
a:v
a:f
o:v
o:f
Fig. 5.10. Effects of FeTPPS on macrophages. Rat pups were exposed for 14 days to air
(a) and received daily s.c. injections of saline vehicle (a:v) or FeTPPS (a:f) or were
exposed to 60% O2 (o) and received daily s.c. injections of saline vehicle (o:v) or
FeTPPS (o:f). Exposure to 60% O2 resulted in an influx of macrophages, as assessed by
CD-68 immunoreactivity (brown stain) which was not apparently affected by treatment
with FeTPPS. Scale bar = 100 μm.
103
5.8 vi Effects of FeTPPS on total and nitrated α-smooth muscle actin
Analysis of values for α-smooth muscle actin revealed a significant increase in
total α-smooth muscle actin (Fig. 5.11 A) following exposure to 60% O2, which was
attenuated by concurrent injections of FeTPPS. A significant increase in nitrated α-
smooth muscle actin (Fig. 5.11 B) following exposure to 60% O2 was attenuated by
concurrent injections of FeTPPS. Treatment with FeTPPS also caused a significant
reduction in nitrated α-smooth muscle actin in air-exposed animals.
5.8 vii Effects of FeTPPS on nitrated PDGFR-α
As a pilot project to determine if FeTPPS protected against nitration of critical
growth factor/growth factor receptor regulators of alveologenesis, nitrated proteins from
lung lysates were immunoprecipitated, then probed for immunoreactive PDGFR-α (Fig.
5.12). There was a significant increase in nitrated PDGFR-α content following exposure
to 60% O2, which was attenuated by concurrent treatment with FeTPPS.
5.9 Discussion
Consistent with previous observations [213, 246, 247], 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 [257]. Based on the literature, this
effect seems to be specific, and I, like others, have observed no apparent adverse side-
effects. It has been previously reported that the PHT observed in this model of chronic
104
a:v a:f o:v o:f0.0
0.4
0.8
1.2
1.6
Nitr
ated
α-s
moo
thm
uscl
eac
tin
Nitratedα-SMA 42 kDa
a:v a:f o:v o:f
Ponceau
A Bα−SMA
GAPDH
a:v a:f o:v o:f
α-s
moo
thm
uscl
eac
tinno
rmal
ized
toG
APD
H
0.0
0.4
0.8
1.2
1.6
*
a:v a:f o:v o:f
42 kDa
37 kDa
*§
Fig. 5.11 Effect of FeTPPS on 60% O2-induced changes in total and nitrated lung α-
smooth muscle actin content. Rat pups were exposed for 14 days to air (a) and received
daily s.c. injections of saline vehicle (a:v) or FeTPPS (a:f) or were exposed to 60% O2 (o)
and received daily s.c. injections of saline vehicle (o:v) or FeTPPS (o:f). (A) Exposure to
60% O2 resulted in an increase in total α-smooth muscle actin. (B) Nitrated
immunoprecipitated α-smooth muscle actin was decreased in air-exposed pups that had
received FeTPPS and was increased by exposure to 60% O2. The latter was attenuated by
treatment with FeTPPS. *Significantly different (P < 0.05) from a:v and o:f groups by 2-
way ANOVA. §Significantly different (P < 0.05) from the a:v group by 2-way ANOVA.
Values are means ± SEM for pooled homogenates from 4 litters.
105
PDGFR-α 170 kDa
a:v a:f o:v o:f
Nitr
ated
PDG
FR-α
con
tent
0
10
20
30
40
*
a:v a:f o:v o:f
Fig. 5.12. Effect of FeTPPS on 60% O2-induced nitration of PDGFR-α. Rat pups were
exposed for 14 days to air (a) and received daily s.c. injections of saline vehicle (a:v) or
FeTPPS (a:f) or were exposed to 60% O2 (o) and received daily s.c. injections of saline
vehicle (o:v) or FeTPPS (o:f). Exposure to 60% O2 resulted in an increase in nitrated
immunoprecipitable PDGFR-α, which was attenuated in 60% O2-exposed pups that had
received FeTPPS. Representative blots are shown. *Significantly different (P < 0.05)
from a:v and o:f groups by 2-way ANOVA. Values are means ± SEM for pooled
homogenates from 3 litters.
