Pulmonary vascular development begins during the embryonic phase of lung development. Endodermal lung buds arise from the ventral aspect of the foregut by the fifth week of gestation. The pulmonary trunk, derived from the truncus arteriosus, divides into the aorta and pulmonary trunks by 8 weeks of gestation by growth of the spiral aortopulmonary septum.1 The pulmonary trunk connects to the pulmonary arch arteries, which are derived from the sixth branchial arch arteries. The mesenchyme surrounding the lung bud then develops into the vascular network. In the human lung, the pre-acinar vascular branching pattern is present by the 20th week of fetal life.2 The intra-acinar arteries form later in fetal life and after birth during the alveolar phase of lung development.3 Development of the pulmonary veins parallels that of the arteries, but they arise separately from the loose mesenchyme of the lung septa and subsequently connect to the left atrium.4 Angiogenesis and vasculogenesis are the two primary morphogenetic processes that contribute to the formation of the pulmonary vasculature. Vasculogenesis is the de novo organization of blood vessels produced by the migration and differentiation of angioblasts or endothelial progenitor cells. These angioblasts migrate, adhere, and form vascular tubes that become arteries, veins, or lymphatics depending on the local growth factors within the mesenchyme.5 Endothelial precursor cells, after differentiation into the endothelium, contribute either to the expression of smooth muscle phenotype in the surrounding mesenchyme or they recruit existing smooth muscle cells to the forming vessel. Angiogenesis refers to the budding, sprouting, and branching of the existing vessels to form new ones. The relative contribution of vasculogenesis and angiogenesis to lung vascular growth during each stage of lung development remains controversial. Evidence indicates that vasculogenesis and angiogenesis are not necessarily sequential processes and that both may occur early in lung development, perhaps giving rise to heterogeneous cell populations in the vasculature.6 In addition, a process of vascular fusion has been described that connects the angiogenic and vasculogenic vessels to allow for expansion of the vascular network.1,4 As the human fetal lung develops, lung septation and alveolarization begin at around 32 to 36 weeks’ gestation and continue well into postnatal life. During this process, vascular growth and branching are tightly coupled with the growth and branching of the airway epithelium.7 During the final stages of vascular development, the pulmonary capillaries surround the thinning alveolar walls, providing the increased alveolar and capillary surface areas necessary for efficient gas exchange at birth. Lung blood vessels actively promote alveolar growth during development and contribute to the maintenance of alveolar structures throughout postnatal life; disrupted development of one system will have important consequences on the development of the other. Antenatal or postnatal events that affect the developmental program of the fetal or newborn lung may contribute to defective pulmonary vascular development. As gestation and fetal lung growth progress, the number of small pulmonary arteries increases, both in absolute terms and per unit volume of the lung.8 For example, in fetal lambs, lung weight increases fourfold during the last trimester, whereas the number of small blood vessels in the lungs increases 40-fold. This dramatic increase in surface area of small blood vessels prepares the lungs to accept the tenfold increase in blood per unit of lung that occurs at birth. This increase in the capacity of the pulmonary arteries also indicates that vascular constriction must play a strategic role in maintaining high pulmonary vascular tone during fetal life. Numerous local growth and transcription factors regulate fetal lung vascular growth, many of which are favored by the low-oxygen intrauterine environment. Vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), fibroblast growth factors, platelet-derived growth factor, angiopoietin and others play an important role in vascular development and cell differentiation in the developing lung.9,10 Growth factor function is also regulated through specific receptors, including the bone morphogenetic protein receptor 2 (BMPR2), a receptor for the TGF-β superfamily. Mutations of BMPR2 are an important association with familial and idiopathic pulmonary hypertension in adults and children,11 