Pulmonary Vascular Development

Structural Development

Pulmonary vascular development begins during the embryonic phase of lung development and is highly coordinated with airway growth ( Fig. 72.1 ). 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. 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. The intra-acinar arteries form later in fetal life and after birth during the alveolar phase of lung development. 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.

Angiogenesis and vasculogenesis are the two primary morphogenetic processes that form the pulmonary vasculature. Vasculogenesis is the de novo organization of blood vessels produced by the migration and differentiation of endothelial progenitor cells or angioblasts. These cells migrate, adhere, and form vascular tubes that become arteries, veins, or lymphatics depending on the local growth factors within the mesenchyme. 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 existing vessels to form new ones. Vasculogenesis and angiogenesis are not necessarily sequential processes and both may occur early in lung development, perhaps giving rise to heterogeneous cell populations in the vasculature. 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.

Fetal lung septation and alveolarization begin at around 32-36 weeks’ gestation in the human fetus 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. The number of small pulmonary arteries increases, both in absolute terms and per unit volume of the lung. For example, in fetal lambs, lung weight increases by fourfold during the last trimester, while the number of small blood vessels in the lungs increases fortyfold. 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. During the final stages of vascular development, the pulmonary capillaries surround the thinning alveolar walls, providing the increased alveolar and capillary surface areas necessary to support gas exchange at birth. Lung blood vessels actively promote alveolar growth during development and contribute to the maintenance of alveolar structures throughout postnatal life, and 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.

The hypoxic conditions of fetal life support lung vascular growth. Hypoxia inducible factors (HIFs) are viewed as the “master regulators” of the transcriptional response to hypoxia and are involved in angiogenesis, survival, and metabolic pathways. HIFs 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. HIF-1 regulates genes involved in angiogenesis (e.g., vascular endothelial growth factor), oxygen transport (e.g., erythropoietin), and energy metabolism (e.g., glycolytic enzymes), among others. Deletion of HIF-1 or HIF-2 produces embryonic lethality, and knockout of HIF-1α in the smooth muscle cells of mice results in pulmonary hypertension.

Vascular endothelial growth factor (VEGF) is expressed in vascular endothelial and smooth muscle cells and in airway epithelium in the fetal lung and is a key mediator of pulmonary vascular development. VEGF transcription is regulated by HIF, and VEGF signaling is transduced via two transmembrane tyrosine kinase receptors, VEGFR-2 and VEGFR-1 expressed on vascular endothelium. Experimental inactivation of VEGF or its receptors before birth results in embryonic lethality characterized by deficient organization of endothelial cells and vascularization; VEGFR-1 and VEGFR-2 inhibitors (e.g., SU5416) impair alveolar development in fetal and newborn rodent models, producing pathologic findings similar to those seen in clinical bronchopulmonary dysplasia (BPD). 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). In adult rats, chronic VEGF receptor inhibition causes pulmonary hypertension and enlarges the air spaces, suggesting that normal VEGF function is required for the maintenance of the pulmonary vasculature and alveolar structures even after lung development is completed.

VEGF-induced lung angiogenesis is in part mediated by nitric oxide (NO). Nitric oxide is best known for its role as a pulmonary vasodilator, but it also plays a key role in lung vascular growth during fetal and neonatal life. Lung endothelial nitric oxide synthase (eNOS) mRNA and protein are present in early fetal life in rats and sheep and increase with advancing gestation. The expression and activity of eNOS are regulated by multiple factors, including hemodynamic forces, hormonal stimuli (e.g., estradiol), paracrine factors (including VEGF), substrate and cofactor availability, oxygen tension, and others. The lungs of late fetal and neonatal eNOS-deficient mice have striking abnormalities of vascularization and are more susceptible to failed vascular growth following exposure to mild hypoxia. Inhibition of VEGF receptors decreased lung eNOS protein expression and NO production, and lung vascular growth is restored by treatment with inhaled NO. However, in neonatal mice that are eNOS deficient, recombinant human VEGF protein treatment restores lung structure after exposure to mild hyperoxia, suggesting that VEGF operates in part through mechanisms independent of eNOS.

Numerous other transcription factors important to lung vascular development have been identified. For example, the Forkhead Box (Fox) family of transcription factors regulate expression of genes involved in cellular proliferation and differentiation. Newborn mice with low FOXF1 levels die with defects in lung vascularization and alveolarization, and endothelial-specific deletion of FOXF1 produces embryonic lethality, growth restriction, and vascular abnormalities in the lung, placenta, and retina. FOXF1 haploinsufficiency is found in 40% of infants with alveolar capillary dysplasia, a lethal disorder of lung vascular development.

