Impact of Advances in Physiology, Biochemistry, and Molecular Biology on Pulmonary Disease in Neonates

JEFFREY A. WHITSETT and MILDRED T. STAHLMAN

Divisions of Neonatology and Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio; and Division of Neonatology, Vanderbilt University Medical Center, Nashville, Tennessee

	INTRODUCTION

TOP INTRODUCTION CONCLUSION REFERENCES

During the last several decades, clinicians actively involved in the care of neonates have both witnessed and contributed to remarkable improvements in survival and reductions in morbidity from the complications of premature birth. These advances are derived from a vast expansion of fundamental knowledge of the physiology, biochemistry, and more recently, the molecular biology of the fetus and neonate. Since perinatal survival is intrinsically linked to the ability of the infant to support respiration at birth, the maturation of the pulmonary system represents a formidable physiologic hurdle for the premature infant. Understanding the principles of cardiopulmonary adaptation began to have an impact on perinatal survival in the 1950s, a process that continues as we approach the year 2000. During this half century, revolutionary advances in physiology, biochemistry, and molecular biology and genetics fueled clinical progress in neonatology. Infants previously denied care because of hopeless conditions are now routinely assured survival. The present work will identify important areas of basic science research that have contributed directly and indirectly to the improvements in our understanding of perinatal lung function, and consequently, to the reduction of morbidity and mortality associated with perinatal adaptation. These areas include: (1) cardiopulmonary physiology; (2) surfactant biology; (3) lung cell differentiation; (4) pulmonary vascular biology; (5) host defenses; and (6) perinatal nutrition.

	ELUCIDATION AND APPLICATION OF THE PRINCIPLES OF ACID-BASE BALANCE

Improved understanding of hemoglobin-oxygen interactions and gas exchange, which occurred in the 1950s and 1960s and were applied to neonatal care, provided a cornerstone for later clinical successes. Blood gas measurements permitted the diagnosis of metabolic and respiratory acidosis associated with the birth process and with postnatal adaption to air breathing (1). Intervention for the stressed fetus in utero and the ability to provide adequate cardiopulmonary support ex utero, contributed substantively to improved perinatal outcomes. Measurements of blood pressure, perfusion, cardiac output, oxygen saturation, pH, PCO₂ and PO₂ provided a rational basis for therapies that enhanced survival. These early studies, focused on perinatal respiratory adaptation, allowed a previously unattainable rationality to the observation and care of premature and newborn infants during the sometimes prolonged period of cardiopulmonary instability accompanying premature or complicated birth. Arterial-venous access, microchemistry, support of blood pressure, thermal regulation, and nutrition all contributed to the improvements in perinatal care.

	PULMONARY MATURATION: ANATOMY, PHYSIOLOGY, BIOCHEMISTRY, AND MOLECULAR BIOLOGY

During the sixth to eighth week of human gestation, the lungs begin as an outpouching of primordial cells from the foregut endoderm, which then invades the splanchnic mesenchyme. The lung is formed by a process of branching morphogenesis, controlled by cell-cell, cell-matrix, and autocrine-paracrine interactions that mediate cellular proliferation and differentiation (2). At approximately 24 wk of gestation in the human fetus, respiratory epithelial cells begin to undergo distinct morphologic changes associated with increased synthesis of phosphytidylcholine, phosphytidylglycerol, and surfactant proteins that are now known to be critical components of the surfactant complex required to reduce surface tension at the air- liquid interface after birth. Perinatal survival hinges on the maturation of the surfactant system that allows adaptation to air breathing.

The elucidation of the factors controlling respiratory epithelial maturation, surfactant synthesis, secretion, and function, while far from complete, has been a major achievement in basic and clinical science in the past several decades. From a clinical perspective, advances in respiratory physiology, particularly in understanding the important role of surfactant in the maintenance of lung volume and compliance, provided the basis for early physiologic interventions; these included the use of continuous positive airway pressure (CPAP) and mechanical ventilation with both conventional and high-frequency oscillation, strategies that are now routinely used in the care of neonates.