but have not been evident in neonatal pulmonary hypertension.12 Vascular endothelial growth factor (VEGF) is expressed in vascular endothelial and smooth muscle cells and in airway epithelium in the fetal lung, and is central to its vascular development. Two distinct transmembrane tyrosine kinase receptors, VEGF receptors 1 and 2, are expressed on vascular endothelium. Experimental inactivation of VEGF or its receptors results in embryonic lethality characterized by deficient organization of endothelial cells and vascularization, and targeted inactivation in the early postnatal period increases mortality and impairs lung vascular development.13 In the clinical setting, decreased VEGF and VEGFR-1 mRNA and protein is observed in the lungs of premature neonates who died with bronchopulmonary dysplasia (BPD).14 In turn, lung VEGF expression is regulated by members of the hypoxia inducible factor (HIF) family of transcription factors.15,16 Hypoxia inducible factors are heterodimers consisting of oxygen-sensitive α-subunits (HIF-1α, HIF-2α) and constitutively expressed β subunits. Hypoxia stabilizes the α subunit leading to nuclear accumulation and activation of multiple target genes responsible for lung growth, including VEGF.10 Deletion of HIF-1 or HIF-2 produces embryonic lethality, and smooth muscle cell-specific knockout of HIF-1α in mice results in pulmonary hypertension.17 Nitric oxide (NO) is a well-known pulmonary vasodilator, but it also plays a strategic role in lung vascular growth during fetal and neonatal life. For example, lungs of late fetal and neonatal eNOS-deficient mice have striking abnormalities of vascularization,18 and are more susceptible to failed vascular growth following exposure to mild hypoxia.19 Studies also suggest that VEGF-induced lung angiogenesis is in part mediated by NO. For instance, inhibition of VEGF receptors decreased lung eNOS protein expression and NO production, and lung vascular growth could be restored by treatment with inhaled NO.20,21 Multiple mechanisms maintain high pulmonary vascular resistance and low pulmonary blood flow in the fetus, including low oxygen tension, low basal production of vasodilator products (e.g., PGI2, NO), and increased production of vasoconstrictors (e.g., endothelin-1, leukotrienes). The fetal pulmonary circulation also exhibits a marked “myogenic response” as gestation progresses, meaning that the vasculature responds to vasodilatory stimuli with active vasoconstriction. The low oxygen environment of the fetus plays a strategic role in maintaining high pulmonary vascular resistance (PVR). Maternal hyperoxygenation has no effect on human fetal pulmonary blood flow before 26 weeks’ gestation, but produces significant increases between 31 to 36 weeks of gestation, suggesting a developmentally regulated capacity of the pulmonary circulation to sense and respond to changes in oxygen tension.22 Because oxygen regulates activity of enzymes such as nitric oxide synthase, the low oxygen environment of the fetus may maintain low production of vasoactive mediators such as nitric oxide and prostacyclin. For example, maternal hyperoxygenation activates endothelial nitric oxide synthase and increases pulmonary blood flow to postnatal levels in fetal lambs.23 Pulmonary artery endothelial cells produce multiple vasoactive mediators that maintain the normal patterns of fetal pulmonary circulation. Although it is assumed that vasoconstrictors help maintain high pulmonary vascular tone in utero, surprisingly little is known about their specific roles throughout gestation. Some of the proposed fetal pulmonary vasoconstrictors include endothelin-1 and vasoconstrictor products of arachidonic acid metabolism such as thromboxane and leukotrienes. For instance, during fetal life, inhibition of ET-1 causes pulmonary vasodilation.24 Low basal production of vasodilators such as nitric oxide or prostacyclin may maintain high PVR, although pharmacologic inhibition of NO decreases pulmonary blood flow and increases PVR, particularly during the latter part of gestation. Serotonin may also contribute to high PVR in the normal fetus.25 Evidence suggests a critical role for the RhoA/Rho kinase signal transduction pathway,26 a central downstream pathway that promotes vasoconstriction through inactivation of myosin light chain phosphatase, thus increasing calcium sensitivity of the smooth muscle cell. Hypoxia activates RhoA, which increases Ca2+ sensitivity of the contractile myofilaments in the vascular smooth muscle. Rho kinase activity