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Growth of the vascular tree
PAEDIATRIC RESPIRATORY REVIEWS (2000) 1, 321–327 doi:10.1053/prrv.2000.0071, available online at http://www.idealibrary.com on
MINI-SYMPOSIUM: GROWTH OF THE LUNG
Growth of the vascular tree A. A. Hislop and C. M. Pierce 1
Vascular Biology and Pharmacology Unit, Institute of Child Health, 30 Guildford Street, London WC1N 1EH, Great Ormond Street Hospital, London WC1N 3JH, UK
2
KEYWORDS pulmonary circulation, development, pulmonary hypertension, endothelium, nitric oxide, endothelin.
Summary Major changes occur in the pulmonary circulation at birth. Failure of normal adaptation leads to pulmonary hypertension of the newborn.The basis for this may be abnormal growth of the lung in utero or a failure of the mechanisms which lead to vasodilatation in the newborn period.This article describes normal development of the pulmonary arteries and veins, their branching pattern and structure and discusses the factors which may control this growth. Endothelial function and dysfunction are crucial in adaptation, and new experimental studies are aiding our understanding of the control and action of factors produced by the endothelium, e.g. nitric oxide, prostacyclin and endothelin.The study of infants with pulmonary hypertension, together with the experimental studies, will aid in producing effective methods of controlling increased pulmonary artery pressure in the newborn period. © 2000 Harcourt Publishers Ltd
INTRODUCTION During fetal life little blood flows through the pulmonary circulation (8–10% of total cardiac output). Oxygenation of the blood occurs via the placenta, blood being diverted away from the lungs. This diversion is achieved by there being a high resistance in the fetal pulmonary circulation. Immediately after birth, with the onset of breathing, there is a drop in pulmonary artery resistance, allowing blood flow through the pulmonary circulation to increase 8–10fold. Failure of the pulmonary circulation to sustain this pressure drop leads to a failure of postnatal adaptation. The mechanisms involved in this failure are not fully understood, but there are several suggestions, some of which result from abnormal development of the blood vessels in utero. It is therefore important to understand normal development of the pulmonary circulation (Table 1).
NORMAL DEVELOPMENT Branching pattern of pulmonary arteries and veins The primary function of the lung is gas exchange and blood vessels are arranged to accommodate efficient gas Correspondence to: A. A. Hislop, Vascular Biology and Pharmacology Unit, Institute of Child Health, 30 Guildford Street, London WC1N 1EH, UK 1526–0550/00/040321 + 07 $35.00/0
Table 1 Timetable of normal blood vessel growth. ●
●
● ●
● ● ●
● ●
From 4–16 weeks arteries and veins develop by vasculogenesis from the mesenchyme around airway buds. Pre-acinar arteries and veins complete by 17 weeks’ gestation. Blood gas barrier established by 24 weeks’ gestation. Intra-acinar arteries and veins develop with the alveoli in late fetal life and up to 2 years of age. Increase in size of arteries and veins as lung grows. Arterial wall thickness high in fetal life. After birth rapid dilatation of pulmonary arteries and then wall thinning. Gradual extension of muscle during childhood. Innervation of pulmonary artery smooth muscle cells as they appear.
exchange by producing a large surface area within the alveolar region. Blood flow must be sufficient and at a linear velocity with the counter current to allow gas exchange to occur. The blood vessels also carry, produce and control the concentration of vasoactive mediators. During development a complex branching pattern of blood vessels develops from a single avascular bud and is connected to the heart at the right ventricle and at the left atrium. In the adult lung the arteries run alongside the airways and branch with them, suggesting that their growth might be regulated by the airways. In addition to these © 2000 Harcourt Publishers Ltd
322
conventional arteries alongside the airway branches there are extra, smaller “supernumerary” arteries, 2–3 times in number which supply the alveolar region more directly. These can carry up to 40% of total blood flow and increase in relative number towards the periphery of the lung. The veins have a similar number of branches both conventional and supernumerary, but they run independently of the airways between acini and segments. Early studies on the development of the arteries and veins showed that they grow at the same time as the airways, so that all pre-acinar vessels are present by the 17th week of gestation, the end of the pseudoglandular stage.1 The supernumerary arteries and veins which will be associated with the alveoli (not developed by this time) also grow during this period. This suggests a genetic influence on blood vessel development. The branching pattern in siblings is more similar to each other than to that of the general population, also suggesting a genetic influence on the branching pattern. In the canalicular stage (17–27 weeks’ gestation) further branching of the arteries and veins accompanies the development of the respiratory airways. At this time the capillaries in the mesenchyme come to lie under the epithelium, which is differentiating into Type I and II pneumonocytes. This produces a blood–gas barrier for gas exchange similar in thickness to that seen in the adult. Recent studies in man using specific antibodies have shown that the pulmonary blood vessels initially form de novo as endothelial tubes in the mesenchyme around the peripheral airways, the process of vasculogenesis, rather than by inward growth of existing arteries (angiogenesis).2 Initially endothelial cell precursors have been identified without a lumen in the splanchnic mesenchyme around the foregut and lung bud at 4 weeks’ gestation. Endothelial tubes then form and coalesce, and a circulation between the right ventricle and left atrium can be recognized in man by 5 weeks’ gestation when the lung has only one airway generation. As the airways divide towards the periphery, the intrapulmonary arteries form by continuous coalescence of endothelial tubes alongside the airway. The veins derive independently from the same mesenchyme and run between the airways. As alveoli form in late fetal life (after 30 weeks’ gestation) and after birth, there is a rapid increase in the number of small preand post-capillary vessels. After 2 years of age the lung grows by increasing the surface area of the alveoli and the area of the capillary bed increases alongside this growth. Between birth and adulthood the surface area of the lung increases about 20-fold and the capillary volume 35-fold to maintain efficient gas exchange. This increase may be by sprouting or alternatively by intussusception of tissue within an existing sheet, so increasing its surface area and complexity.3 At birth the overall pattern of the arteries and veins is the same pattern as in the adult (Fig. 1). The vessels increase in size with age as the lung increases in volume and the number and volume of vessels at the periphery increases.
A. A. HISLOP AND C. M. PIERCE
Figure 1 Post-mortem arteriogram of a fetus of 39 weeks’ gestation. The arteries have been injected with barium sulphate (mag × 0.75).