106
neonatal lung injury is mediated by increases in both 8-isoprostane and its downstream
regulator of vascular tone, endothelin-1 [190, 212]. The attenuation of the 60% O2-
induced increases in 8- isoprostane and preproendothelin-1 by FeTPPS is entirely
consistent with a pivotal role for peroxynitrite-derived hydroxyl radical-induced 8-
isoprostane formation [259-261].
Vessel formation in the lung is mediated by a number of growth regulating
factors, including VEGF, its receptor VEGF-R2, and Ang-1 [262]. I have previously
reported reduced lung contents of both VEGF and Ang-1 after exposure to 60% O2 [246].
This was again observed, along with a reduced VEGF-R2 content. The reduced contents
of all three growth regulators induced by 60% O2 were reversed by treatment with
FeTPPS. Peroxynitrite under some conditions may stimulate VEGF expression [263], and
it is recognized to be a normal component of the VEGF signaling pathway [264].
However, peroxynitrite has also been shown by others to reduce VEGF synthesis under
hyperoxic conditions [265]. I had originally speculated that sustained vasoconstriction of
the pulmonary vessels in response to peroxynitrite [204] could contribute to the vascular
pruning observed after exposure to 60% O2. To this must be added an effect of
peroxynitrite on lung growth factor content. In addition, peroxynitrite may interfere with
the VEGF signaling pathway through nitration of PI 3-kinase [266]. VEGF-mediated
vessel formation is also essential for normal alveologenesis [28], and peroxynitrite-
mediated reduced tissue concentrations of VEGF, VEGFR2, and Ang-1 may explain the
observed impairment of alveologenesis, which was restored by treatment with FeTPPS.
The reduced lung contents of these angiogenic growth factors probably reflect their
down-regulation by 60% O2, rather than their inactivation, suggesting a possible effect of
107
nitration or the hydroxyl radical on an upstream regulator. Addressing this issue awaits
characterization of such upstream regulators in the postnatal lung.
The observations that concurrent treatment with FeTPPS and 60% O2 resulted in a
reduced mean linear intercept and mean linear intercept variance, and an increased
alveolar surface area and total alveolar number, are in keeping with previous studies in
which other interventions reduced both phagocyte influx and tissue nitration [161, 246,
247]. This suggests that the factor impairing alveolar development after exposure to 60%
O2, both in this study and in the cited previous studies, was peroxynitrite or a
peroxynitrite-derived factor. The mechanism by which peroxynitrite impairs alveolar
formation is uncertain. One mechanism alluded to above is reduced VEGF and Ang-1
pathway signaling. An alternative, or additional, explanation is that peroxynitrite could
impair alveolar formation by the nitration of α-smooth muscle actin. Secondary crest
formation requires that fibroblasts at sites of future secondary crest formation become α-
smooth muscle-containing myofibroblasts, which is under the control of the growth factor
PDGF-AA [25]. Not only is there a reduction in immunoreactive PDGF-AA after
exposure to 60% O2 [26], but nitration of α-smooth muscle actin may prevent
myofibroblasts from functioning normally in secondary crest formation. My pilot results
also suggest the possibility that the PDGF pathway could be disrupted through nitration
of its receptor PDGFR-α, though confirmation that nitration of PDGFR-α causes a loss of
function is required. It is also possible that reducing peroxynitrite concentrations is
protective against lung injury by limiting nitration of antioxidant enzymes and its
attendant reductions in enzyme activity [267, 268].
