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BackgroundAlthough studies involving preterm infants ≤34 weeks gestation report a decreased incidence of patent ductus arteriosus after antenatal betamethasone, studies involving younger gestation infants report conflicting results.MethodsWe used preterm baboons, mice, and humans (≤276/7 weeks gestation) to examine betamethasone's effects on ductus gene expression and constriction both in vitro and in vivo.ResultsIn mice, betamethasone increased the sensitivity of the premature ductus to the contractile effects of oxygen without altering the effects of other contractile or vasodilatory stimuli. Betamethasone's effects on oxygen sensitivity could be eliminated by inhibiting endogenous prostaglandin/nitric oxide signaling. In mice and baboons, betamethasone increased the expression of several developmentally regulated genes that mediate oxygen-induced constriction (K+ channels) and inhibit vasodilator signaling (phosphodiesterases). In human infants, betamethasone increased the rate of ductus constriction at all gestational ages. However, in infants born ≤256/7 weeks gestation, betamethasone's contractile effects were only apparent when prostaglandin signaling was inhibited, whereas at 26-27 weeks gestation, betamethasone's contractile effects were apparent even in the absence of prostaglandin inhibitors.ConclusionsWe speculate that betamethasone's contractile effects may be mediated through genes that are developmentally regulated. This could explain why betamethasone's effects vary according to the infant's developmental age at birth.

ObjectiveTo identify genes affected by advancing gestation and racial/ethnic origin in human ductus arteriosus (DA).Study designWe collected 3 sets of DA tissue (n = 93, n = 89, n = 91; total = 273 fetuses) from second trimester pregnancies. We examined four genes, with DNA polymorphisms that distribute along racial lines, to identify "Caucasian" and "non-Caucasian" DA. We used real time polymerase chain reaction to measure RNA expression of 48 candidate genes involved in functional closure of the DA, and used multivariable regression analyses to examine the relationships between advancing gestation, "non-Caucasian" race, and gene expression.ResultsMature gestation and non-Caucasian race are significant predictors for identifying infants who will close their patent DA when treated with indomethacin. Advancing gestation consistently altered gene expression in pathways involved with oxygen-induced constriction (eg, calcium-channels, potassium-channels, and endothelin signaling), contractile protein maturation, tissue remodeling, and prostaglandin and nitric oxide signaling in all 3 tissue sets. None of the pathways involved with oxygen-induced constriction appeared to be altered in "non-Caucasian" DA. Two genes, SLCO2A1 and NOS3, (involved with prostaglandin reuptake/metabolism and nitric oxide production, respectively) were consistently decreased in "non-Caucasian" DA.ConclusionsProstaglandins and nitric oxide are the most important vasodilators opposing DA closure. Indomethacin inhibits prostaglandin production, but not nitric oxide production. Because decreased SLCO2A1 and NOS3 expression can lead to increased prostaglandin and decreased nitric oxide concentrations, we speculate that prostaglandin-mediated vasodilation may play a more dominant role in maintaining the "non-Caucasian" patent DA, making it more likely to close when inhibited by indomethacin.

The cancer chemopreventive activity of green tea and its major polyphenolic constituent, epigallocatechin-3-gallate (EGCG) have been attributed to its antioxidant, antiproliferative and antiangiogenic effects. Several new molecular targets for EGCG's anticarcinogenic activity have been proposed in the recent literature. However, the understanding of the molecular mechanisms of EGCG's activity in vivo have been confounded by its low oral bioavailability and low plasma levels. Studies of EGCG would be greatly aided by the availability of synthetic analogs of EGCG designed to understand the contributions of the A, B, and D-rings and the phenolic hydroxyl groups of EGCG to its molecular mechanisms of action. We recently reported the de novo synthesis of a D-ring analog of EGCG, with the objective of using such analogs to understand the molecular mechanisms of EGCG action. We report here the first studies with a synthetic D-ring analog of EGCG. We examined the ability of the synthetic D-ring analog to inhibit tumor cell proliferation in breast carcinoma cells. We also investigated the effect of the analog on stress-induced VEGF production in breast carcinoma cells using Northern analysis and quantitative RT-PCR. We report here that the synthetic D-ring analog inhibits breast cancer cell growth in vitro with potencies equivalent to those of EGCG. Our results also show that, like EGCG, the synthetic analog inhibits hypoxia- and serum starvation-induced production of VEGF mRNA in breast cancer cells. Such synthetic analogs are valuable for understanding the structure-function relationship of EGCG and identifying relevant mechanisms of the chemopreventive action of EGCG.