maintains high PVR in the fetal lung, and its inhibition dilates the perinatal circulation by a mechanism independent of NO.26,28 Pulmonary veins are now recognized to function as more than passive conduits; instead, they are reactive vessels that contribute to the regulation of total pulmonary vascular tone and resistance.29,30 In perinatal sheep, both endogenous and exogenous NO produce greater increases in the intracellular cyclic guanosine monophosphate (cGMP) content and relaxation in pulmonary veins than in arteries, effects that are oxygen-dependent and modulated by cGMP.31 Pulmonary veins are also the primary sites of action of certain vasoconstrictors such as endothelin-1 and thromboxane,32,33 and pulmonary venous constriction in turn increases microvascular pressures and promotes pulmonary edema. At birth, the fetal pulmonary circulation must rapidly adapt to direct blood flow to the lungs as the organ of gas exchange. A rapid and dramatic decrease in pulmonary vascular resistance allows half of the combined ventricular output to be redirected from the placenta to the lung, leading to an eight- to tenfold increase in pulmonary blood flow. This increase in pulmonary blood flow increases pulmonary venous return and left atrial pressure, promoting functional closure of the one-way valve of the foramen ovale. Systemic vascular resistance also increases at birth, in large part owing to removal of the low-resistance vascular bed of the placenta. It has been proposed recently that delayed cord clamping may improve the pulmonary vascular adaptation by allowing PVR to fall before the rise in systemic afterload.34 The largest drop in pulmonary vascular resistance occurs shortly after birth, although resistance continues to drop over the first several months of life until it reaches the low levels normally found in the adult circulation. As pulmonary vascular resistance drops and oxygen tension rises, blood flow through the patent ductus arteriosus reverses and the ductus arteriosus functionally closes. This effectively separates the pulmonary and systemic circulations and establishes the normal postnatal circulatory pattern. The stimuli most important in decreasing PVR are lung inflation with gas and an increase in oxygen tension. Each independently decreases PVR and increases pulmonary blood flow, with the largest effects seen when the two events occur simultaneously. Mechanical distention of the lungs initiates the process of rapid structural adaptation of the pulmonary vessels. The external diameter of the nonmuscular arteries increases, and the prominent endothelial cells assume a flattened appearance (Figure 80-1). There is an increase in cell length and surface-to-volume ratio as the cells “spread” within the vessel wall to increase lumen diameter and lower resistance.35 This process is likely facilitated by the paucity of interstitial connective tissue, allowing for greater plasticity of the vessel. In postmortem arterial-injected specimens, the number of nonmuscular arteries that fill with injection material increases rapidly during the first 24 hours, suggesting that there is a rapid increase in the number of precapillary arteries “recruited” into the pulmonary circulation after birth.36 An increase in oxygen tension will reduce PVR independent of the effects of lung inflation. This oxygen response emerges at approximately 70% gestation in the fetal lamb and continues to develop as gestation progresses.37 The full vasodilatory effect of oxygen can be achieved with relatively modest increases in arterial concentrations: Pao2 levels of approximately 50 mm Hg in the near-term fetal lamb will decrease pulmonary vascular resistance and increase pulmonary blood flow to levels comparable to postnatal lambs. 23 In addition to facilitating vasodilation, oxygen may also promote the rapid endothelial spreading and remodeling after birth.38 Finally, the initial increase in pulmonary blood flow increases shear stress in the pulmonary vasculature, which further promotes rapid vasodilation in the pulmonary circulation of the newborn and late-gestation fetus.39 The mechanisms of shear stress–mediated responses are complex, but involve stimulation of K+ channels and activation of NO synthases.40,41 Numerous vasoactive factors interact to facilitate the drop in pulmonary vascular resistance at birth, and increased vasodilator activity is probably more important than decreased vasoconstrictors. Of these, nitric oxide has emerged as a central mediator of pulmonary vascular tone