Ventilatory support for critically ill infants in the 1960s and 1970s saved many infants who would have previously succumbed from the problems of prematurity. Unfortunately, the rescue of this population was accompanied by an increase in the incidence of "new" diseases associated with extreme prematurity, including necrotizing enterocolitis (NEC), intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), and bronchopulmonary dysplasia (BPD), the latter a chronic lung disease complicating therapy of the respiratory distress syndrome (RDS), prematurity, or other forms of severe lung injury. The development of strategies designed to avoid lung dysfunction in the preterm infant represented a further advance and came from several investigational directions: (1) the enhancement of biochemical and functional maturation of the respiratory epithelium in the preterm infant in utero; (2) the delay in the delivery of premature infants until lung maturation; and (3) the development of replacement surfactant for prophylaxis or rescue therapy of lung disease after premature birth.

	ANTENATAL GLUCOCORTICOIDS FOR PREVENTION OF RDS

The recognition in the late 1960s and early 1970s that exogenous glucocorticoids, when administered to the mother several days before giving birth, accelerated lung maturation and improved lung function was a major advance in prenatal care. Studies in the premature lamb by Liggins (3), in which glucocorticoid treatment of the ewe markedly improved respiratory function of the lamb after premature birth, were rapidly applied clinically, and led to the widespread and now routine use of maternal glucocorticoids for the prevention of RDS (4). Glucocorticoids increase lung phosphytidylcholine by enhancing various enzymes within the metabolic pathways involved in the synthesis of surfactant lipids. Glucocorticoids also enhance the maturation of the alveolar portion of the respiratory epithelium, increasing type II cell differentiation and causing a thinning of the pulmonary mesenchyme. The biochemical changes induced by prenatal glucocorticoids improve lung function and decrease the risk of respiratory distress in infants. Perinatal glucocorticoid therapy, when used in conjunction with avoidance of asphyxia and shock at birth, resulted in marked improvements in clinical outcomes of premature infants, chiefly by decreasing the incidence and severity of pulmonary disease and death from RDS. Improvements in lung function observed following prenatal therapy with glucocorticoids are synergized by postnatal surfactant therapy (5).

	SURFACTANT BIOLOGY

Knowledge of the biochemistry, physiology, and anatomy of the surfactant system provided an important milestone in the clinical care of neonates (see review by Clements and Avery in this supplement). Seminal observations in the 1950s and 1960s by Pattle (6) and Clements (7) clarified the important role of surfactant in stabilizing the alveolus, and in maintaining lung volumes and gas exchange by reducing surface tension at the air-liquid interface. Reduction of surface tension is accomplished by phosphatidylcholine and other phospholipids that form an interface between air and liquid at the surface of the alveoli. A complex biological system has evolved that controls the synthesis, storage, secretion, function, and catabolism of surfactant in the lung. Fundamental observations regarding the surfactant system led to the application of surfactant replacement therapy, using both synthetic lipids and organic solvent extracts of pulmonary surfactant obtained from animals, which are now routinely used for the prevention and treatment of RDS worldwide.

	MOLECULAR BIOLOGY OF THE SURFACTANT SYSTEM

Advances in molecular biology created the ability to isolate proteins, cDNAs, and genes and to generate recombinant proteins critical to lung function; these provided the basic tools with which to explore the molecular and cellular biology of the surfactant system. The application of molecular biology to the study of surfactant proteins also made possible important advances in an understanding of lung morphogenesis, differentiation, and function that had been previously unapproachable.