Studies performed in sheep and baboons have shown that after birth, the normoxic muscle media of ductus arteriosus (DA) becomes profoundly hypoxic as it constricts and undergoes anatomic remodeling. We used isolated fetal lamb DA (pretreated with inhibitors of prostaglandin and nitric oxide production) to determine why the immature DA fails to remain tightly constricted during the hypoxic phase of remodeling. Under normoxic conditions, mature DA constricts to 70% of its maximal active tension (MAT). Half of its normoxic tension is due to [Ca.sup.2+] entry through calcium L-channels and store-operated calcium (SOC) channels. The other half is independent of extracellular [Ca.sup.2+] and is unaffected by inhibitors of sarcoplasmic reticulum (SR) [Ca.sup.2+] release (ryanodine) or reuptake [cyclopiazonic acid (CPA)]. The mature DA relaxes slightly during hypoxia (to 60% MAT) due to decreases in calcium L-channel-mediated [Ca.sup.2+] entry. Inhibitors of Rho kinase and tyrosine kinase inhibit both [Ca.sup.2+] -dependent and [Ca.sup.2+] -independent DA tension. Although Rho kinase activity may increase during gestation, immature DA develop lower tensions than mature DA, primarily because of differences in the way they process [Ca.sup.2+.] Calcium L-channel expression increases with advancing gestation. Under normoxic conditions, differences in calcium L-channel-mediated [Ca.sup.2+] entry account for differences in tension between immature (60% MAT) and mature (70% MAT) DA. Under hypoxic conditions, differences in both calcium L-channel-dependent and calcium L-channel-independent [Ca.sup.2+] entry, account for differences in tension between immature (33% MAT) and mature (60% MAT) DA. Stimulation of [Ca.sup.2+] entry through reverse-mode [Na.sup.+]/[Ca.sup.2+] exchange or CPA-induced SOC channel activity constrict the DA and eliminate differences between immature and mature DA during both hypoxia and normoxia. normoxia: hypoxia: oxygen concentration: Y27632: tyrosine kinase: calcium channels: gestation: calcium L-channels: store-operated calcium channels: RhoA: RhoB: Rho kinase

Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days + 14 days pro re nata [O.sub.2] model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1[alpha], but not HIF-2[alpha], mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-l-expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age-matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD. prolyl hydroxylase domain-containing protein; angiogenesis; alveolization; bronchopulmonary dysplasia; prematurity

After birth, constriction of the full-term ductus arteriosus induces oxygen, glucose and ATP depletion, cell death, and anatomic remodeling of the ductus wall. The immature ductus frequently fails to develop the same degree of constriction or anatomic remodeling after birth. In addition, the immature ductus loses its ability to respond to vasoconstrictive agents, like oxygen or indomethacin, with increasing postnatal age. We examined the effects of premature delivery and postnatal constriction on the immature baboon ductus arteriosus. By 6 days after birth, surrogate markers of hypoxia (HIF1[alpha]/VEGF mRNA) and cell death [dUTP nick-end labeling (TUNEL)-staining] increased, while glucose and ATP concentrations (bioluminescence imaging) decreased in the immature ductus. TUNEL-staining was significantly related to the degree of glucose and ATP depletion. Glucose and ATP depletion were directly related to the degree of ductus constriction; while TUNEL-staining was logarithmically related to the degree of ductus constriction. Extensive cell death (>15% TUNEL-positive cells) occurred only when there was no Doppler flow through the ductus lumen. In contrast, HIF1[alpha]/VEGF expression and ATP concentrations were significantly altered even when the immature ductus remained open after birth. Decreased ATP concentrations produced decreased oxygen-induced contractile responses in the immature ductus. We hypothesize that ATP depletion in the persistently patent immature newborn ductus is insufficient to induce cell death and remodeling but sufficient to decrease its ability to constrict after birth. This may explain its decreasing contractile response to oxygen, indomethacin, and other contractile agents with increasing postnatal age. baboon; hypoxia; glucose; glycogen; HIF1[alpha]

VEGF regulates remodeling during permanent anatomic closure of the ductus arteriosus. Am J Physiol Regulatory Integrative Comp Physiol 282: R199-R206, 2002; 10.1152/ajpregu.00298.2001.--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. nitric oxide; neointima; hypoxia; vasa vasorum

Nonselective cyclooxygenase (COX) inhibitors are potent tocolytic agents that have adverse effects on the fetal ductus arteriosus. To test the hypothesis that COX-2 inhibitors may not affect the ductus if the predominant COX isoform is COX-1, ductus arteriosus from late-gestation fetal lambs was obtained and analyzed. Results showed that both COX-1 and COX-2 were expressed in fetal lamb ductus arteriosus, contrary to the initial hypothesis.