108
The mechanism or mechanisms have yet to be defined by which 60% O2, in the
presence of agents that suppress phagocyte influx [161, 246, 247], increases alveolar
formation and, in this study, the expression of Ang-1. Before this study, it was assumed
that the underlying mechanism relates to the concentration of reactive oxygen species
(ROS) to which lung cells are exposed. Superoxide, for example, can stimulate DNA
synthesis by lung cells at low concentrations and inhibit it at high concentrations [269]. It
seemed possible that once the dominant phagocyte-derived contribution to ROS in the
60% O2-exposed lung [161] was removed, the residual mitochondria-derived ROS, which
are increased by exposure to hyperoxic conditions [270], achieved a concentration in the
range that stimulates DNA synthesis. The results of this study suggest that such a
mechanism is unlikely. FeTPPS did not affect the phagocyte influx induced by 60% O2
and would not be expected to directly affect ROS formation by phagocytes.
As alluded to above, the FeTPPS-mediated protection against the development of
PHT could be explained by a pivotal role for peroxynitrite-derived hydroxyl radical-
induced 8-isoprostane formation. That FeTPPS protects against 60% O2-mediated
impairment of alveologenesis could also be the basis of its preventive effects on PHT and
vascular pruning. It is widely believed that capillary development is inextricably linked to
alveolar development and that impaired vascular development, results in impaired
alveolar development, with a likely critical central role for VEGF/VEGF-R2 [271].
Consistent with this concept are such observations as protection against hyperoxia-
induced impaired alveolar formation by the phosphodiesterase-5 inhibitor, sildenafil,
which restores VEGF-R2 [272, 273]. Peroxynitrite may also impair alveologenesis
through a peroxynitrite-mediated synthesis of TGF-β by lung fibroblasts [274]. Along
109
with the recent observation that Akt activation protects against oxidant-mediated
impairment of alveolar development [275], these findings suggest a possible mechanism
by which FeTPPS could protect against 60% O2-mediated impairment of alveolar
formation. The nitrates formed by FeTPPS could release nitric oxide to increase cGMP
with a resultant downstream increase in Akt and thus account for the observed restoration
of VEGF-R2.
My observations suggest a much more significant role for peroxynitrite in CNLI
than I had initially anticipated. It must be acknowledged that this study does not
distinguish between the beneficial effects exerted by FeTPPS-mediated inhibition of
nitration and the beneficial effects of FeTPPS-induced nitrate formation [276].
Particularly intriguing was the observation that the phagocyte influx in response to 60%
O2 was not associated with any detected adverse effects, other than those mediated by
peroxynitrite. Perhaps protection against peroxynitrite-mediated antioxidant enzyme
inactivation [267, 268] by FeTPPS could contribute.
Although one must be careful about extrapolating mechanisms from an animal
model to the human situation, a number of the above findings in the animal model are
also observed in the human infant developing CNLI. Apart from the lung parenchymal
thickening, the impaired alveolarization, and the PHT in infants with established CNLI,
plasma 8-isoprostane is elevated in those infants destined to develop CNLI [277], as is
endothelin-1 in their bronchoalveolar lavage fluids [278]. Plasma 3-nitrotyrosine content
is increased in the first month of life in human infants developing CNLI [172], consistent
with sustained peroxynitrite-mediated oxidative stress contributing to the development of
the lung pathology [173]. They also have reduced levels of VEGF in their
110
bronchoalveolar lavage fluid [279]. Taken together, the above findings strongly suggest
that the role of peroxynitrite in the development of CNLI should be further explored.
111
Chapter 6
Future directions and preliminary findings
112
6.1 Future directions
My studies, as outlined above, have reiterated the critical role that macrophages
play in the development of the CNLI seen in neonatal rats exposed to 60% O2. An as yet
unresolved question is the identity of the macrophage chemotaxin(s) responsible for their
influx.