Surface tension at the air-liquid interface in the alveolus of the lung creates a collapsing force that may lead to atelectasis; this, in turn, causes hypoxemia and respiratory failure. Surface tension is normally reduced by pulmonary surfactant, a complex mixture of phospholipids and proteins, that forms a distinct phospholipid-rich interface between alveolar gas and the fluid lining terminal air spaces. While it is well recognized that phospholipids, particularly phosphatidylcholine, represent the major component of surface-active material, the critical surfactant-like propertiesrapid spreading and stability during dynamic expansion and compression required to maintain appropriate surface tension reduction during the respiratory cycleare not achieved by phospholipids alone. Avery and Mead (8) recognized that the lungs of premature infants who died from RDS lacked the lipid-rich surfactant material. Measurement of phospholipids in the amniotic fluid is routinely used to assess the risk for RDS of premature infants; either the lecithin-to-sphingomyelin ratio (L/S) or the phospholipid concentration can be used to test for pulmonary maturity (9). The recognition that the hydrophobic surfactant-associated proteins expressed by respiratory epithelial cells were also critical to surfactant-like properties of lung phospholipid represented the next important advance in the understanding of the surfactant system (10). Progress in the knowledge of the surfactant system provided the basis for the now widespread use of exogenous surfactant replacement therapy for prevention and treatment of RDS (11, 12). The scientific study of the surfactant system not only generated molecular reagents (cDNAs, genes, proteins, and antibodies) useful in understanding the structure, function, and biology of surfactant, it also generated tools with which to explore the nature of lung epithelial cell differentiation and gene expression per se.

	SURFACTANT PROTEINS

Four distinct "surfactant"-associated proteins were identified and purified from lung surfactant and ultimately termed surfactant proteins A (SP-A), B (SP-B), C (SP-C), and D (SP-D) (13). Knowledge of their primary structure and immunologic properties made possible the cloning of the cDNAs and genes that encoded the proteins, and provided, in turn, the complete primary structure of the proteins; consequently, strong inferences could be made regarding their potential functions. Surfactant proteins include two distinct classes of polypeptides: (1) SP-A and SP-D, which are large, complex glycosolated proteins, now classified as members of the collectin family of Ca⁺-dependent lectins sharing homology with mannose-binding protein and conglutinin; and (2) SP-B and SP-C, which are extemely hydrophobic, small peptides that confer the surfactant- like properties to lung phospholipids. SP-B and SP-C are the only proteins detected in various animal-based surfactant replacement preparations used for therapy of RDS, including Survanta, Curosurf, Alveofact, and Infrasurf (10). The absence of active SP-B and SP-C in SP-B gene-targeted mice and in infants with hereditary SP-B deficiency demonstrates the critical role of these proteins in lung function at birth (15). Mutations in the SP-B gene cause severe RDS, atelectasis, and respiratory failure in the immediate neonatal period.

While in vitro studies initially suggested that SP-A and SP-D play a role in surfactant function and metabolism, new and increasing evidence supports the concept that they function primarily as components of the innate host defense system in the lung (17). SP-A and SP-D bind to a variety of lung pathogens, including bacteria, viruses, mycoplasma, and fungi, thereby enhancing their opsonization and/or killing by alveolar macrophage. Gene-targeted mice lacking SP-A are susceptible to infection by bacteria, including the common neonatal respiratory pathogens, group B -hemolytic streptococcus, but the mice have no evidence of primary abnormalities of the surfactant system (18, 19). The cDNAs, RNAs, and antibodies generated for study of surfactant proteins have been useful reagents, which have promoted advances in the clinical diagnosis and fostered studies of surfactant homeostasis in various clinical settings.

	SURFACTANT REPLACEMENT

Fujiwara and colleagues (11) demonstrated that the intratracheal administration of an organic solvent extract of cow lung, supplemented with surfactant-like phospholipids, was extremely useful in improving lung function in premature infants with severe RDS. This seminal clinical study, supplemented by extensive studies in laboratory animal models, provided the basis for the now widespread use of exogenous surfactant for prevention and rescue of infants with RDS. Surfactant replacement has profoundly reduced the morbidity and mortality of premature infants worldwide and represents a major advance in the care of infants (20). Exogenous surfactant decreased mortality, pneumothorax, and air leak, and dramatically diminished the severity of bronchopulmonary dysplasia (BPD), the lung disease that often accompanies the therapy of RDS (12).