Mediators of Fetal Pulmonary Vascular Tone

Pulmonary hypertension is a normal state during fetal life and a necessity for survival on placental support. In the fetal lamb, pulmonary arterial blood has a PO ₂ of approximately 18 mm Hg and oxygen saturation of 50%. Elevated pulmonary vascular resistance permits only ~16% of the combined ventricular output to be directed to the pulmonary vascular bed. Most of the fetal right ventricular output bypasses the lung via the foramen ovale and the ductus arteriosus and is directed to the descending aorta. The blood is then oxygenated in the placenta and returns to the body through the umbilical vein, with a PO ₂ of ~32 to 35 mm Hg in lambs.

Multiple mechanisms maintain high PVR and low pulmonary blood flow in the fetus, including low oxygen tension, low basal production of vasodilator products (such as PgI ₂ and NO), and increased production of vasoconstrictors. 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 key role in maintaining high pulmonary vascular resistance. Maternal hyperoxygenation has no effect on human fetal pulmonary blood flow prior to 26 weeks’ gestation but produces significant increases between 31 to 36 weeks of gestation, suggesting that the capacity of the pulmonary circulation to sense and respond to changes in oxygen tension is developmentally regulated. 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.

Pulmonary artery endothelial cells produce multiple vasoactive mediators that maintain the normal patterns of fetal pulmonary circulation. Proposed fetal pulmonary vasoconstrictors include endothelin-1 and lipid mediators, such as thromboxane and leukotrienes C ₄ and D ₄ , and platelet-activating factor, but none have been shown to play a central regulatory role. Endothelin-1 (ET-1) is produced by vascular endothelium and acts on the ET-A receptors in the smooth muscle cell to induce vasoconstriction by increasing ionic calcium concentrations. A second endothelial receptor, ET-B, on the endothelial cell stimulates NO release and vasodilation. PreproET-1 mRNA (the precursor to ET-1) has been identified in fetal rat lung early in gestation, and high circulating ET-1 levels are present in umbilical cord blood. Although capable of both vasodilator and constrictor responses, ET-1 appears to primarily act as a pulmonary vasoconstrictor in the fetal pulmonary circulation.

Endogenous serotonin (5-HT) production also appears to contribute to the high PVR of the fetus. In fetal lambs, infusions of 5-HT increase PVR, and infusions of ketanserin, a 5-HT 2A receptor antagonist, decrease fetal PVR. Conversely, brief infusions of selective serotonin reuptake inhibitors (SSRI), such as sertraline and fluoxetine, produce potent and sustained elevations of PVR. These findings suggest that 5-HT causes pulmonary vasoconstriction and contributes to maintenance of high PVR in the normal fetus through stimulation of 5-HT 2A receptors and Rho kinase activation. These findings have clinical implications for SSRI treatment for maternal depression.

Additional evidence suggests a critical role for the RhoA/Rho kinase signal transduction pathway, 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 Ca ³⁸⁺ 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.

As gestation progresses, NO and cGMP become central to the emergence of pulmonary vascular reactivity. Chronic inhibition of VEGF receptors downregulates eNOS and induces pulmonary hypertension in the late gestation fetus, indicating the importance of both in the development and function of the developing pulmonary vasculature. Inhibition of eNOS increases basal PVR as early as 0.75 gestation (112 days) in the fetal lamb, indicating that endogenous NOS activity contributes to vasoregulation during late gestation. The response to NO is dependent on activity of its receptor molecule, soluble guanylate cyclase, in the smooth muscle cell ( Fig. 72.2 ). In the fetal lamb, sGC mRNA levels are low through the second trimester and markedly increase toward the end of the third trimester. Intracellular cGMP levels are also tightly regulated by cGMP-specific phosphodiesterase (PDE5) activity. PDE5 expression and activity increase during late gestation, and it plays a critical role in pulmonary vasoregulation during the perinatal period. Overall, prostaglandins appear to play a less important role than NO in regulating fetal and transitional pulmonary vascular tone.

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. In perinatal sheep, both endogenous and exogenous NO increase intracellular cGMP content and relaxation to a greater degree in pulmonary veins than in arteries, effects that are oxygen dependent. Pulmonary veins are also the primary sites of action of certain vasoconstrictors such as endothelin-1 and thromboxane, and pulmonary venous constriction in turn increases microvascular pressures and promotes pulmonary edema.