Neutrophil elastase activity has been considered to be important in the
development of “new” BPD [280], with reduced elastase inhibitory capacity in infants
that developed BPD [101]. In a cohort study, tracheal lavage fluid of infants that later
developed BPD, showed higher neutrophil counts and elastase activity [281]. An anti-
neutrophil-elastase approach has already been used in the treatment of acute lung injury
[282, 283], septic acute respiratory distress syndrome [284] and bacterial pneumonia with
systemic inflammatory response syndrome [285]. My findings, in tandem with those of
others in this model of CNLI, logically point towards a role played by a chemotactic
stimulus for macrophage influx. The well recognized macrophage chemotaxins MCP-1,
MIP-1α and MIP-1β are not increased in the neonatal rat model of CNLI [247]. This led
me to speculate that elastin fragments may be the macrophage chemotaxin involved, as
has been observed in a murine model of emphysema [286]. This can be tested by the use
of a neutrophil elastase inhibitor in the 60% O2 model of CNLI. I have conducted a pilot
study using the neutrophil elastase inhibitor, elafin, to determine an effective dose that
prevents macrophage influx. An initial time point of 6 days was designed to capture
initial stage of macrophage influx and the same blocks were also used for Hart’s staining.
However, this will be a project on 14-day model. In future experiments I plan to verify
elafin’s effect by using an EnzCheck elastase assay kit (Invitrogen). However, this and
113
the following experiments are beyond the scope of the present thesis. Following the
exposure of neonatal pups to air or 60% O2 for 14d ± daily elafin or vehicle, lung tissue
thickening, secondary crest formation with BrdU-labelling (to assess for proliferating
cells), alveolar numbers, vessel density and medial wall thickness would be assessed.
Inflammatory cells’ densities would be assessed by direct counts of immunostained
macrophages and neutrophils. Post-fixation lung volumes, lung weights, body weights
and lung weight/body weight would be measured. Protein nitration would be assessed by
immunohistochemistry and Western blot. Since neutrophil elastase also cleaves VEGF
[287], quantification of VEGF, VEGF-R2 and Ang-1 content in lung homogenates would
be performed by ELISA. Elastin fragments would be assessed by competitive ELISA
[288], and a comparison with immunohistochemistry for elastin, and western blots for α-
elastin and tropoelastin, be performed. Elastin fibre density would be quantified by
threshold analysis of Hart’s stained tissue sections [289]. Based on previous results from
blocking macrophage influx [213] and my current findings demonstrating prevention of
60% O2-exposure-associated protein nitration by a peroxynitrite decomposition catalyst
(FeTPPS), it is anticipated that inhibition of neutrophil elastase will prevent macrophage
influx as well as protein nitration. It will also offer protection against impaired alveolar
development, pulmonary vessel pruning and PHT. Elafin might also have a direct
independent effect on PHT [290]. Its likely target epitope is in the family of
hexapeptides, VGVAPG [286], which binds to the cell surface elastin receptor, and can
be neutralized with a commercially available antibody BA4 [291] to confirm these
findings.
114
6.2 Clinical relevance
As previously reported, [189] in the 60%-O2-model of CNLI, a non-specific
antiprotease (α1-antitrypsin) was protective against the pulmonary vascular and
parenchymal injury, perhaps due to an anti-elastase effect. To determine whether the
protective effect was indeed due to elastase inhibition would require additional studies
with a specific anti-elastase such as elafin. However, in a clinical trial, α1-antitrypsin did
not protect human infants from developing BPD [292]. This could be explained by
elastase not playing a critical role, but was more likely due to inadequate dosing, based
on the pharmacokinetics of α1-antitrypsin in the preterm human [293]. Therefore anti-
elastase therapy remains worthy of continued investigation as a protective therapy against
BPD.
6.3 An elastase inhibitor prevents 60% O2-mediated macrophage influx in neonatal
rats.
6.4 Aims/objectives
To study the effect of elafin, a neutrophil elastase inhibitor, on 60% O2-induced
chronic neonatal lung injury.
6.5 Hypothesis
Neutrophils mediate PHT and parenchymal injury by inducing macrophage
chemotaxis through generation of macrophage-chemotactic elastin fragments by
neutrophil elastase. Macrophage depletion prevents PHT as well as protein nitration.
