Physiology of the Fetal Pulmonary Circulation

Along with the rapid and dramatic progression of lung vascular growth and structure during development, the fetal pulmonary circulation also undergoes maturational changes in function. PVR is high throughout fetal life, especially in comparison with the low resistance of the systemic circulation. As a result, the fetal lung receives, 3-8% of combined ventricular output, with most of the right ventricular output crossing the ductus arteriosus (DA) to the aorta. Pulmonary arterial pressure and blood flow increase with advancing gestational age, along with increasing lung vascular growth (29). Despite this rise in vascular surface area, PVR increases with gestational age when corrected for lung or body weight, suggesting that vascular tone actually increases during late gestation and is high before birth. Studies of the human fetus support physiological data from fetal lambs (65). Based on multiple Doppler assessments of pulmonary artery velocity waveforms, fetal pulmonary artery impedance decreases slightly during the early third trimester, but does not decrease further during the latter stage of the third trimester despite ongoing vascular growth (65).

Several mechanisms contribute to high basal PVR in the fetus, including low oxygen tension, relatively low basal production of vasodilator products (such as PGI2 and NO), increased production of vasoconstrictors (including ET-1 or leukotrienes), and altered smooth muscle cell reactivity (such as enhanced myogenic tone). In addition to high PVR, the fetal pulmonary circulation is also characterized by progressive changes in responsiveness to vasoconstrictor and vasodilator stimuli (or vasoreactivity). In the ovine fetus, the pulmonary circulation is initially poorly responsive to vasoactive stimuli during the early canalicular period, and responsiveness to several stimuli increases during late gestation. For example, the pulmonary vasoconstrictor response to hypoxia, and the vasodilator response to increased fetal Po2 and acetylcholine increase with gestation (52, 70). As observed in the sheep fetus, human studies also demonstrate maturational changes in the fetal pulmonary vascular response to increased Pa02 (65). Maternal hyperoxiadoes not increase pulmonary blood flow between 20-26 weeks gestation, but increased Pa02 caused pulmonary vasodilation in the 31-36 week fetus. These findings suggest that in addition to structural maturation and growth of the developing lung circulation, the vessel wall also undergoes functional maturation, leading to enhanced vasoreactivity during fetal life.

Mechanisms that contribute to progressive changes in pulmonary vasoreactivity during development are uncertain, but are likely due to maturational changes in endothelial cell function, especially with regard to NO production (1, 2, 4, 9). Lung endothelial NO synthase (eNOS, type III) mRNA and protein is present in the early fetus and increases with advancing gestation in utero and during the early postnatal period in rats and sheep (26, 56, 59). The timing ofthis increase in lung eNOS content immediately precedes and parallels changes in the capacity to respond to endothelium-dependent vasodilators, as assessed by in vivo and in vitro studies (4, 39). The timing of this increase in lung endothelial NOS content coincides with the capacity to respond to endothelium-dependent vasodilator stimuli, such as oxygen and acetylcholine. In contrast, fetal pulmonary arteries are already quite responsive to exogenous NO much earlier in gestation (4, 39). Overall, the ability of exogenous NO to dilate fetal pulmonary arteries is greater at less mature gestational ages than responsiveness to vasodilator stimuli that require the endothelium to release endogenous NO. These findings suggest that the ability of the endothelium to produce or sustain production of NO in response to specific stimuli during maturation lags the capacity of fetal pulmonary smooth muscle to relax to NO. This may account for clinical observations that extremely premature newborns are highly responsive to inhaled NO (7).