at birth. NO stimulates soluble guanylate cyclase activity and increases cyclic guanosine monophosphate (cGMP) in vascular smooth muscle, producing smooth muscle relaxation via mechanisms involving decreased phosphorylation of myosin light chain (Figure 80-2).42 Pulmonary expression of all three isoforms of nitric oxide synthase (NOS) and its receptor molecule, soluble guanylate cyclase, increase late in gestation, preparing the lung for pulmonary vasodilation. Acute or chronic inhibition of NOS in fetal lambs produces pulmonary hypertension following delivery, illustrating the critical importance of the NO-cGMP pathway in facilitating normal transition.43,44 Expression of cGMP-specific phosphodiesterases also increases during late lung development, which maintains a tight regulation of intracellular cGMP concentrations and signal transduction at birth.45 Prostacyclin is a second central vasodilator that is upregulated in response to ventilation of the lung. Cyclooxygenase (COX) and prostacyclin synthase generate prostacyclin from arachidonic acid. Cyclooxygenase-1 in particular is upregulated during late gestation, leading to an increase in prostacyclin production in late gestation and early postnatal life.46,47 Prostacyclin stimulates adenylate cyclase to increase intracellular cyclic adenosine monophosphate (cAMP) levels, which, similar to cGMP, produce vasorelaxation through a decrease in intracellular calcium concentrations (see Figure 80-2). Despite pharmacologic inhibition of cyclooxygenase, PVR decreases with the onset of air-breathing life, indicating that while prostaglandin I2 (PGI2) is involved in the decrease in PVR at birth, it is not absolutely required.48,49 The decrease in PVR caused by PGI2 at birth is modest in comparison with that induced by NO.50 Persistent pulmonary hypertension (PPHN) describes the failure of normal pulmonary vascular adaptation at birth, and is characterized by elevated pulmonary vascular resistance and right-to-left extrapulmonary shunting of deoxygenated blood that produces severe hypoxemia.51 The PPHN syndrome complicates the course of approximately 10% of term and preterm infants with respiratory failure, and carries a significant risk of death, pulmonary morbidity, and neurodevelopmental impairment.52 Persistent pulmonary hypertension is often thought of as falling into one of three categories: (1) the abnormally constricted pulmonary vasculature caused by lung parenchymal diseases such as meconium aspiration syndrome, respiratory distress syndrome, or pneumonia; (2) the lung with normal parenchyma and remodeled pulmonary vasculature, also referred to as idiopathic PPHN; or (3) the hypoplastic vasculature as seen in congenital diaphragmatic hernia. Although idiopathic pulmonary hypertension is responsible for only 10% to 20% of all infants with PPHN, severe cases of PPHN associated with parenchymal disease are almost always complicated by a significant degree of vascular remodeling. Significant pulmonary vascular remodeling occurs antenatally in infants presenting with early, severe PPHN, characterized by vessel wall thickening and smooth muscle hyperplasia. An important feature includes extension of the smooth muscle to the level of the intra-acinar arteries (Figure 80-3), which does not normally occur until much later in postnatal development.53 Because it is difficult to gain sufficient mechanistic insights in the clinical setting, animal models have provided many of the insights into the antenatal and postnatal vascular abnormalities associated with PPHN. Potential causes of antenatal remodeling of the pulmonary vasculature are strategic environmental exposures or genetic risk factors. Maternal use of salicylates was one of the earliest triggers identified for PPHN,54 and earlier reports suggested a strong association between clinically significant PPHN and the maternal use of NSAIDs (aspirin, ibuprofen, naproxen).55,56 However, a recent large multicenter epidemiologic study found no significant association between PPHN risk and maternal use of nonaspirin NSAIDs or ibuprofen use during third trimester.57 Conflicting evidence exists whether maternal selective serotonin reuptake inhibitor (SSRI) use during pregnancy increases the risk of persistent pulmonary hypertension of the newborn. In animal studies, newborn rats exposed in utero to fluoxetine developed pulmonary vascular remodeling, abnormal oxygenation, and higher mortality when compared with vehicle-treated controls.58 Brief infusions of sertraline and fluoxetine in