115
Since macrophage-derived peroxynitrite mediates both PHT and impaired alveologenesis
in a rat model of hyperoxia-induced CNLI, a neutrophil elastase inhibitor has the
potential to completely prevent this injury by preventing macrophage influx and
peroxynitrite formation.
6.6 Elafin
Elafin is a 6-kD naturally-occurring specific inhibitor of neutrophil elastase, a
serine protease synthesized in the neutrophils and stored in the azurophilic granules.
Elafin has been identified in bronchial secretions [294], skin [295], intestinal epithelium
of the large intestine [296] and endometrium during menstruation [297]. It is secreted
predominantly at mucosal sites and is shown to be modulated in multiple pathological
conditions suggesting that it has a role in the adaptive immune system [298].
6.7 Specific materials and experimental procedures
6.7 i In vivo interventions
Rat pups (10–12 per litter) were exposed to air or 60% O2 in paired chambers for
up to 6 days. Half the litters in 60% O2 were injected with 2, 4, 6 or 8 mg/kg s.c. elafin on
a daily basis.
6.8 Preliminary results
6.8 i Elafin prevented the 60% O2-induced macrophage induction
Unpublished observations from Dr. Tanswell’s laboratory had demonstrated a
marked reduction in macrophage counts upon blocking the neutrophil influx with a
116
CXCR2 receptor antagonist. A rational approach was to assess for elafin’s effects, if any,
on the counts of macrophages in 60% O2-exposed lungs. Analysis for values of
macrophage densities revealed that at 8 mg/kg/s.c. dose, elafin had completely (while 2
and 4 mg/kg/s.c. dose had partially) attenuated the significant increase in macrophage
densities in 60% O2-exposed pups (Fig. 6.1).
6.8 ii Elafin partially reversed the reduced lung elastin content in 60% O2-exposed
pups
Because I had anticipated that the use of a neutrophil elastase inhibitor would
prevent the degradation of elastin in 60% O2-exposed pups, a logical approach was to
next evaluate the elastin content in lungs of pups that received daily injections of Elafin
in the presence of 60% O2. As anticipated, visual observation of Hart’s elastin-stain
sections, suggested that daily injections of elafin had partially reversed the reduced
elastin content in the lungs of 60% O2-exposed pups (Fig. 6.2).
6.9 Discussion
After having developed preliminary evidence consistent with a role for
neutrophil-derived elastase in initiating the macrophage influx, which in turn is required
for peroxynitrite generation to cause 60% O2-mediated CNLI, a logical approach is to
study whether therapeutic elafin will prevent the lung injury in this model. Quantitation
of the tissue fraction to assess for parenchymal thickening, followed by lung
morphometric analysis to assess alveolar and pulmonary vascular development, and the
117
Elafin (mg/kg/d s.c.)
0 0 2 4 8
Mac
roph
ages
/fiel
d
0
2
4
6
8
10 ** *
Air
60% Oxygen
# #
Fig. 6.1. Preliminary data to assess the effect of s.c. elafin once daily for 6 days on lung
macrophage accumulation in 60% O2. *Significantly different (P < 0.05) from air group
by 1-way ANOVA. #Significantly (P < 0.05) different from elafin at both 0 and 8
mg/kg/d by 1-way ANOVA. Values are from 10 images/section and 4 sections from 2
animals.
118
Air 60% O2 60% O2 + 8mg/kg/d
Fig. 6.2. Hart’s stain of day-6 lung tissue, after subtraction of background, to show
elastin in pups exposed to air or to 60% O2 ± s.c. elafin 8 mg/kg/d. The loss of elastin in
60% O2 appears to be partially reversed in elafin-treated pups.
119
measurement of elastin fibre density by threshold analysis [289], will establish elafin’s
protective effect, if any. If the damaging pathway and the injury are prevented as a result
of this intervention, it would seem reasonable to pursue elafin as a therapeutic option
rather than hypercapnia or FeTPPS. Since elafin is a naturally occurring protein, and the
human recombinant protein is commercially available, this intervention reduces the risk
of toxicity.