Structure of the pulmonary artery wall Smooth muscle cells are found around the newly formed arteries soon after they line up alongside the airways. Muscle cells appear to derive from the bronchial smooth muscle cells of the adjacent airway. As the arteries increase in size further putative muscle cells are recruited from the mesenchyme and increase the thickness of the muscle wall. Elastic laminae and collagen are laid down between the layers of muscle cells.2 Halfway through fetal life the structure of the pulmonary arteries is the same as it is in the adult.1 The proximal half of the pulmonary arterial pathway has vessels with an elastic structure; that is, with more than seven layers of smooth muscle cells separated by collagen and elastic laminae. The elastin provides the vessel with distensibility and the collagen provides rigidity. Beyond this proximal half there is a gradual decrease in the number of layers of muscle and in the arteries at the periphery there is a single layer of muscle cells between elastic laminae. The veins are relatively thin walled, with only two to three layers of muscle even at the hilum. Both arteries and veins are surrounded by a thick adventitia made up of collagen. During fetal life the pulmonary vascular resistance is high. This is associated with a relatively small lumen in the arteries and a thick muscle wall. After birth there is a decrease in wall thickness. This is rapid in small arteries: however, the decrease in relative wall thickness in the large elastic arteries is achieved over a period of months rather than days. It is likely to be as a result of an increase in size of the artery with no increase in muscle cells. After this, as the blood vessels increase in size there is a concomitant increase in size of smooth muscle cells and connective tissue to maintain the same percentage wall thickness. During early childhood the peripheral pulmonary arteries are relatively non-muscular and with time there is a gradual
GROWTH OF THE VASCULAR TREE
extension of muscle to the periphery as arteries increase in size. Arteries have a nervous supply which appears early, at the same time as that of the airways. It extends peripherally alongside the smooth muscle cells.4 There is no rapid change in the structure of the pulmonary veins after birth but muscle increases in the wall as they increase in size.1
FACTORS CONTROLLING ARTERIAL GROWTH The development of the pulmonary arterial tree is dependent upon a series of interactions in both directions between different cell types and connective tissue in the endoderm (airways) and mesenchymal matrix.5 Extracellular matrix components induce endothelial differentiation. Fibronectin, laminin and the basement membrane collagens IV and V play an early role in formation of endothelial tubes. Endothelial tubes are maintained by cell adhesion molecules such as E-selectin and platelet endothelial cell adhesion molecule-1 (PECAM). Antibodies to these prevent formation of endothelial tubes in culture. The formation of the capillary tubes around the lung buds suggest that there are growth factors produced from the airways which drive vasculogenesis. Vascular endothelial growth factor (VEGF) is found in the bronchial epithelium and its receptors are found on the endothelial cells in the developing human lung. Another growth factor, fibroblast growth factor (FGF), induces formation of endothelial tubes in avian mesenchymal cells while other growth factors do not. FGF is found in both epithelium and mesenchyme of mice, with the receptor found in the mesenchyme only. After tube formation a different set of signals is involved in the development of the muscle wall. A proposed model was put forward by Folkman and D’Amore.6 They suggested that undifferentiated mesenchymal cells produced angiopoietin bound to TIE2 receptors on the endothelial cell. This releases a signal such as platelet-derived growth factor (PDGF) or heparin binding epidermal growth factor (HB-EGF) which attracts the mesenchymal cells. Once alongside the endothelial cells, they commit to becoming smooth muscle cells. They then differentiate to a more mature cell type by accumulation of contractile and cytoskeletal properties. The large pulmonary arteries have over half of the wall made up from collagen and elastin produced by the smooth muscle cells. The majority of the elastin is produced during fetal life, while the collagen which produces rigidity (collagen I) increases rapidly after birth.7
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bronchial smooth muscle cells. There is little connective tissue around the cells. Immediately after birth, as flow increases, there is a decrease in relative wall thickness. In the small muscular arteries this is achieved in hours by the realignment and thinning of the endothelial cells and smooth muscle cells within the vessel wall.4 This process is helped by the small amount of connective tissue and the relatively synthetic phenotype of the muscle cells. There is no net loss of muscle cells during this period of adaptation. Studies on the mechanisms leading to this thinning have depended heavily on experimental studies using the sheep and the pig both for in vivo and in vitro studies. These have recently been reviewed.7–9 Changes in the wall structure immediately after birth involve remodelling of the actin cytoskeleton. There is an abrupt but transient reduction in the number of contractile myofilaments due to disassembly of actin filaments to their monomeric form. By 2–3 weeks of age they have reformed in porcine pulmonary arteries.4 Pulmonary vascular resistance falls when the lung is ventilated. Experimental studies have shown that pulmonary resistance will do this whether the lung is ventilated by oxygen or nitrogen, but there is a greater response to oxygen. The endothelial cells play a critical role in modulating vascular tone in the systemic and pulmonary circulation throughout life. They also play a part in growth and structural changes and are particularly important in the changes that occur during adaptation to extra-uterine life. The sudden increase in flow after birth increases sheer stress on the endothelium, this also stimulates the release of nitric oxide (NO) and prostacyclin, which are vasodilators. Stretch also stimulates the production of endothelin (ET-1), a vasoconstrictor, by the endothelial cells.
Nitric oxide Stimulation of the endothelial cells by oxygen, sheer stress, bradykinin or acetylcholine leads to production of NO (Fig. 2). NO is produced during the conversion of
MECHANISMS INVOLVED IN ADAPTATION AFTER BIRTH During fetal life the pulmonary arteries have a relatively thick wall maintaining the high pulmonary vascular resistance. Though the wall is thick, the smooth muscle cells do not have as mature a contractile structure as
Figure 2
Diagram representing the nitric oxide pathway.
