Permanent closure of the full-term newborn ductus arteriosus (DA) occurs only if profound hypoxia develops within the vessel wall during luminal obliteration. We used fetal and newborn baboons and lambs to determine why the immature DA fails to remodel after birth. When preterm newborns were kept in a normoxic range (Pa(O(2)): 50-90 mmHg), 86% still had a small patent DA on the sixth day after birth; in addition, the preterm DA wall was only mildly hypoxic and had only minimal remodeling. The postnatal increase in Pa(O(2)) normally induces isometric contractile responses in rings of DA; however, the excessive inhibitory effects of endogenous prostaglandins and nitric oxide, coupled with a weaker intrinsic DA tone, make the preterm DA appear to have a smaller increment in tension in response to oxygen than the DA near term. We found that oxygen concentrations, beyond the normoxic range, produce an additional increase in tension in the preterm DA that is similar to the contractile response normally seen at term. We predicted that preterm newborns, kept at a higher Pa(O(2)), would have increased DA tone and would be more likely to obliterate their lumen. We found that preterm newborns, maintained at a Pa(O(2)) >200 mmHg, had only a 14% incidence of patent DA. Even though DA constriction was due to elevated Pa(O(2)), obliteration of the lumen produced profound hypoxia of the DA wall and the same features of remodeling that were observed at term. DA wall hypoxia appears to be both necessary and sufficient to produce anatomic remodeling in preterm newborns.

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Nonselective cyclooxygenase (COX) inhibitors are potent tocolytic agents; however, they also have adverse fetal effects such as constriction of the fetal ductus arteriosus. Recently, selective COX-2 inhibitors have been used in the management of preterm labor in the hope of avoiding fetal complications. However, both COX-1 and -2 are expressed by cells of the ductus arteriosus. We used fetal lambs (0.88 gestation) to assess the ability of selective COX-2 inhibitors celecoxib and NS398 to affect the ductus arteriosus. Both selective COX-2 inhibitors decreased PGE(2) and 6ketoPGF(1alpha) production in vitro; both inhibitors constricted the isolated ductus in vitro. The nonselective COX-1/COX-2 inhibitor indomethacin produced a further reduction in PG release and an additional increase in ductus tension in vitro. We used a prodrug of celecoxib to achieve 1.4 +/- 0.6 microg/ml, mean +/- standard deviation, of the active drug in vivo. This concentration of celecoxib produced both an increase in pressure gradient and resistance across the ductus; celecoxib also decreased fetal plasma concentrations of PGE(2) and 6ketoPGF(1alpha). Indomethacin (0.7 +/- 0.2 microg/ml) produced a significantly greater fall in ductus blood flow than celecoxib and tended to have a greater effect on ductus resistence in vivo. We conclude that caution should be used when recommending COX-2 inhibitors for use in pregnant women, because COX-2 appears to play a significant role in maintaining patency of the fetal ductus arteriosus.

The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal P-globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switchIng.The timing of the fetal (y-globin) to adult (,f-globin) hemoglobin switch appears to be on a set developmental clock in many mammalian species (1, 2). Prolonging fetal y-globin gene expression is of considerable interest for therapeutic potential in ameliorating the P-globin chain diseases such as sickle cell anemia and ,3-thalassemia (3) and would be of value for investigating developmental control mechanisms. Fetuses that develop in the presence of maternal diabetes have a markedly delayed fetal-to-adult globin gene switch before birth (4, 5). In these subjects, elevated plasma concentrations of a labile analogue of butyric acid, a-amino-n-butyric acid, are reported (6). We examined the effects of this metabolite on globin gene expression in neonatal and fetal erythroid cell cultures in vitro and found that it increased y-globin gene expression and decreased P3-globin gene expression (7).Therefore, we sought to examine the effect of butyric acid in an in vivo fetal animal model and found that butyrate infusions into the fetal lamb indeed delay the biologic clock for fetal-to-adult globin gene switching. at frequent intervals for arterial blood gas analysis, hemoglobin, and analysis of globin chain synthesis and electrophoresis as described (9, 10). Metabolic, renal, and hepatic functions were monitored weekly. Results in butyratetreated fetuses were compared to results in 12 control fetuses, which were similarly catheterized in utero, including 3 that were infused with normal saline. MATERIALS AND METHODS RESULTSIn control fetuses, f3-globin chain production began at about gestation day 112 and steadily increased to 45% of non-aglobin at gestation day 125 and to 80-100% of non-a-globin by term at gestation days 140-145 (Fig. 1). We infused sodium butyrate into four fetal lambs; in three, the globin gene switch was delayed. Two of these three animals, which had about 10-15% P-globin synthesis at the beginning of the infusion, produced at term about half of the amount of adult hemoglobin produced by the control group. In the third lamb, adult globin synthesis was very low (4%) at the start of the butyrate infusion and remained...