The above data are preliminary in nature and require quantitation using an
adequate sample size. However, use of a neutrophil elastase inhibitor appears to reduce
elastin degradation. This would be expected to reduce the formation of elastin fragments
that I hypothesize are chemotactic for macrophages. The resultant preliminary
observation of a prevention of macrophage influx gives weight to this hypothesis and it is
hoped that, by preventing the influx of macrophages, elafin will provide the lung
complete protection against CNLI and its associated complications.
120
Chapter 7
Conclusions
121
7.1 Summary of findings.
My studies showed therapeutic hypercapnia to be completely protective against
60% O2-mediated parenchymal thickening. Not only did therapeutic hypercapnia
completely prevent 60% O2-mediated protein nitration (by preventing macrophage
influx) it also prevented PHT as evidenced by the prevention of medial (smooth muscle)
hypertrophy of pulmonary vessels. The small pulmonary vessel pruning caused by 60%
O2 was restored to normal as were the levels of VEGF-A and Ang-1, while enhanced
secondary crest and subsequent alveolar formation increased the estimated gas exchange
surface area.
The subsequent study to determine the role of peroxynitrite, the likely nitrating
agent in my first study, revealed a completely protective role by the peroxynitrite
decomposition catalyst, FeTPPS. In addition to the restoration of small pulmonary vessel
counts (and associated mediators of angiogenesis), FeTPPS also prevented the PHT. The
increases in total 8-isoprostane and preproendothelin-1 were attenuated by treatment with
FeTPPS, though this attenuation was only partial for preproendothelin-1. FeTPPS
normalized the tissue fraction as well as the secondary crest density. Total alveolar
number as well as alveolar surface area were significantly increased by FeTPPS. A lack
of effect on increased inflammatory cells suggested that FeTPPS attenuated an
inflammatory-cell-derived product (peroxynitrite), which was critical to injury. The
nitration of both PDGFR-α and α-smooth muscle actin was prevented by the intervention
with FeTPPS.
122
7.2 Relevance.
These two studies demonstrate the critical role that macrophages play in
generating lung injury through their contribution to peroxynitrite formation. The benefits
of preventing an influx of inflammatory cells (therapeutic hypercapnia), and/or averting
their adverse effects (peroxynitrite decomposition catalyst, elafin), do suggest that there
are promising potential therapeutic interventions for human infants developing CNLI.
7.3 Limitations of this study.
My first study, using therapeutic hypercapnia as a protective strategy against
CNLI, was the first of its kind to demonstrate hypercapnia-mediated inhibition of
inflammation, and prevention of the associated injury. However, level of hypercapnia that
was achieved surpassed the clinically accepted norms for neonatal practice. While
extreme levels of passive hypercapnia have been tolerated by adults [242, 243], such
levels are definitely not justifiable in developing neonates. Studying only one time point
(d-14) precluded me from specifically delineating the role and levels of the different
angiogenic factors involved in vascular growth. Additionally, I did not determine whether
a shorter duration, or a lesser degree, of hypercapnia might have yielded results similar to
those observed in my current studies. My use of therapeutic hypercapnia prevented
macrophage influx, and limited inflammation, but did not define whether these results
were direct consequences of therapeutic hypercapnia itself, or whether hypercapnic
acidosis might have been responsible.
Use of the peroxynitrite decomposition catalyst completely prevented the neonatal
lung injury, but I did not determine whether the beneficial effects were mediated by
123
inhibition of nitration, or by FeTPPS-induced nitrate formation. Although this study
identified that nitration of PDGFR-α by peroxynitrite was prevented by the use of
FeTPPS, it did not address other pertinent growth factors and their respective receptors
that might have been involved.