	RESPIRATORY EPITHELIAL CELL BIOLOGY: GENE REGULATION AND GENE ABLATION

In addition to the advances provided by insights into the structure and function of the surfactant proteins, the reagents generated in this study have provided an important entre into the study of lung biology per se. Surfactant protein genes are expressed almost exclusively in subsets of respiratory epithelial cells beginning early in fetal lung development. Analysis of the regulatory regions of the surfactant protein genes, particularly those of SP-B and SP-C, led to the recognition that all of the surfactant proteins and CCSP, a protein synthesized primarily in Clara cells in the conducting airways, share common regulatory motifs dependent on the interactions between cis- active elements in the target genes and nuclear transcription proteins that bind to the cis-acting regulatory regions in each of the genes (21). Several transcription factors, e.g., thyroid transcription factor-1 (TITF-1) and HNF-3, and other family members of the HNF family, were found to be critical regulators of surfactant gene expression (22). Although TITF-1 and HNF-3 play important roles in the regulation of the expression of surfactant proteins needed at birth, both are also involved in the commitment of progenitor cells of the foregut endoderm to form the respiratory epithelium, and are therefore critical determinants of lung morphogenesis per se. Targeted deletion of HNF-3 blocks node and foregut endoderm formation, whereas ablation of the TITF-1 gene causes thyroid and pulmonary agenesis, demonstrating their critical role in lung morphogenesis (23, 24). The regulatory regions of the surfactant protein and CCSP genes (the latter expressed in nonciliated tracheal and bronchiolar epithelial cells) have been useful in adding or deleting genes of interest in transgenic animals, a procedure useful in identifying the function of genes involved in lung morphogenesis and function in vivo (25).

	TRANSGENIC ANIMALS

Advances in the production of transgenic mice, in which genes are added or deleted, represents an extraordinary advance in the study of lung biology: creating animal models of lung disease provides insight into the pathogenesis and therapy of lung disease in humans. Transgenic animal models have provided direct evidence concerning a probable genetic basis of human lung disease that had previously been regarded as idiopathic. Mutations in the cystic fibrosis transmembrane regulator protein (CFTR) gene cause lethal gastrointestinal abnormalities in mice and cystic fibrosis in humans (26). Disruption of the gene that regulates granulocyte macrophage colony-stimulating factor (GM-CSF) or its receptor causes severe pulmonary alveolar proteinosis (PAP), owing to the inability of the macrophage to catabolize surfactant proteins and lipids in transgenic mice (27, 28). Recent evidence supports the concept that mutations in GM-CSF receptor signaling causes pulmonary alveolar proteinosis in humans. Gene targeting of the surfactant protein B gene in mice caused respiratory failure at birth, which included features identical to those observed in infants dying from hereditary SP-B deficiency, now known to be caused by various mutations in the SP-B gene (15, 16). Such models provide insight into the pathogenesis of many forms of lung disease and provide models for developing new therapies for both genetic and acquired lung disease.

	ADVANCES IN HUMAN GENETICS

Advances in molecular biology are facilitating the identification of gene mutations causing inherited lung disease and are proceeding at an extraordinary pace. This process is being facilitated by the Human Genome Project, and has profound medical and social implications for the future. Nearly every day, genes that cause or contribute to congenital malformations and inherited diseases are identified, thus creating reagents useful in prenatal and postnatal diagnosis. These are useful to study interactions among various genes that contribute to complex traits and that affect genetic susceptibility for common diseases, including those of the lung. Major advances in the understanding of cystic fibrosis (CF), which is caused by genetic abnormalities in the cystic fibrosis transmembrane regulator protein (CFTR), have contributed to our understanding of the relationship between CFTR, sodium and choloride transport, and the pathogenesis of lung disease in humans (26). This knowledge and genetically engineered animal models are useful in testing novel therapies to correct CF. Diagnosis of CF is now made at the molecular level, prenatally or postnatally, and provides the basis both for understanding and predicting the pathogenesis of CF lung disease and for the early therapeutic interventions that may improve clinical outcomes.