Although most studies ofthe perinatal lung have focused on the role ofeNOS in vasoregulation, the other NOS isoforms, including neuronal NOS (nNOS; type I) and inducible NOS (iNOS; type II), have been identified by immunostaining in the rat, sheep and human fetal lung (6, 62-64, 72, 81). Lung nNOS mRNA and protein increases in parallel with eNOS expression during development in the fetal rat. Inducible (type II) NOS has also been detected in the ovine fetal lung, and is predominantly expressed in airway epithelium and vascular smooth muscle, with little expression in vascular endothelium. Whether the "non-endothelial" (types I and II) isoforms contribute to the physiologic responses of NO-dependent modulation of fetal pulmonary vascular tone has been controversial. Treatment of pregnant rats with an iNOS-selective antagonist caused constriction of the great vessels (main pulmonary artery and thoracic aorta) and DA in fetal rats. Selective iNOS and nNOS antagonists increase fetal PVR and inhibit shear stress vasodilation at doses that do not inhibit acetylcholine -induced pulmonary vasodilation (6, 62-64). These findings support the speculation that iNOS and nNOS may also modulate pulmonary vascular tone in utero and at birth (see below).

NOS expression and activity are affected by multiple factors, including oxygen tension, hemodynamic forces, hormonal stimuli (e.g., estradiol), paracrine factors (including vascular endothelial growth factor), substrate and cofactor availability, superoxide production (which inactivates NO), and others (Fig. 1) (24, 47, 57, 58). Recent studies have shown that estradiol acutely releases NO and upregulates eNOS expression in fetal pulmonary artery endothelial cells (47). However, in vivo findings on the effects of estradiol differ from these observations of isolated endothelial cells in vitro. Although estradiol does not cause acute fetal pulmonary vasodilation in vivo, prolonged estradiol treatment (24-72 hours) causes marked vasodilation, which is sustained despite cessation of estradiol infusion (57, 58). In contrast with estradiol, vascular endothelial growth factor (VEGF) acutely releases NO and causes pulmonary vasodilation in vivo. Chronic inhibition of VEGF receptors downregulates eNOS and induces pulmonary hypertension in the late gestation fetus (24). These findings illustrate that diverse hormonal and paracrine factors can regulate NOS expression and activity and affect lung vascular maturation during development.

Figure 1. Physiology of the NO regulation in the developing lung.

In addition to transcriptional and translational regulation, multiple factors regulate NO production through alterations in NOS activity. NOS is a heterodimer with both reductase and oxygenase domains. When there is an abundance of availability of substrates such as L-arginine and the pteridine cofactor tetrahydrobiopterin, NADPH oxidation and NO synthesis remain coupled and NO production is favored. When concentrations of one or more factors are decreased, eNOS is uncoupled and generates superoxide. Under certain conditions, NOS may generate reactive oxygen species (superoxide and H202) rather than NO. The balance of NO vs. superoxide production depends on numerous factors. Heat shock protein 90 (Hsp90), a chaperone molecule, has been recently described as a factor that associates with NOS, stabilizes biopterin binding, and thus facilitates NO release. Konduri et al. have shown that association of Hsp90 with NOS is required for NO production in response to ATP in pulmonary arteries isolated from late-gestation fetal lambs (42).

Vascular responsiveness to endogenous or exogenous NO is also dependent upon several smooth muscle cell enzymes, including soluble guanylate cyclase

(sGC), cGMP-specific phosphodiesterase (PDE5), and cGMP kinase (16, 28, 79, 81, 88). NO stimulates sGC by binding to the prosthetic heme of the enzyme, causing up to a 400-fold activation of the purified enzyme. Several studies have shown that sGC, which produces cGMP in response to NO activation, is active before 0.7 of term gestation in the ovine fetal lung. Similar to the pattern of expression for eNOS, sGC levels are high late in gestation, and are greater than those observed in the adult lung.

Cyclic nucleotide phosphodiesterases (PDE) constitute the only known pathway for the hydrolysis of cGMP, and control the intensity and duration of its signal transduction. At least eleven families of PDE isoenzymes have been identified, and at least four PDE isoenzymes have been identified in human pulmonary arteries. PDE5, a cGMP-binding, cGMP-specific isoform is found in especially high concentrations in the lung, and is active in the fetus. In the fetal lung, PDE5 expression has been localized to vascular smooth muscle; and similar to NOS and sGC, PDE5 activity is high in fetal lung in comparison with the postnatal lung. Infusions of selective PDE5 antagonists, including zaprinast, dipyridamole, E4021 and DMPPO, cause potent and sustained fetal pulmonary vasodilation. Thus, PDE5 activity appears to play a critical role in pulmonary vasoregulation during the perinatal period, and must be accounted for in assessing responsiveness to endogenous NO and related vasodilator stimuli. While most studies have focused on PDE5, there are many PDE families and isoforms that vary in their specificity for binding or metabolizing cGMP, cAMP or both. PDE are likely important mediators of cross talk between cGMP and cAMP signaling pathways, and other PDE isoforms may be important in the response to NO.