fetal lambs directly increased PVR, which was sustained for at least 1 hour after the infusion was stopped.25 To date, six retrospective population-based studies have presented a range of findings: Three of the studies reported a positive association with adjusted odds ratio of 2.1 to 6.1, although little information is provided on the severity of PPHN.59–61 Three additional retrospective cohort studies found no association.62–64 In addition, other reviews have highlighted the challenges of distinguishing the impact of SSRI use from the impact of depression (including an increased risk of prematurity) on the risk for PPHN.65 The FDA currently concludes that the evidence is currently insufficient to conclude that SSRI use in pregnancy causes PPHN and recommends that health care professionals treat depression during pregnancy as clinically appropriate. Unlike pulmonary hypertension in children or adults, PPHN is rarely familial, and few genetic risk factors have been identified. Children with Down syndrome (trisomy 21) commonly develop pulmonary hypertension in association with structural heart defects, and an elevated incidence of PPHN without associated cardiac disease has also been reported.66,67 In a Dutch cohort with excellent early ascertainment of Down syndrome, PPHN was documented in 5.2% of the infants.68 Infants with Down syndrome are similarly overrepresented in the extracorporeal membrane oxygenation (ECMO) registry maintained by the Extracorporeal Life Support Organization, and their survival to discharge is significantly decreased compared with the general population.69 A recent single-center study reported the results of rigorous genotype analysis of 88 neonates with documented PPHN.12 No differences were noted in most candidate genes, including BMPR2 and nitric oxide synthase. However, PPHN was significantly associated with genetic variants for corticotropin releasing hormone receptor-1 (CRHR1) and CRH-binding protein and with significantly increased levels of 17-hydroxyprogesterone. These data are supported by animal data indicating that antenatal and postnatal steroids reduce oxidant stress and normalize nitric oxide synthase and phosphodiesterase function in experimental PPHN.70,71 Disruptions in the production or function of vasoactive mediators at birth will also lead to pulmonary vasoconstriction and/or remodeling. Strong evidence from animal models and human infants indicates that disruptions of the NO-cGMP, prostacyclin-cAMP, and endothelin signaling pathways are among those that play an important role in the vascular dysfunction associated with PPHN. The NO-cGMP pathway has been a topic of particularly intense investigation over the last decade, in part because of the ability to deliver inhaled nitric oxide gas as a therapeutic agent. Decreased expression and activity of eNOS have been documented in animal models,72,73 and decreased eNOS expression has been reported in umbilical venous endothelial cell cultures from human infants with meconium staining who develop PPHN.74 Furthermore, PPHN is associated with disrupted eNOS activity through “uncoupling” of the enzyme, which reduces synthesis of nitric oxide and promotes production of reactive oxygen species such as superoxide. Downstream vascular abnormalities include reduced levels of the critical second messenger, cGMP. For instance, in vascular smooth muscle from PPHN lambs, reduced cGMP concentrations are generated in response to NO, in part because of reduced activity of soluble guanylate cyclase and increased activity of cGMP-specific phosphodiesterase (PDE5).75–78 Phosphodiesterase-5 is highly expressed in the perinatal lung, and its activity is elevated in fetal lambs with chronic intrauterine pulmonary hypertension,76,79 and striking increases in activity emerge in response to mechanical ventilation and oxygen therapy.76,80 Less is known about the role of abnormal prostacyclin-cAMP signaling in PPHN. Some data suggest that abnormal prostacyclin synthesis and downstream adenylate cyclase signaling occur, analogous to the changes reported for NO-cGMP signaling.81,82 In addition, elevated production of the vasoconstrictor arachidonic acid metabolite, thromboxane, plays a role in pulmonary hypertension produced by chronic hypoxia.83 Circulating levels of the potent vasoconstrictor endothelin-1 (ET-1) are elevated in lambs and newborn infants with PPHN.84,85 Endothelin-1 effects are mediated through two receptors, ET-A receptors on smooth muscle cells that mediate vasoconstriction and ET-B