	PULMONARY VASCULAR BIOLOGY

In utero, systemic venous blood returning to the right atrium and ventricle is diverted across the patent foramen ovale and patent ductus arteriosus, largely bypassing the pulmonary vascular bed, a situation termed the "fetal circulation." Immediately after birth, changes in the heart and systemic vascular pressure, expansion of the lung, and oxygenation all contribute to establish the postnatal circulation, in which the output from the right ventricle is diverted to the pulmonary arteries. Changes in cardiac output, systemic arterial and pulmonary arterial blood pressures, and closure of both the ductus arteriosus and foramen ovale are all necessary for cardiopulmonary adaptation following birth. This physiologic transition often fails to occur in preterm and ill neonates. Failure to reduce adequately pulmonary arterial pressure in the immediate postnatal period complicates pulmonary adaptation in various diseases, including meconium aspiration, asphyxia, pneumonia, sepsis, and prematurity. Persistence of the patent ductus arteriosus (PDA) complicates the cure of RDS in premature infants. The PDA is now easily diagnosed and can be treated with indomethacin (29). Pulmonary hypertension often accompanies and complicates neonatal cardiovascular adaptation at birth and can cause respiratory and cardiovascular failure. Pulmonary vascular pressures remain elevated in some infants in the absence of clear stimuli, a syndrome initially termed persistent fetal circulation (PFC) or idiopathic pulmonary hypertension (IPH). Thus, advances in our understanding of the molecular, physiological, and anatomical bases of neonatal pulmonary hypertension have provided new therapies that have an important impact on perinatal care.

In vitro studies of vessel contractility in the 1970s led to the identification of a factor released locally, initially termed endothelium-derived relaxing factor (EDRF), that dilated pulmonary and systemic blood vessels. Later, EDRF was identified as nitric oxide (NO), a gas produced locally by the enzyme nitric oxide synthase(s) (30). Activation of nitric oxide synthase generates NO which, in turn, modulates many cellular processes, including inflammation, vascular permeability and tone, immune response, and cell signaling. Since NO is active only locally and is metabolized near the site of synthesis, inhalation of NO has been explored as a therapy to selectively reduce pulmonary vascular pressures in patients with many types of lung disease. Inhaled NO has been tested clinically for the treatment of term infants with pulmonary hypertension (both idiopathic and following meconium aspiration) and is under active investigation for the therapy of infants with RDS and bronchopulmonary dysplasia (31, 32). Extracorporeal membrane oxygenation (ECMO), in which the pulmonary circulation is bypassed, has also been highly successful in rescuing infants with lethal forms of primary pulmonary hypertension and other forms of respiratory failure. ECMO provides substitute oxygenation until the infant's lung and cardiovascular bed recover from the particular injury (33). These scientific advances have led to powerful new therapies that are decreasing both morbidity and mortality of neonates in the intensive care unit.

	INFLAMMATION AND HOST DEFENSES

Even when the premature infant survives the initial problems associated with RDS and lung immaturity, pulmonary infection and sepsis, primarily from bacterial pathogens acquired at birth and postnatally, represent an important cause of morbidity and mortality. The infant's susceptibility to infection likely rests with the immaturity of both innate and acquired immune systems. Fortunately, improvements in antibiotics in the 1950s and 1960s have made an important impact on the treatment of infants with severe bacterial infections such as pneumonia, sepsis, and meningitis. With advances in neonatal respiratory support contributing to markedly improved survival of ever smaller immature infants, who often require prolonged hospitalization, intubation and indwelling catheters, the surviving infants are increasingly subject to nosocomial infection. Nearly one-quarter of extremely premature infants acquire serious bacterial infections in the nursery. The emergence of multiple antibiotic resistance among common bacterial pathogens now represents a serious threat to infants in the neonatal intensive care unit. Further improvements in neonatal outcome will depend upon advances in the development of novel therapies, whether powerful antibiotics or molecules that augment innate and acquired host defenses.