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L-arginine to L-citrulline by the action of nitric oxide synthase (NOS). A number of co-factors, including the presence of calcium, are required. There are three NOS isoforms, but that produced by the endothelium (eNOS, Type III) is the most important in the pulmonary circulation. NO formed by the endothelium diffuses into the underlying smooth muscle cell and causes vasodilatation by the action of soluble guanylate cyclase to produce cGMP (Fig. 2). The protein and mRNA for eNOS has been demonstrated in fetal lambs from 70 days’ gestation (term 145 days) when its concentration is greater than postnatally.8 In the porcine lung the amount of eNOS is greater in the fetus and the newborn than in the adult, with a marked transient increase at 2–3 days after birth. Pulmonary veins have more eNOS than the pulmonary arteries.10 There is a basal release of NO in fetal lambs and pigs which is greater in veins than arteries. In sheep, blocking the NOS activity prior to delivery attenuates the post-natal drop in pulmonary vascular resistance.8 The NOS activity is greater in mid gestation than in the newborn or adult, suggesting that it may be important for lung growth and endothelial development. Stimulated NO release varies with age, type of vessel and the species studied. Oxygen upregulates the NOS gene in human endothelial cells. In newborn piglets pulmonary arteries do not respond by relaxation to acetylcholine until after 3 days of age, whilst the veins do. This may be related to the NOS or to the muscarinic receptors.9 In newborn lambs there is also an attenuated response to acetylcholine. In the peripheral lung of piglets NOS activity can be stimulated at birth, but it is absent in fetal lung tissue 1 week pre-term. This suggests that there is an inhibitor during fetal life, possibly the endogenous inhibitor of NOS dimethylarginine (ADMA),11 which is increased in systemic hypertension and in pulmonary hypertensive children. A lack of NOS, as seen in eNOS-null mice, does not lead to hypertension at sea level but there is an increase in their vasoconstrictive response to hypoxia. This suggests that NOS is less important for baseline tone than vasoconstriction.12 Hypertension produced in lambs in utero by closure of the ductus arteriosus reduced the production and activity of eNOS and also reduced the vascular smooth muscle response to NO. Similar changes are seen in the hypertensive newborn piglet.
Prostacyclin Prostacyclin is known to dilate the fetal vascular bed, its synthesis increases in smooth muscle cells and endothelial cells during fetal life.7 Increasing oxygen tensions in fetal lamb pulmonary arteries increase prostacyclin production but in intact lungs the plasma level does not increase. Prostacyclin production in lambs decreases within hours of birth: however, hypoxia increases its production and gene expression. Prostacyclin causes relaxation via
A. A. HISLOP AND C. M. PIERCE
Figure 3
Diagram representing the endothelin pathway.
adenylate cyclase which decreases towards term; therefore, prostacyclin may be more important in preterm babies.
Endothelin Endothelin is a vasoactive and a growth factor produced by the endothelium. In experimental studies ET-1 constricts fetal lamb arteries and veins and increases the pulmonary artery pressure in newborn lambs.7,8 Plasma ET-1 is high in newborn and hypertensive infants, decreasing with the increase in pulmonary arterial pressure. In the piglet it is high at birth and falls by 3 days of age. In piglets kept hypertensive after birth, this fall is reduced. ET-1 has two receptors (Fig. 3). ETA receptors are found on smooth muscle cells where their stimulation leads to vasoconstriction. ETB receptors are found on the same cells, stimulation leading to vasoconstriction, but others are found on the endothelial cells where they lead to release of NO or prostacyclin and thus vasodilatation. In a study on piglet lungs the ETB receptors appeared on the endothelium 1–3 days after birth but did not appear in hypertensive piglets.13 The number of ETA contractile receptors also increased in hypertensive piglets. Endothelin is also increased in the sheep model of hypertension with a failure in the vasodilator effect of ET-1. In the sheep an ETA blocker attenuated the structural changes in the pulmonary artery and enhanced vasodilation at birth.9 ETA antagonists may have a part in the treatment of hypertensive lung disease in infants.
ABNORMAL PULMONARY CIRCULATION Abnormalities in the pulmonary circulation may be as a result of abnormal development of the vascular tree in utero, the branching pattern or the structure of the vessel
GROWTH OF THE VASCULAR TREE
325
Figure 4 Photomicrographs of a pulmonary artery from the respiratory region of the lung in a normal infant (A) and one with pulmonary hypertension (B).The muscle wall is thick and the lumen narrow in the hypertensive vessel (mag × 525). Table 2
Pulmonary hypertension in infancy.
Primary
Secondary
PPHN
Hypoxia Prematurity, BPD
Meconium aspiration Sepsis
Congenital heart disease
Hypoxia Unknown
Hypoplastic lungs, e.g. CDH Primary pulmonary hypertension
wall. They can also be due to a failure in the postnatal adaptation after birth or secondary to congenital heart disease or premature delivery (Table 2). In most of these the pathological appearance is an increase in pulmonary arterial smooth muscle in the walls of small pulmonary arteries (Fig. 4).
Hypoplastic lungs The pulmonary arteries appear to be controlled by the airways; thus, any reduction in airway branching is accompanied by a reduction in arteries. Thus, in hypoplastic lungs increased pulmonary resistance may be secondary to an abnormal or hypoplastic development of the vascular tree. In congenital diaphragmatic hernia (CDH), where the number of pre-acinar airway and arterial branches is reduced, there is a reduced number of alveoli and arteries. In most cases of CDH there is pulmonary
hypertension which may be related to the reduced capillary bed or may be a structural abnormality in the vasculature. Both lungs have arteries with an increase in smooth muscle cells in the media and an increase in connective tissue in the adventitia, both of which decrease after treatment with extra corporeal membrane oxygenation (ECMO).14 Many cases of CDH are resistant to NO therapy, implying a fundamental abnormality in the structure of the artery walls. In renal agenesis and dysplasia, which are associated with lung hypoplasia, there is a primary reduction in airway and arterial branches but there is no increase in arterial wall thickness.
Primary pulmonary hypertension Primary pulmonary hypertension (PPH) is a rare childhood disease with a poor prognosis; 6% are familial. Recent studies have located a gene to chromosome 2q31–3215 and it seems that some forms of familial PPH show gene anticipation.
Persistent pulmonary hypertension of the newborn (PPHN) The most common cause of pulmonary hypertension in the newborn is PPHN where there is failure of the pulmonary circulation to adapt normally in full-term infants. There is a high pulmonary vascular resistance and marked
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vasolability, usually associated with right to left shunting of blood and hypoxaemia. There is a high morbidity and mortality associated with PPHN. It can be secondary to meconium aspiration, sepsis or hypoxia. Many infants with PPHN unresponsive to NO treatment have idiopathic lung hypoplasia with failure in development of a normal alveolar region. Infants with PPHN have decreased eNOS mRNA, reduced levels of cGMP and are deficient in arginine but have increased levels of the vasoconstrictor ET-1.