Anatomic remodeling and permanent closure of the newborn ductus arteriosus appears to require the development of intense hypoxia within the constricted vessel wall. Hypoxic ductus smooth muscle cells express vascular endothelial cell growth factor (VEGF). We studied premature baboons and sheep to determine the effects of VEGF inhibition (in baboons) and VEGF stimulation (in sheep) on ductus remodeling in vivo. For study of VEGF inhibition, 13 premature newborn baboons (68% gestation) were treated with inhibitors of both prostaglandin and nitric oxide production to constrict the ductus and induce ductus wall hypoxia. Six received a neutralizing monoclonal antibody against VEGF (A.4.6.1, mAbVEGF), while seven did not. Both groups developed the same degree of ductus constriction, tissue hypoxia, and VEGF expression. The mAbVEGF treatment produced a significant ( P < 0.05) reduction in ductus vasa vasorum ingrowth and neointima formation (due to both a decrease in luminal endothelial cell proliferation and a decrease in smooth muscle cell migration into the neointima). For study of VEGF stimulation, nine sheep fetuses (70% gestation) had their ductus wall injected with either VEGF ( n = 6) or vehicle ( n = 4) in vivo. VEGF administration produced a significant ( P < 0.05) increase in vasa vasorum ingrowth and neointima formation. We conclude that VEGF plays an important role in the formation of neointimal mounds and vasa vasorum ingrowth during permanent ductus closure.

Use of cyclooxygenase (COX) inhibitors to delay preterm birth is complicated by in utero constriction of the ductus arteriosus and delayed postnatal closure. Delayed postnatal closure has been attributed to loss of vasa vasorum flow and ductus wall ischemia resulting from constriction in utero. We used the murine ductus (which does not depend on vasa vasorum flow) to determine whether delayed postnatal closure may be because of mechanisms independent of in utero constriction. Acute inhibition of both COX isoforms constricted the fetal ductus on days 18 and 19 (term) but not earlier in gestation; COX-2 inhibition constricted the fetal ductus more than COX-1 inhibition. In contrast, mice exposed to prolonged inhibition of COX-1, COX-2, or both COX isoforms (starting on day 15, when the ductus does not respond to the inhibitors) had no contractile response to the inhibitors on days 18 or 19. Newborn mice closed their ductus within 4 h of birth. Prolonged COX inhibition on days 11-14 of gestation had no effect on newborn ductal closure; however, prolonged COX inhibition on days 15-19 resulted in delayed ductus closure despite exposure to 80% oxygen after birth. Similarly, targeted deletion of COX-2 alone, or COX-1/COX-2 together, impaired postnatal ductus closure. Nitric oxide inhibition did not prevent the delay in ductus closure. These data show that impaired postnatal ductus closure is not the result of in utero ductus constriction or upregulation of nitric oxide synthesis. They are consistent with a novel role for prostaglandins in ductus arteriosus contractile development.