Functionally, the NO-cGMP cascade plays several important physiologic roles in vaso-regulation of the fetal pulmonary circulation. These include: a) modulation ofbasal PVR in the fetus (3); b) mediating the vasodilator response to specific physiologic and pharmacologic stimuli (3, 17); and c) opposing the strong myogenic tone in the normal fetal lung (77). Intrapulmonary infusions of NOS inhibitors increase basal PVR by 35% at least as early as 0.75 gestation (112 days; canalicular period) in the fetal lamb, suggesting that endogenous NOS activity appears to contribute to vasoregulation throughout late gestation. NOS inhibition also selectively blocks pulmonary vasodilation to such stimuli as acetylcholine, 02, and shear stress in the normal fetus. Recent studies further suggest that NO release plays an additional role in modulating high intrinsic or myogenic tone in the fetal pulmonary circulation. The fetal lung circulation is characterized by its ability to oppose sustained pulmonary vasodilation during prolonged exposure to vasodilator stimuli. For example, increased Pa02 increases fetal pulmonary blood flow during the first hour of treatment; however, blood flow returns toward baseline values overtime despite maintaining high Pa02 (9). Similar responses are observed during acute hemodynamic stress (shear stress) caused by partial compression of the DA (1) or with infusions of several pharmacologic agents (2). These findings suggest that unique mechanisms exist in the fetal pulmonary circulation that oppose vasodilation and maintain high

PVR in utero.

We have speculated that this transient vasodilator response reflects the presence of an augmented myogenic response within the fetal pulmonary circulation. The myogenic response is commonly defined by the presence of increased vasoconstriction caused by acute elevation of intravascular pressure or "stretch stress." Past in vitro studies demonstrated the presence of a myogenic response in sheep pulmonary arteries, and that fetal pulmonary arteries have greater myogenic activity than neonatal or adult arteries. More recent studies of intact fetal lambs have shown that high myogenic tone is normally operative in the fetus, and contributes to maintaining high PVR in utero (49, 77, 78), and that acute inhibition ofNO production unmasks a potent myogenic response. Further work suggests that downregulation of NO, as observed in experimental pulmonary hypertension, may further increase myogenic activity, increasing the risk for unopposed vasoconstriction in response to stretch stress at birth.

Since eNOS protein is present at a stage of lung development when blood flow is absent or minimal, it has been hypothesized that NO may potentially contribute to angiogenesis during early lung development (26). There are conflicting data regarding the effects of eNOS activity in promoting new vessel formation in experimental models of angiogenesis. Although NO can inhibit endothelial cell mitogenesis and proliferation, NO has also been shown to mediate the angiogenic effects of substance P and vascular endothelial growth factor in vitro. Growing bovine aortic endothelial cells in culture express greater eNOS mRNA and protein than confluent cells, but NOS inhibition does not affect their rate of proliferation in vitro. Recent studies have shown that NOS inhibition blocks VEGF-induced tube formation by fetal pulmonary artery endothelial cells in vitro, suggesting that NO may modulate lung vascular development. In addition angiogenesis, NO also modulates vascular wall structure by decreasing smooth muscle cell proliferation in vitro. Several studies have examined the role ofNO in vascular growth and remodeling, but its effects vary between experimental settings, and the effects of NO on lung growth and structure in vivo are still controversial.