A major complication following cardiac surgery in young babies (with marked increase in medial wall thickness but no intimal damage) is hypertensive crises. There is sporadic increase in pulmonary artery pressure above baseline to above systemic level; ET-1, thromboxane and catecholamines all increase. These babies are now treated with NO and other pulmonary vasodilators. The use of phosphodiesterase inhibitors to improve the levels of cGMP may change the way in which these patients are treated in the future.
Secondary pulmonary hypertension
TREATMENT
Hypertension can be secondary to congenital heart disease or hypoxia, as seen in infants with chronic lung disease after premature delivery and artificial ventilation. The clinical course is variable depending upon the cause.
There have been significant advances in the treatment of infants with pulmonary hypertension by the use of intravenous and inhaled vasodilators like prostacyclin, dipyridamole and NO. The use of dipyridamole and prostacyclin is limited by the significant systemic vasodilator effect. Inhaled NO is a selective pulmonary vasodilator. Several randomized controlled trials have shown improved oxygenation and a reduction in the need for extra corporeal membrane oxygenation (ECMO) in neonates with PPHN or hypoxic respiratory failure but without a reduction in mortality.17 Long-term studies are needed to assess the neuro-developmental consequences of NO therapy. The primary mechanism of pulmonary vasodilatation is via the NO-cGMP signal transduction pathway, therefore alternative treatments by agents such as Sildenafil, a selective phosphodiesterase inhibitor to maintain the level of cGMP or addition of the substrate for NO, L-Arginine, may potentiate vasodilatation. Experimental studies also suggest that inhibitors or blockers of the ET-1 receptor ETA may help in vasodilatation.
Premature delivery Premature babies with hyaline membrane disease are probably born with a normal pulmonary circulation. However, those which go on to develop chronic lung disease may, as a result of hypoxia or possibly as a result of hyperoxia during artificial ventilation, have an increase in arterial and venous smooth muscle. Those with clinically measured pulmonary hypertension show a greater increase in pulmonary vascular smooth muscle than those without right ventricular hypertrophy.4 Babies that are born during the canalicular phase, (24–27 weeks’ gestation) now survive and in these there is failure of the normal development of the capillary bed in the alveolar region. This hypoplasia and dysplasia may have a long-term effect. Little is known about the effect of antenatal or postnatal glucocorticoids on the pulmonary arteries. They lead to premature thinning of the alveolar walls and may therefore affect the vascular bed. Experimental studies showed that neither glucocorticoid nor surfactant treatment improved the capillary development after very early premature delivery.16
Congenital heart disease There is no evidence that the branching pattern of the pulmonary circulation is abnormal in children with congenital heart disease, except those with pulmonary atresia where the lungs are supplied from the systemic circulation. In children with an increase in flow or exposure to systemic pressures, normal adaptation probably does not occur and there is gradual development of obstructive pulmonary vascular disease with eventual intimal damage and dilatation lesions. The speed of onset of disease depends upon the type of cardiac anomaly. Some become inoperable within months but operative procedures before intimal damage has occurred allow resistance to return to normal.4
PRACTICE POINTS Pulmonary hypertension in infancy may be primary, secondary to other disease or may be persistent hypertension of the newborn (PPHN). Primary ● ●
Needs long-term support and treatment. May eventually need transplantation.
Secondary ● ●
Aims at primary prevention. Needs long-term support and treatment.
PPHN ●
●
Aim at support throughout the neonatal period with modern intensive care techniques. It is potentially a fully reversible condition.
GROWTH OF THE VASCULAR TREE
REFERENCES 1. Hislop AA, Reid LM. Formation of the pulmonary vasculature. In: Hodson WA (ed). Development of the Lung Monograph. New York: Marcel Dekker, 1977: 37–86. 2. Hall SM, Hislop AA, Pierce C, Haworth SG. Prenatal origins of human intrapulmonary arteries: formation and maturation. Am J Respir Cell Mol Biol 2000; 23: 194–203. 3. Burri PH. Structural aspects of prenatal and postnatal development and growth of the lung. In: McDonald JA (ed). Lung Growth and Development. New York: Marcel Dekker, 1997: 1–35. 4. Haworth SG. Pulmonary hypertension in childhood. Eur Respir J 1993; 6: 1037–1043. 5. Morrell NW, Weiser MCM, Stenmark KR. Development of the Pulmonary Vasculature. In: Gaultier C, Bourbon JR, Post M. (eds). Lung Development. Oxford: Oxford University Press, 1999: 152–195. 6. Folkman J, D’Amore PA. Blood vessel formation: what is its molecular basis? Cell 1996; 87: 1153–1155. 7. Dukarm RC, Steinhorn RH, Morin III FC. The normal pulmonary vascular transition at birth. Clin Perinatol 1996; 23: 711–726. 8. Abman S, Kinsella JP, Mercier J-C. Nitric oxide and endothelin in the developing pulmonary circulation: physiologic and clinical implications. In: Gaultier C, Bourbon JR, Post M (eds). Lung Development. Oxford: Oxford University Press, 1999: 196–220. 9. Haworth SG. The pathophysiology of persistent pulmonary hypertension of the newborn. Semin Neonatol 1997; 2: 13–23. 10. Hislop AA, Springall DR, Buttery LDK, Pollock JS, Haworth SG. Abundance of endothelial nitric oxide synthase in newborn intrapulmonary arteries. Arch Dis Child 1995; 12: F17–F21.