The lung synthesizes and secretes a variety of molecules mediating innate (native) host defense and acquired immunity. These include polypeptides, e.g., defensins, surfactant proteins A and D, complement immunoglobulins, lysozymes, and compounds like hydrochlorous acid, nitric oxide, and reactive oxygen metabolites, which all contribute to the ability of the infant to clear bacterial pathogens from the lung or other tissues. Understanding the nature and mechanisms by which these molecules contribute to local host defense may be an important adjunct to understanding the pathogenesis of infectious lung disease in vulnerable newborns and in high-risk older infants, such as those with CF and inherited immune defects.

	NUTRITIONAL SUPPORT OF THE NEONATE: ROLE OF RETINOIDS

Support of nutritional needs of the preterm infant during the postnatal period of recovery has been critical to improvements in clinical outcome. Total parenteral nutrition, with improvements in delivery of trace minerals, vitamins, lipids, and amino acids, has markedly improved the nutritional status of critically ill neonates as they recover from lung disease. Evidence from clinical studies and from animal models supports the importance of dietary retinoids in lung morphogenesis and repair.

Over 50 years ago, Wolbach and Howe (34) first demonstrated that vitamin A deprivation caused squamous metaplasia of conducting airway epithelium that could be reversed by vitamin A repletion. Subsequent studies showed that human premature newborns had low retinoid liver stores and blood levels at birth (35, 36), and that, if they developed respiratory distress which precluded adequate oral intake, these levels remained low or declined still further. Accordingly, the use of supplemental therapeutic vitamin A was suggested. This rationale was strengthened by the pathological findings of squamous metaplasia of the conducting airways in chronically ill infants who developed BPD, findings similar to those occurring in vitamin A-depleted animals. Although dosage regimens have varied in clinical studies, and studied populations have varied in their initial retinoid levels, the results have strongly suggested that the course of chronic lung disease in very-low-birth-weight infants can be ameliorated by supplemental vitamin A (37, 38).

Arrest in alveolar septation is a frequent finding in infants who died of chronic lung disease in the post-surfactant replacement era, many of whom received corticosteroids to promote extubation. Recent studies by the Massaros (39) of rat pups, whose alveolarization process had been arrested by corticosteroids, showed that retinoic acid administration caused a dramatic increase in the numbers of alveoli, through the proliferation of secondary septae. Clinical trials testing the effects of vitamin A supplementation on neonatal lung disease are ongoing.

	SUMMARY

TOP INTRODUCTION CONCLUSION REFERENCES

Revolutionary advances in the physiology, biochemistry, cell and molecular biology, and genetics of the lung have synergized advances in our understanding of the pathogenesis of both inherited and acquired lung disease. Fortunately, for many commonthough previously lethaldiseases, this knowledge has been rapidly translated into new diagnostic abilities and into highly effective therapies that have reduced morbidity and mortality of infants with RDS and other forms of lung disease in the neonatal period. While clinical hurdles remain, the fundamental scientific advances of the last half century have transformed the once bleak outcome offered premature infants and term infants with severe respiratory disease to one of clear optimism. Further improvements in neonatal care will rest on continued novel observations made through basic science and their rapid translation into applications for both prevention and treatment of respiratory disease in newborn infants.

	Footnotes

Correspondence and requests for reprints should be addressed to Jeffrey A. Whitsett, M.D., Children's Hospital Medical Center, Divisions of Neonatology and Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail: whitj0{at}chmcc.org

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TOP INTRODUCTION CONCLUSION REFERENCES

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