7.4 Current concepts of the pathways involved in 60%-O2-induced CNLI
Current concepts of the pathways involved in the CNLI induced in the 60%-O2-
model are shown in Fig. 7.1. Essentially, following exposure to 60% O2, distal lung
epithelial cells release interleukin-1 which in turn increases cytokine-induced neutrophil
chemoattractant (CINC-1), initiating a neutrophil influx. The ensuing increase in ROS
impairs alveologenesis by their effects on growth factors. Release of neutrophil elastase
causes elastin fragmentation. The resulting elastin fragments cause macrophage
chemotaxis. Macrophage influx results in PHT through sequential involvement of
peroxynitrite, hydroxyl radical mediated lipid peroxidation, 8-isoprostane and endothelin-
1. Peroxynitrite, at the same time, also impairs alveologenesis by several potential
mechanisms. Firstly, by nitrating growth factors and/or their associated receptors and
secondly, by increasing the production TGF-β, eventually leading to aberrant lung
growth. Because this model mimics the histological features of “new” BPD, and the
cascade of events that set off the oxidant injury involves an influx of neutrophils followed
by macrophages, a rational approach is to prevent the influx of macrophages by
eliminating chemotactic elastin fragments.
124
CINC-1
60% O2
Elas
tase
ROS GFs
F-Isoprostanes
ET-1
ONOO
IL-1
AberrantLung Growth
Elastin
ElastinFragments
PMNLINFLUX
AMINFLUX Pulmonary
Hypertension
NO .NO .
O2.-O2.-
ArachidonicAcid
O2.-O2.-
_
GF/GF-Rnitration
TGF-β
Fig. 7.1. Concepts of the pathways involved in the 60% O2-induced rat model of CNLI.
Reproduced by permission of Dr. K. Tanswell. AM = alveolar macrophages, CINC-1 =
cytokine-induced neutrophil chemoattractant-1, ET-1 = endothelin-1, GFs = growth
factors, GF-R = growth factor receptors, IL-1 = interleukin-1, NO• = nitric oxide,
ONOO¯ = peroxynitrite anion, O2•- = superoxide, PMNL = polymorphonuclear
leukocytes and ROS = reactive oxygen species.
125
7.5 Implications for CNLI
My studies raise an important question with respect to the use of therapeutic
hypercapnia in the management of CNLI. Was the target PaCO2 in the multicentre trials
too low to observe beneficial effects? Caution is advised in translating my entire findings
to the human infant, since the PaCO2 values that I used in my studies were significantly
higher than that those in routine practice in neonatology units. Still, future studies could
be devised aiming for less stringent levels that might still yield beneficial outcomes.
My study on the use of a peroxynitrite decomposition catalyst yielded exciting
results. The absolute protection provided by the peroxynitrite decomposition catalyst was
unanticipated, but begs the question of whether peroxynitrite has a similar and
unrecognized role in human infants. My findings, and the observation of plasma
nitrotyrosine in infants destined to develop CNLI, justifies further study of the
therapeutic potential for peroxynitrite decomposition catalysts in a larger animal model
such as the 125-day gestation baboon.
Both the studies demonstrated stimulated alveolar growth in the presence of 60%
O2 but only addressed the changes in the markers of angiogenesis. Probing other growth
factors in these experiments would have likely yielded enough information to propose
future experiments targeting these factors in this model of BPD.
7.6 Clinical implications
My study of therapeutic hypercapnia suggests that neonates are responsive to the
protective effects of hypercapnia. What remains to be determined is where the effective
threshold lies in human infants, and whether that threshold is at a level which can be
126
applied safely. The use of a peroxynitrite decomposition catalyst in clinical practice is far
from being applicable at this time. It does have the potential advantage over therapeutic
hypercapnia that it does not dampen phagocyte influx, but it remains to be seen whether
the phagocytes remain bacteriocidal. Neutrophil elastase inhibitors are already emerging
in trials related to adult lung injury. Their use in neonatal practice, and especially in the
prevention of development of CNLI in premature infants, is an exciting prospect.
127
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