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11. MacAllister RJ, Fickling SA, Whitley GStJ, Vallance P. Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis. Br J Pharmacol 1994; 112: 43–48. 12. Fagan KA, Fouty BW, Tyler RC, Morris Jr KG, Hepler LKSK, LeCras TD, Abman SH, Weinberger HD, Huang PL, McMurtry IF, Rodman DM. The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresonsive to mild hypoxia. J Clin Invest 1999; 103: 291–299. 13. Noguchi Y, Hislop AA, Haworth SG. Influence of hypoxia on endothelin-1 binding sites in neonatal porcine pulmonary vasculature. Am J Physiol 1997; 272: H669–H678. 14. Shehata SMK, Tibboel D, Sharma HS, Mooi WJ. Impaired structural remodelling of pulmonary arteries n newborns with congenital diaphragmatic hernia: a histological study of 29 cases. J Pathol 1999; 189: 112–118. 15. Nichols WC, Koller DL, Slovis B, Foroud T, Terry VH, Arnold ND, Siemieniak DR, Wheeler L, Phillips JAI, Newman JH, Conneally PM, Ginsburg D, Lloyd JE. Localization of the gene for familial primary pulmonary hypertension to chromosome 2q31–32. Nat Genet 1997; 15: 277–280. 16. Coalson JJ. Pathology of chronic lung disease of early infancy. In: Bland RD, Coalson JJ. (eds). Chronic lung disease in early infancy. New York: Marcel Dekker, 2000: 85–124. 17. Clark RH, Kueser TJ, Walker MW, Southgate WM, Huckaby JL, Perez JA, Roy BJ, Keszler M, Kinsella JP. Low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. Clinical Inhaled Nitric Oxide Research Group. New England J Med 2000; 342: 469–474.
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MINI-SYMPOSIUM: GROWTH OF THE LUNG
Growth of the vascular tree A. A. Hislop and C. M. Pierce 1
Vascular Biology and Pharmacology Unit, Institute of Child Health, 30 Guildford Street, London WC1N 1EH, Great Ormond Street Hospital, London WC1N 3JH, UK
2
KEYWORDS pulmonary circulation, development, pulmonary hypertension, endothelium, nitric oxide, endothelin.
Summary Major changes occur in the pulmonary circulation at birth. Failure of normal adaptation leads to pulmonary hypertension of the newborn.The basis for this may be abnormal growth of the lung in utero or a failure of the mechanisms which lead to vasodilatation in the newborn period.This article describes normal development of the pulmonary arteries and veins, their branching pattern and structure and discusses the factors which may control this growth. Endothelial function and dysfunction are crucial in adaptation, and new experimental studies are aiding our understanding of the control and action of factors produced by the endothelium, e.g. nitric oxide, prostacyclin and endothelin.The study of infants with pulmonary hypertension, together with the experimental studies, will aid in producing effective methods of controlling increased pulmonary artery pressure in the newborn period. © 2000 Harcourt Publishers Ltd
INTRODUCTION During fetal life little blood flows through the pulmonary circulation (8–10% of total cardiac output). Oxygenation of the blood occurs via the placenta, blood being diverted away from the lungs. This diversion is achieved by there being a high resistance in the fetal pulmonary circulation. Immediately after birth, with the onset of breathing, there is a drop in pulmonary artery resistance, allowing blood flow through the pulmonary circulation to increase 8–10fold. Failure of the pulmonary circulation to sustain this pressure drop leads to a failure of postnatal adaptation. The mechanisms involved in this failure are not fully understood, but there are several suggestions, some of which result from abnormal development of the blood vessels in utero. It is therefore important to understand normal development of the pulmonary circulation (Table 1).
NORMAL DEVELOPMENT Branching pattern of pulmonary arteries and veins The primary function of the lung is gas exchange and blood vessels are arranged to accommodate efficient gas Correspondence to: A. A. Hislop, Vascular Biology and Pharmacology Unit, Institute of Child Health, 30 Guildford Street, London WC1N 1EH, UK 1526–0550/00/040321 + 07 $35.00/0
Table 1 Timetable of normal blood vessel growth. ●
●
● ●
● ● ●
● ●
From 4–16 weeks arteries and veins develop by vasculogenesis from the mesenchyme around airway buds. Pre-acinar arteries and veins complete by 17 weeks’ gestation. Blood gas barrier established by 24 weeks’ gestation. Intra-acinar arteries and veins develop with the alveoli in late fetal life and up to 2 years of age. Increase in size of arteries and veins as lung grows. Arterial wall thickness high in fetal life. After birth rapid dilatation of pulmonary arteries and then wall thinning. Gradual extension of muscle during childhood. Innervation of pulmonary artery smooth muscle cells as they appear.
exchange by producing a large surface area within the alveolar region. Blood flow must be sufficient and at a linear velocity with the counter current to allow gas exchange to occur. The blood vessels also carry, produce and control the concentration of vasoactive mediators. During development a complex branching pattern of blood vessels develops from a single avascular bud and is connected to the heart at the right ventricle and at the left atrium. In the adult lung the arteries run alongside the airways and branch with them, suggesting that their growth might be regulated by the airways. In addition to these © 2000 Harcourt Publishers Ltd
322
conventional arteries alongside the airway branches there are extra, smaller “supernumerary” arteries, 2–3 times in number which supply the alveolar region more directly. These can carry up to 40% of total blood flow and increase in relative number towards the periphery of the lung. The veins have a similar number of branches both conventional and supernumerary, but they run independently of the airways between acini and segments. Early studies on the development of the arteries and veins showed that they grow at the same time as the airways, so that all pre-acinar vessels are present by the 17th week of gestation, the end of the pseudoglandular stage.1 The supernumerary arteries and veins which will be associated with the alveoli (not developed by this time) also grow during this period. This suggests a genetic influence on blood vessel development. The branching pattern in siblings is more similar to each other than to that of the general population, also suggesting a genetic influence on the branching pattern. In the canalicular stage (17–27 weeks’ gestation) further branching of the arteries and veins accompanies the development of the respiratory airways. At this time the capillaries in the mesenchyme come to lie under the epithelium, which is differentiating into Type I and II pneumonocytes. This produces a blood–gas barrier for gas exchange similar in thickness to that seen in the adult. Recent studies in man using specific antibodies have shown that the pulmonary blood vessels initially form de novo as endothelial tubes in the mesenchyme around the peripheral airways, the process of vasculogenesis, rather than by inward growth of existing arteries (angiogenesis).2 Initially endothelial cell precursors have been identified without a lumen in the splanchnic mesenchyme around the foregut and lung bud at 4 weeks’ gestation. Endothelial tubes then form and coalesce, and a circulation between the right ventricle and left atrium can be recognized in man by 5 weeks’ gestation when the lung has only one airway generation. As the airways divide towards the periphery, the intrapulmonary arteries form by continuous coalescence of endothelial tubes alongside the airway. The veins derive independently from the same mesenchyme and run between the airways. As alveoli form in late fetal life (after 30 weeks’ gestation) and after birth, there is a rapid increase in the number of small preand post-capillary vessels. After 2 years of age the lung grows by increasing the surface area of the alveoli and the area of the capillary bed increases alongside this growth. Between birth and adulthood the surface area of the lung increases about 20-fold and the capillary volume 35-fold to maintain efficient gas exchange. This increase may be by sprouting or alternatively by intussusception of tissue within an existing sheet, so increasing its surface area and complexity.3 At birth the overall pattern of the arteries and veins is the same pattern as in the adult (Fig. 1). The vessels increase in size with age as the lung increases in volume and the number and volume of vessels at the periphery increases.