Inflammatory processes play a crucial role in the pathogenesis of atherosclerosis and other vascular disorders. We hypothesized that ischemia of the ductus arteriosus might initiate an active inflammatory response that could play a role in ductus remodeling and permanent closure. To test this hypothesis, we studied effects of postnatal ductus construction on inflammatory processes and remodeling in late-gestation fetal and newborn baboons, and preterm newborn baboons. After postnatal ductus constriction, the expression of several genes known to be essential for atherosclerotic remodeling [vascular cell adhesion molecule (VCAM)-1, E-selectin, IL-8, macrophage colony stimulating factor-1, CD154, interferon-␥, IL-6, and tumor necrosis factor-␣] was increased in the ductus wall. We were unable to detect intercellular adhesion molecule (ICAM)-1, ICAM-2, Pselectin, monocyte chemoattractant protein-1, or IL-1 by either real-time PCR or immunohistochemistry. VCAM-1, which is newly expressed by luminal cells of the closed ductus, is an important ligand for the mononuclear cell adhesion receptor VLA4. After postnatal constriction, VLA4 ϩ monocytes/ macrophages (CD68 ϩ and CD14 ϩ ) and, to a lesser extent, T-lymphocytes adhered to the ductus wall. Neutrophils and platelets were not observed. The extent of postnatal neointimal remodeling (both endothelial cell layering and subendothelial space thickening) was associated with the degree of mononuclear cell adhesion. Similarly, the extent of vasa vasorum ingrowth correlated with the invasion of CD68 ϩ cells, from the adventitia into the muscle media. Based on these data, we conclude that the inflammatory response following postnatal ductus constriction may be as necessary for ductus remodeling as it is for atherosclerotic remodeling. Closure of the full-term ductus arteriosus occurs in two phases. First, smooth muscle constriction obstructs the ductus' lumen. Then, anatomic remodeling permanently occludes the lumen. The initial constriction appears to be the required stimulus for anatomic closure. During constriction, loss of luminal and vasa vasorum flow produce a zone of ischemichypoxia in the ductus' muscle media that induces the following anatomic changes: luminal endothelial proliferation, subendothelial thickening, ingrowth of vasa vasorum, and cell death (1). The preterm newborn is capable of remodeling its ductus, just like the full-term newborn, if it can impede its luminal flow and develop the same degree of ischemic-hypoxia as found at term (2).Although the cell death and ingrowth of vasa vasorum in the ductus wall appear to be due to ATP depletion (3,4) and VEGF induction (5), respectively, the mechanism(s) responsible for the neointimal changes are still unknown. It is now clear that inflammatory processes play a crucial role in the pathogenesis of several vascular disorders (6 -9). The most studied model of

Myocardial growth during fetal life is accomplished by proliferation of the number of myocytes (hyperplasia). Shortly after birth, normal growth of the heart is predominantly due to increase in cell size (hypertrophy), and myocytes largely lose the capability to replicate. This change is characterized by a decrease in myocardial DNA concentration and an increase in protein/DNA concentration ratio. Among many of the events associated with birth is an increase in plasma cortisol concentrations in the few days before delivery of the fetus. To determine the possible role of cortisol in the postnatal change in myocardial growth, we measured DNA and protein concentrations in the free walls of the left (LV) and right (RV) ventricles in normal fetal lambs, normal newborn lambs, and in fetal lambs in which cortisone was infused for 72-80 h into the left coronary artery, which we showed does not perfuse the RV free wall. Normally, fetal RV DNA is higher than LV DNA concentration, and DNA/protein ratio is lower in RV than in LV. It is suggested that this could be related to the greater load on the RV. Postnatally, protein concentrations increase progressively, but DNA remains the same in both ventricles, and protein/DNA ratios increase. Cortisol, infused to achieve normal prenatal levels in LV myocardium, markedly decreases LV DNA without affecting RV DNA concentrations. The present study indicates that cortisol inhibits myocyte replication and that cortisol simulates the change in myocardial growth pattern normally occurring after birth. It raises concerns regarding prenatal administration of glucocorticoids to mothers to mature the fetal lungs before preterm delivery.