receptors on endothelial cells that mediate vasodilation. There is evidence that the balance of ET receptors is shifted to the vasoconstrictor (ET-A) pathways.86 In addition, endothelin may affect vascular tone by increasing production of reactive oxygen species such as superoxide and hydrogen peroxide, which also act as vasoconstrictors.87 Therefore, the elevated endothelin levels in PPHN may increase vasoconstriction through preferential stimulation of ET-A receptors and through increased production of superoxide. Oxidant stress plays an important role in the pathogenesis of PPHN both antenatally and following birth. An increase in reactive oxygen species such as superoxide and hydrogen peroxide in the smooth muscle and adventitia of pulmonary arteries has been demonstrated in neonatal animal models of pulmonary hypertension.88,89 Possible sources for elevated concentrations of reactive oxygen species include mitochondrial oxidant stress, increased expression and activity of NADPH oxidases, uncoupled NOS activity, and a reduction in superoxide dismutase (SOD) activity.89–93 Once present in the lung, elevated concentrations of reactive oxygen species (ROS) promote vasoconstriction, increase production of other constrictors such as isoprostanes and peroxynitrite, increase vascular smooth muscle cell proliferation, and blunt cGMP accumulation through increased activity of cGMP-specific phosphodiesterases.94 See Chapters 74 and 78. Congenital diaphragmatic hernia (CDH) affects approximately 1 in 2500 to 3000 pregnancies when factoring in prenatal diagnosis, and represents approximately 8% of all major congenital anomalies. Congenital diaphragmatic hernia includes abnormal diaphragm development, herniation of abdominal viscera into the chest, and a variable degree of lung hypoplasia. Herniation occurs most often in the posterolateral segments of the diaphragm, and 80% of the defects occur on the left side. Severe CDH develops early in the course of lung development, and an arrest in the normal pattern of airway branching occurs in both lungs, resulting in reduced lung volume and impaired alveolarization. A similar developmental arrest occurs in pulmonary arterial branching, resulting in reduced cross-sectional area of the pulmonary vascular bed, thickened media and adventitia of small arterioles, and abnormal medial muscular hypertrophy extending distally to the level of the acinar arterioles.95 Although in utero lung compression by herniated viscera has been implicated as the primary mechanism responsible for producing the lung abnormalities of CDH, some evidence suggests that decreased pulmonary blood flow alone causes lung hypoplasia.96 After birth, PVR often remains at suprasystemic levels, causing extrapulmonary right-to-left shunting across the foramen ovale and ductus arteriosus and profound hypoxemia. High PVR in the newborn with CDH is related to multiple factors, including the small cross-sectional area of pulmonary arteries, structural vascular remodeling, vasoconstriction with altered reactivity, and LV dysfunction causing pulmonary venous hypertension.97,98 The mediators of altered pulmonary vascular reactivity in CDH are not well understood, although substantial evidence points to disruptions in NO-cGMP and endothelin signaling.99 Abnormalities of cardiac development and function also play an important role in the pathophysiology of CDH. The size of the left ventricle, left atrium, and intraventricular septum are hypoplastic in infants who die of CDH relative to age-matched controls,100 perhaps because of low fetal and postnatal pulmonary blood flow as well as compression by the hypertensive right ventricle. Left ventricular hypoplasia and dysfunction increase left atrial and pulmonary venous pressures, and the resulting pulmonary venous hypertension diminishes the clinical response to inhaled NO during the first few days of life. Some infants may have exceptionally severe left ventricular dysfunction that leads to dependence on the right ventricle for systemic perfusion; this subset may benefit from clinical strategies that maintain patency of the ductus arteriosus.
Pulmonary Vascular Development
Fetal Pulmonary Vascular Development
Structural Development
Mediators of Fetal Pulmonary Vascular Tone
Pulmonary Vascular Transition
Vasoactive Mediators of the Pulmonary Vascular Transition
Abnormalities of Pulmonary Vascular Development
Persistent Pulmonary Hypertension of the Newborn
Congenital Diaphragmatic Hernia
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