A. A. HISLOP AND C. M. PIERCE
Figure 1 Post-mortem arteriogram of a fetus of 39 weeks’ gestation. The arteries have been injected with barium sulphate (mag × 0.75).
Structure of the pulmonary artery wall Smooth muscle cells are found around the newly formed arteries soon after they line up alongside the airways. Muscle cells appear to derive from the bronchial smooth muscle cells of the adjacent airway. As the arteries increase in size further putative muscle cells are recruited from the mesenchyme and increase the thickness of the muscle wall. Elastic laminae and collagen are laid down between the layers of muscle cells.2 Halfway through fetal life the structure of the pulmonary arteries is the same as it is in the adult.1 The proximal half of the pulmonary arterial pathway has vessels with an elastic structure; that is, with more than seven layers of smooth muscle cells separated by collagen and elastic laminae. The elastin provides the vessel with distensibility and the collagen provides rigidity. Beyond this proximal half there is a gradual decrease in the number of layers of muscle and in the arteries at the periphery there is a single layer of muscle cells between elastic laminae. The veins are relatively thin walled, with only two to three layers of muscle even at the hilum. Both arteries and veins are surrounded by a thick adventitia made up of collagen. During fetal life the pulmonary vascular resistance is high. This is associated with a relatively small lumen in the arteries and a thick muscle wall. After birth there is a decrease in wall thickness. This is rapid in small arteries: however, the decrease in relative wall thickness in the large elastic arteries is achieved over a period of months rather than days. It is likely to be as a result of an increase in size of the artery with no increase in muscle cells. After this, as the blood vessels increase in size there is a concomitant increase in size of smooth muscle cells and connective tissue to maintain the same percentage wall thickness. During early childhood the peripheral pulmonary arteries are relatively non-muscular and with time there is a gradual
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extension of muscle to the periphery as arteries increase in size. Arteries have a nervous supply which appears early, at the same time as that of the airways. It extends peripherally alongside the smooth muscle cells.4 There is no rapid change in the structure of the pulmonary veins after birth but muscle increases in the wall as they increase in size.1
FACTORS CONTROLLING ARTERIAL GROWTH The development of the pulmonary arterial tree is dependent upon a series of interactions in both directions between different cell types and connective tissue in the endoderm (airways) and mesenchymal matrix.5 Extracellular matrix components induce endothelial differentiation. Fibronectin, laminin and the basement membrane collagens IV and V play an early role in formation of endothelial tubes. Endothelial tubes are maintained by cell adhesion molecules such as E-selectin and platelet endothelial cell adhesion molecule-1 (PECAM). Antibodies to these prevent formation of endothelial tubes in culture. The formation of the capillary tubes around the lung buds suggest that there are growth factors produced from the airways which drive vasculogenesis. Vascular endothelial growth factor (VEGF) is found in the bronchial epithelium and its receptors are found on the endothelial cells in the developing human lung. Another growth factor, fibroblast growth factor (FGF), induces formation of endothelial tubes in avian mesenchymal cells while other growth factors do not. FGF is found in both epithelium and mesenchyme of mice, with the receptor found in the mesenchyme only. After tube formation a different set of signals is involved in the development of the muscle wall. A proposed model was put forward by Folkman and D’Amore.6 They suggested that undifferentiated mesenchymal cells produced angiopoietin bound to TIE2 receptors on the endothelial cell. This releases a signal such as platelet-derived growth factor (PDGF) or heparin binding epidermal growth factor (HB-EGF) which attracts the mesenchymal cells. Once alongside the endothelial cells, they commit to becoming smooth muscle cells. They then differentiate to a more mature cell type by accumulation of contractile and cytoskeletal properties. The large pulmonary arteries have over half of the wall made up from collagen and elastin produced by the smooth muscle cells. The majority of the elastin is produced during fetal life, while the collagen which produces rigidity (collagen I) increases rapidly after birth.7
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bronchial smooth muscle cells. There is little connective tissue around the cells. Immediately after birth, as flow increases, there is a decrease in relative wall thickness. In the small muscular arteries this is achieved in hours by the realignment and thinning of the endothelial cells and smooth muscle cells within the vessel wall.4 This process is helped by the small amount of connective tissue and the relatively synthetic phenotype of the muscle cells. There is no net loss of muscle cells during this period of adaptation. Studies on the mechanisms leading to this thinning have depended heavily on experimental studies using the sheep and the pig both for in vivo and in vitro studies. These have recently been reviewed.7–9 Changes in the wall structure immediately after birth involve remodelling of the actin cytoskeleton. There is an abrupt but transient reduction in the number of contractile myofilaments due to disassembly of actin filaments to their monomeric form. By 2–3 weeks of age they have reformed in porcine pulmonary arteries.4 Pulmonary vascular resistance falls when the lung is ventilated. Experimental studies have shown that pulmonary resistance will do this whether the lung is ventilated by oxygen or nitrogen, but there is a greater response to oxygen. The endothelial cells play a critical role in modulating vascular tone in the systemic and pulmonary circulation throughout life. They also play a part in growth and structural changes and are particularly important in the changes that occur during adaptation to extra-uterine life. The sudden increase in flow after birth increases sheer stress on the endothelium, this also stimulates the release of nitric oxide (NO) and prostacyclin, which are vasodilators. Stretch also stimulates the production of endothelin (ET-1), a vasoconstrictor, by the endothelial cells.