Although other vasodilator products, including PGI2, are released upon stimulation of the fetal lung (e.g., increased shear stress), basal prostaglandin release appears to play a less important role than NO in fetal pulmonary vasoregulation. Cyclooxygenase inhibition has minimal affect on basal PVR and does not increase myogenic tone in the fetal lamb, PGI2 vasodilation is blocked after NOS inhibition in the fetal lamb. The physiologic roles of other dilators, including adrenomedullin, adenosine and endothelium-derived hyperpolarizing factor (EDHF), are uncertain. EDHF is a short-lived product ofcytochrome P450 activity that is produced by vascular endothelium, and has been found to cause vasodilation through activation of Ca2+-activated K+ channels in vascular smooth muscle in vitro (18). K+-channel activation appears to modulate basal PVR and vasodilator responses to shear stress and increased oxygen tension in the fetal lung, and may be particularly important in relaxation responses in resistance vessels. Whether this is partly related to EDHF activity remains unknown.

Carbon monoxide (CO) is another gaseous molecule that is produced by heme-oxygenase and has been shown to have several vascular effects, including vasodilation in the adult systemic and pulmonary vascular beds. CO may act in part through activation of sGC, increasing cGMP content in vascular smooth muscle, and causing vasodilation, as described for NO. Despite several studies that suggest an important role in vasoregulation in some models, CO has yet to be shown to play an important physiologic role in the perinatal lung. For example, inhaled CO treatment of the late gestation fetal lamb had no affect on PVR, and infusions of a heme-oxygenase inhibitor did not alter basal pulmonary vascular tone. Further studies are needed to clarify the physiologic importance of CO in the developing lung circulation.

Vasoconstrictors have long been considered as potentially maintaining high PVR in utero. Several candidate products, such as thromboxane A2, leukotrienes C4 and D4, platelet-activating factor and ET-1, have been extensively studied. Thromboxane A2, a potent pulmonary vasoconstrictor that has been implicated in animal models of Group B Streptococcal sepsis, does not appear to influence PVR in the normal fetus. In contrast, inhibition of leukotriene production causes fetal pulmonary vasodilation, suggesting a potential role for lipoxygenase products in vasoregulation (73).

ET-1, a potent vasoconstrictor and co-mitogen that is produced by vascular endothelium, has been demonstrated to play a key role in fetal pulmonary vasoregulation (31). PreproET-1 mRNA (the precursor to ET-1) was identified in fetal rat lung early in gestation, and high circulating ET-1 levels are present in umbilical cord blood. Although ET-1 causes an intense vasoconstrictor response in vitro, its effects in the intact pulmonary circulation are complex. Brief infusions of ET-1 cause transient vasodilation, but PVR progressively increases during prolonged treatment. The biphasic pulmonary vascular effects during pharmacologic infusions of ET-1 are explained by the presence of at least two different ET receptors. The ETB receptor, localized to the endothelium in the sheep fetus, mediates the ET-1 vasodilator response by releasing NO. A second receptor, the ETA receptor, is located on vascular smooth muscle, and when activated, causes marked constriction. Although capable ofboth vasodilator and constrictor responses, ET-1 is more likely to play an important role as a pulmonary vasoconstrictor in the normal fetus (31). This is suggested in extensive fetal studies that have shown that inhibition of the ETA receptor decreases basal PVR and augments the vasodilator response to shear stress-induced pulmonary vasodilation. Thus, ET-1 is likely to modulate PVR through the ETa and ET0 receptors, but its predominant role is as a vasoconstrictor through stimulation of the ETA receptor.

Several studies have shown that NO and ET-1 regulate each other through autocrine feedback loops. Endothelium-derived nitric oxide decreases endothelin production via a cGMP-dependent mechanism in cultured endothelial cells. Other studies have suggested that inhalation of NO increases levels of plasma ET-1 in young lambs, and that ET-1 in turn reduces NOS activity through activation of ETA receptors (48, 87). Activation of ETA receptors has been shown to increase superoxide production in vascular smooth muscle cells, which may provide a mechanism for the effects of ET-1 on NOS activity. These complex relationships are controversial and need further study.

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