Factors that increase the contractile tone of the ductus arteriosus also regulate its anatomic remodeling. Am J Physiol Regulatory Integrative Comp Physiol 281: R291-R301, 2001.--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 ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]: 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] 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 [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], would have increased DA tone and would be more likely to obliterate their lumen. We found that preterm newborns, maintained at a [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] [is greater than] 200 mmHg, had only a 14% incidence of patent DA. Even though DA constriction was due to elevated [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], 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. hypoxia; nitric oxide; prostaglandins; oxygen; newborn

Characterization of [PGE.sub.2] receptors in fetal and newborn lamb ductus arteriosus. Am J Physiol Heart Circ Physiol 280: H2342-H2349, 2001.--Although the role of [PGE.sub.2] in maintaining ductus arteriosus (DA) patency is well established, the specific [PGE.sub.2] receptor subtype(s) (EP) involved have not been clearly identified. We used late gestation fetal and neonatal lambs to study developmental regulation of EP receptors. In the fetal DA, radioligand binding and RT-PCR assays virtually failed to detect [EP.sub.1] but detected [EP.sub.2], [EP.sub.3D], and [EP.sub.4] receptors in equivalent proportions. In the newborn lamb, DA total density was one-third of that found in the fetus and only [EP.sub.2] was detected. Stimulation of [EP.sub.2] and [EP.sub.4] increased cAMP formation and was associated with DA relaxation. Though stimulation of [EP.sub.3] inhibited cAMP formation, it surprisingly relaxed the fetal DA both in vitro and in vivo. This [EP.sub.3]-induced relaxation was specifically diminished by the ATP-sensitive [K.sup.+] ([K.sub.ATP]) channel blocker glibenclamide. In conclusion, [PGE.sub.2] dilates the late gestation fetal DA through pathways that involve either cAMP ([EP.sub.2] and [EP.sub.4]) or [K.sub.ATP] channels ([EP.sub.3]). The loss of [EP.sub.3] and [EP.sub.4] receptors in the newborn DA is consistent with its decreased responsiveness to [PGE.sub.2]. ductus arteriosus; EP receptors; ATP-sensitive potassium channels.

Tissue hypoxia inhibits prostaglandin and nitric oxide production and prevents ductus arteriosus reopening. Am J Physiol Regulatory Integrative Comp Physiol 279: R278-R286, 2000.--Regulation of ductus arteriosus (DA) tension depends on a balance between oxygen-induced constriction and PG and nitric oxide (NO)-mediated relaxation. After birth, increasing [Pa.sub.o2] produces DA constriction. However, as the full-term ductus constricts, it develops severe tissue hypoxia in its inner vessel wall (oxygen concentration [is less than] 0.2%). We used isolated rings of fetal lamb DA to determine why the constricted ductus does not relax and reopen as it becomes hypoxic. We used a modification of the 2-(2-nitro-1H-imidazol-1-yl)-N-2,2,3,3,3-pentafluoropropyl) acetamide (EF5) technique (Clyman RI, Chan CY, Mauray F, Chen YQ, Cox W, Seidner SR, Lord EM, Weiss H, Wale N, Evan SM, and Koch CJ. Pediatr Res 45: 19-29, 1999) to determine mean tissue oxygen concentration. A decrease in the ductus' mean tissue oxygen concentration from 1.4 to 0.1% lowers the isometric tone of the ductus by 15 [+ or -] 10% of its maximal active tension (the maximal tension that can be produced by the ductus). Although decreases in oxygen concentration diminish ductus tension, most of the vasoconstrictor tone in the ductus is independent of ambient oxygen concentration. This oxygen-independent tone is equivalent to 64 [+ or -] 10% of the maximal active tension. At mean tissue oxygen concentrations [is greater than] 0.2%, endogenous PGs and NO inhibit more than 40% of the active tension developed by the ductus. However, when tissue oxygen concentrations drop below 0.2%, the constitutive relaxation of the ductus by endogenous PGs and NO is lost. In the absence of PG and NO production, tension increases to a level normally observed only after treatment of the ductus with indomethacin and nitro-L-arginine methyl ester (inhibitors of PG and NO production). Therefore, under conditions of severe hypoxia (tissue oxygen concentration [is less than] 0.2% oxygen), the loss of PG- and NO-mediated relaxation more than compensates for the loss of oxygen-induced tension. We hypothesize that this increased ductus tone enables the vessel to remain closed as it undergoes tissue remodeling. prostaglandin E.sub.2; prostacyclin; oxygen; vascular smooth muscle