Nitric oxide Stimulation of the endothelial cells by oxygen, sheer stress, bradykinin or acetylcholine leads to production of NO (Fig. 2). NO is produced during the conversion of
MECHANISMS INVOLVED IN ADAPTATION AFTER BIRTH During fetal life the pulmonary arteries have a relatively thick wall maintaining the high pulmonary vascular resistance. Though the wall is thick, the smooth muscle cells do not have as mature a contractile structure as
Figure 2
Diagram representing the nitric oxide pathway.
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L-arginine to L-citrulline by the action of nitric oxide synthase (NOS). A number of co-factors, including the presence of calcium, are required. There are three NOS isoforms, but that produced by the endothelium (eNOS, Type III) is the most important in the pulmonary circulation. NO formed by the endothelium diffuses into the underlying smooth muscle cell and causes vasodilatation by the action of soluble guanylate cyclase to produce cGMP (Fig. 2). The protein and mRNA for eNOS has been demonstrated in fetal lambs from 70 days’ gestation (term 145 days) when its concentration is greater than postnatally.8 In the porcine lung the amount of eNOS is greater in the fetus and the newborn than in the adult, with a marked transient increase at 2–3 days after birth. Pulmonary veins have more eNOS than the pulmonary arteries.10 There is a basal release of NO in fetal lambs and pigs which is greater in veins than arteries. In sheep, blocking the NOS activity prior to delivery attenuates the post-natal drop in pulmonary vascular resistance.8 The NOS activity is greater in mid gestation than in the newborn or adult, suggesting that it may be important for lung growth and endothelial development. Stimulated NO release varies with age, type of vessel and the species studied. Oxygen upregulates the NOS gene in human endothelial cells. In newborn piglets pulmonary arteries do not respond by relaxation to acetylcholine until after 3 days of age, whilst the veins do. This may be related to the NOS or to the muscarinic receptors.9 In newborn lambs there is also an attenuated response to acetylcholine. In the peripheral lung of piglets NOS activity can be stimulated at birth, but it is absent in fetal lung tissue 1 week pre-term. This suggests that there is an inhibitor during fetal life, possibly the endogenous inhibitor of NOS dimethylarginine (ADMA),11 which is increased in systemic hypertension and in pulmonary hypertensive children. A lack of NOS, as seen in eNOS-null mice, does not lead to hypertension at sea level but there is an increase in their vasoconstrictive response to hypoxia. This suggests that NOS is less important for baseline tone than vasoconstriction.12 Hypertension produced in lambs in utero by closure of the ductus arteriosus reduced the production and activity of eNOS and also reduced the vascular smooth muscle response to NO. Similar changes are seen in the hypertensive newborn piglet.
Prostacyclin Prostacyclin is known to dilate the fetal vascular bed, its synthesis increases in smooth muscle cells and endothelial cells during fetal life.7 Increasing oxygen tensions in fetal lamb pulmonary arteries increase prostacyclin production but in intact lungs the plasma level does not increase. Prostacyclin production in lambs decreases within hours of birth: however, hypoxia increases its production and gene expression. Prostacyclin causes relaxation via
A. A. HISLOP AND C. M. PIERCE
Figure 3
Diagram representing the endothelin pathway.
adenylate cyclase which decreases towards term; therefore, prostacyclin may be more important in preterm babies.
Endothelin Endothelin is a vasoactive and a growth factor produced by the endothelium. In experimental studies ET-1 constricts fetal lamb arteries and veins and increases the pulmonary artery pressure in newborn lambs.7,8 Plasma ET-1 is high in newborn and hypertensive infants, decreasing with the increase in pulmonary arterial pressure. In the piglet it is high at birth and falls by 3 days of age. In piglets kept hypertensive after birth, this fall is reduced. ET-1 has two receptors (Fig. 3). ETA receptors are found on smooth muscle cells where their stimulation leads to vasoconstriction. ETB receptors are found on the same cells, stimulation leading to vasoconstriction, but others are found on the endothelial cells where they lead to release of NO or prostacyclin and thus vasodilatation. In a study on piglet lungs the ETB receptors appeared on the endothelium 1–3 days after birth but did not appear in hypertensive piglets.13 The number of ETA contractile receptors also increased in hypertensive piglets. Endothelin is also increased in the sheep model of hypertension with a failure in the vasodilator effect of ET-1. In the sheep an ETA blocker attenuated the structural changes in the pulmonary artery and enhanced vasodilation at birth.9 ETA antagonists may have a part in the treatment of hypertensive lung disease in infants.
ABNORMAL PULMONARY CIRCULATION Abnormalities in the pulmonary circulation may be as a result of abnormal development of the vascular tree in utero, the branching pattern or the structure of the vessel
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Figure 4 Photomicrographs of a pulmonary artery from the respiratory region of the lung in a normal infant (A) and one with pulmonary hypertension (B).The muscle wall is thick and the lumen narrow in the hypertensive vessel (mag × 525). Table 2
Pulmonary hypertension in infancy.
Primary
Secondary
PPHN
Hypoxia Prematurity, BPD
Meconium aspiration Sepsis
Congenital heart disease
Hypoxia Unknown
Hypoplastic lungs, e.g. CDH Primary pulmonary hypertension
wall. They can also be due to a failure in the postnatal adaptation after birth or secondary to congenital heart disease or premature delivery (Table 2). In most of these the pathological appearance is an increase in pulmonary arterial smooth muscle in the walls of small pulmonary arteries (Fig. 4).
Hypoplastic lungs The pulmonary arteries appear to be controlled by the airways; thus, any reduction in airway branching is accompanied by a reduction in arteries. Thus, in hypoplastic lungs increased pulmonary resistance may be secondary to an abnormal or hypoplastic development of the vascular tree. In congenital diaphragmatic hernia (CDH), where the number of pre-acinar airway and arterial branches is reduced, there is a reduced number of alveoli and arteries. In most cases of CDH there is pulmonary
hypertension which may be related to the reduced capillary bed or may be a structural abnormality in the vasculature. Both lungs have arteries with an increase in smooth muscle cells in the media and an increase in connective tissue in the adventitia, both of which decrease after treatment with extra corporeal membrane oxygenation (ECMO).14 Many cases of CDH are resistant to NO therapy, implying a fundamental abnormality in the structure of the artery walls. In renal agenesis and dysplasia, which are associated with lung hypoplasia, there is a primary reduction in airway and arterial branches but there is no increase in arterial wall thickness.
Primary pulmonary hypertension Primary pulmonary hypertension (PPH) is a rare childhood disease with a poor prognosis; 6% are familial. Recent studies have located a gene to chromosome 2q31–3215 and it seems that some forms of familial PPH show gene anticipation.
Persistent pulmonary hypertension of the newborn (PPHN) The most common cause of pulmonary hypertension in the newborn is PPHN where there is failure of the pulmonary circulation to adapt normally in full-term infants. There is a high pulmonary vascular resistance and marked
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vasolability, usually associated with right to left shunting of blood and hypoxaemia. There is a high morbidity and mortality associated with PPHN. It can be secondary to meconium aspiration, sepsis or hypoxia. Many infants with PPHN unresponsive to NO treatment have idiopathic lung hypoplasia with failure in development of a normal alveolar region. Infants with PPHN have decreased eNOS mRNA, reduced levels of cGMP and are deficient in arginine but have increased levels of the vasoconstrictor ET-1.
A major complication following cardiac surgery in young babies (with marked increase in medial wall thickness but no intimal damage) is hypertensive crises. There is sporadic increase in pulmonary artery pressure above baseline to above systemic level; ET-1, thromboxane and catecholamines all increase. These babies are now treated with NO and other pulmonary vasodilators. The use of phosphodiesterase inhibitors to improve the levels of cGMP may change the way in which these patients are treated in the future.
Secondary pulmonary hypertension
TREATMENT
Hypertension can be secondary to congenital heart disease or hypoxia, as seen in infants with chronic lung disease after premature delivery and artificial ventilation. The clinical course is variable depending upon the cause.
There have been significant advances in the treatment of infants with pulmonary hypertension by the use of intravenous and inhaled vasodilators like prostacyclin, dipyridamole and NO. The use of dipyridamole and prostacyclin is limited by the significant systemic vasodilator effect. Inhaled NO is a selective pulmonary vasodilator. Several randomized controlled trials have shown improved oxygenation and a reduction in the need for extra corporeal membrane oxygenation (ECMO) in neonates with PPHN or hypoxic respiratory failure but without a reduction in mortality.17 Long-term studies are needed to assess the neuro-developmental consequences of NO therapy. The primary mechanism of pulmonary vasodilatation is via the NO-cGMP signal transduction pathway, therefore alternative treatments by agents such as Sildenafil, a selective phosphodiesterase inhibitor to maintain the level of cGMP or addition of the substrate for NO, L-Arginine, may potentiate vasodilatation. Experimental studies also suggest that inhibitors or blockers of the ET-1 receptor ETA may help in vasodilatation.
Premature delivery Premature babies with hyaline membrane disease are probably born with a normal pulmonary circulation. However, those which go on to develop chronic lung disease may, as a result of hypoxia or possibly as a result of hyperoxia during artificial ventilation, have an increase in arterial and venous smooth muscle. Those with clinically measured pulmonary hypertension show a greater increase in pulmonary vascular smooth muscle than those without right ventricular hypertrophy.4 Babies that are born during the canalicular phase, (24–27 weeks’ gestation) now survive and in these there is failure of the normal development of the capillary bed in the alveolar region. This hypoplasia and dysplasia may have a long-term effect. Little is known about the effect of antenatal or postnatal glucocorticoids on the pulmonary arteries. They lead to premature thinning of the alveolar walls and may therefore affect the vascular bed. Experimental studies showed that neither glucocorticoid nor surfactant treatment improved the capillary development after very early premature delivery.16
Congenital heart disease There is no evidence that the branching pattern of the pulmonary circulation is abnormal in children with congenital heart disease, except those with pulmonary atresia where the lungs are supplied from the systemic circulation. In children with an increase in flow or exposure to systemic pressures, normal adaptation probably does not occur and there is gradual development of obstructive pulmonary vascular disease with eventual intimal damage and dilatation lesions. The speed of onset of disease depends upon the type of cardiac anomaly. Some become inoperable within months but operative procedures before intimal damage has occurred allow resistance to return to normal.4
PRACTICE POINTS Pulmonary hypertension in infancy may be primary, secondary to other disease or may be persistent hypertension of the newborn (PPHN). Primary ● ●
Needs long-term support and treatment. May eventually need transplantation.
Secondary ● ●
Aims at primary prevention. Needs long-term support and treatment.
PPHN ●
●
Aim at support throughout the neonatal period with modern intensive care techniques. It is potentially a fully reversible condition.
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11. MacAllister RJ, Fickling SA, Whitley GStJ, Vallance P. Metabolism of methylarginines by human vasculature; implications for the regulation of nitric oxide synthesis. Br J Pharmacol 1994; 112: 43–48. 12. Fagan KA, Fouty BW, Tyler RC, Morris Jr KG, Hepler LKSK, LeCras TD, Abman SH, Weinberger HD, Huang PL, McMurtry IF, Rodman DM. The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresonsive to mild hypoxia. J Clin Invest 1999; 103: 291–299. 13. Noguchi Y, Hislop AA, Haworth SG. Influence of hypoxia on endothelin-1 binding sites in neonatal porcine pulmonary vasculature. Am J Physiol 1997; 272: H669–H678. 14. Shehata SMK, Tibboel D, Sharma HS, Mooi WJ. Impaired structural remodelling of pulmonary arteries n newborns with congenital diaphragmatic hernia: a histological study of 29 cases. J Pathol 1999; 189: 112–118. 15. Nichols WC, Koller DL, Slovis B, Foroud T, Terry VH, Arnold ND, Siemieniak DR, Wheeler L, Phillips JAI, Newman JH, Conneally PM, Ginsburg D, Lloyd JE. Localization of the gene for familial primary pulmonary hypertension to chromosome 2q31–32. Nat Genet 1997; 15: 277–280. 16. Coalson JJ. Pathology of chronic lung disease of early infancy. In: Bland RD, Coalson JJ. (eds). Chronic lung disease in early infancy. New York: Marcel Dekker, 2000: 85–124. 17. Clark RH, Kueser TJ, Walker MW, Southgate WM, Huckaby JL, Perez JA, Roy BJ, Keszler M, Kinsella JP. Low-dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. Clinical Inhaled Nitric Oxide Research Group. New England J Med 2000; 342: 469–474.
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