Hypoxia-induced ARHGAP26 deficiency inhibits the proliferation and migration of human ductus arteriosus smooth muscle cell through activating RhoA-ROCK-PTEN pathway.

The Rho family plays crucial roles in O2 -induced vasoconstriction, cell proliferation, and migration. Rho GTPase-activating protein 26 (ARHGAP26) is a GTPase-activating protein for the small GTPases of the Rho family. Our previous studies have demonstrated that ARHGAP26 expression was significantly downregulated in patent human ductus arteriosus (DA) tissue. However, its role underlying the maintenance of DA patency is unclear. In this study, patent (fetal) and constricted (newborn) mouse DA tissues were harvested to confirm the differences in the levels of expression of ARHGAP26. Human DA smooth muscle cells (DASMCs) were isolated and cultured in vitro and used to test the function of ARHGAP26. The expression of ARHGAP26 was significantly lower in patent (fetal) than constricted (newborn) mouse DA. ARHGAP26-knocked-down human DASMCs showed reduced proliferation and migration, which are both crucial to anatomic closure of DA. Moreover, after culturing under hypoxic conditions, the expression of ARHGAP26 in human DASMCs was significantly lower and hypoxia-induced ARHGAP26 deficiency activated the phosphorylation level of phosphatase and tensin homolog (PTEN) in DASMCs by mediating the activity of RhoA and RhoA-associated kinase 1 (ROCK1). Use of Y27632, an inhibitor of ROCK which further reduces the phospholipid activity of PTEN can reverse the inhibitory effect of PTEN on the proliferation and migration of human DASMCs. This provides insight into the molecular regulation of the RhoA-ROCK-PTEN pathway in DA smooth muscle cells, which may be a suitable therapeutic target or diagnostic biomarker for perinatal DA tone management.

B-type natriuretic peptide prevents postnatal closure of ductus arteriosus by both vasodilation and anti-remodeling in neonatal rats.

The physiologic process of postnatal ductus arteriosus (DA) closure consists of vasoconstriction followed by vascular remodeling. We have recently reported that B-type natriuretic peptide (BNP), a potent vasodilator, also has anti-remodeling effects in pulmonary vasculature. However, its effects on DA have not been elucidated. We investigated whether BNP can prevent DA closure, and if so, the underlying mechanisms. Using in vivo studies, we examined effects of BNP (10 mg/kg, ip at birth) on DA closure in neonatal rats within 4 h after birth. We found that in control rats, the DA spontaneously closed at 4 h with a decreased DA diameter, enhanced intimal thickening, and luminal occlusion. BNP prevented DA closure at 4 h with a preserved DA diameter, attenuated intimal thickening, and preserved luminal patency. Ex vivo, BNP attenuated oxygen-induced vasoconstriction of isolated DA rings of newborn rats. These vasodilating effects were blunted by Rp-8-Br-PET-cGMPS, a cGMP inhibitor. In vitro, BNP inhibited angiotensin II (Ang II)-induced proliferation and migration of DA smooth muscle cells (DASMCs). BNP inhibited Ang II-induced mitochondrial reactive oxygen species (ROS) production and calcium overload in DASMCs. Finally, BNP inhibited Ang II-induced ERK1/2 activation. These in vitro effects were antagonized by Rp-8-Br-PET-cGMPS. In conclusion, BNP prevents postnatal DA closure by both vasodilation and anti-remodeling through the cGMP pathway. The mechanisms underlying anti-remodeling effects include anti-poliferation and anti-migration, with attenuation of mitochondrial ROS production and intracellular calcium and ERK1/2 signaling. Therefore, the BNP/cGMP pathway can be a promising therapeutic target for clinical management of DA patency.

CCN3 secreted by prostaglandin E2 inhibits intimal cushion formation in the rat ductus arteriosus.

The ductus arteriosus (DA), an essential fetal shunt between the pulmonary trunk and the descending aorta, changes its structure during development. Our previous studies have demonstrated that prostaglandin E2 (PGE2)-EP4 signaling promotes intimal cushion formation (ICF) by activating the migration of DA smooth muscle cells via the secretion of hyaluronan. We hypothesized that, in addition to hyaluronan, PGE2 may secrete other proteins that also regulate vascular remodeling in the DA. In order to detect PGE2 stimulation-secreted proteins, we found that CCN3 protein was increased in the culture supernatant in the presence of PGE2 in a dose-dependent manner by nano-flow liquid chromatography coupled with tandem mass spectrometry analysis and enzyme-linked immunosorbent assay. Quantitative RT-PCR analysis revealed that PGE2 stimulation tended to increase the expression levels of CCN3 mRNA in DA smooth muscle cells. Immunohistochemical analysis revealed that CCN3 was highly localized in the entire smooth muscle layers and the endothelium of the DA. Furthermore, exogenous CCN3 inhibited PGE2-induced ICF in the ex vivo DA tissues. These results suggest that CCN3 is a secreted protein of the DA smooth muscle cells induced by PGE2 to suppress ICF of the DA. The present study indicates that CCN3 could be a novel negative regulator of ICF in the DA to fine-tune the PGE2-mediated DA remodeling.

Ibuprofen and indomethacin differentially regulate vascular endothelial growth factor and its receptors in ductus arteriosus endothelial cells.

BACKGROUND: Cyclooxygenase inhibitors are widely applied to facilitate ductal closure in preterm infants. The mechanisms that lead to patent ductus arteriosus closure are incompletely understood. Vascular endothelial growth factor plays pivotal roles during ductal closure and remodelling. Aim The aim of this study was to investigate the effects of ibuprofen and indomethacin on the expression of vascular endothelial growth factor and its receptors in a primary rat ductus arteriosus endothelial cell culture. METHODS: Protein expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 1 and 2 was confirmed in rat ductus arteriosus and aorta by immunofluorescence staining. Fetal rat endothelial cells were isolated from ductus arteriosus and aorta using immunomagnetic cell sorting and treated with ibuprofen or indomethacin. mRNA expression levels were assessed by quantitative polymerase chain reaction analysis. RESULTS: In ductal endothelial cells, ibuprofen significantly induced vascular endothelial growth factor and its receptor 2, but not receptor 1, whereas indomethacin did not alter the expression levels of the vascular endothelial growth factor system. In contrast, ibuprofen significantly induced vascular endothelial growth factor and its receptors 1 and 2 in aortic endothelial cells, whereas indomethacin only induced vascular endothelial growth factor receptor 2. CONCLUSION: Our results indicate differential effects of ibuprofen and indomethacin on the expression levels of the vascular endothelial growth factor system in ductus arteriosus endothelial cells. In addition, vessel-specific differences between ductal and aortic endothelial cells were found. Further in vivo studies are needed to elucidate the biological significance of these findings.

Gamma-secretase Inhibitor Prevents Proliferation and Migration of Ductus Arteriosus Smooth Muscle Cells through the Notch3-HES1/2/5 Pathway.

Patent ductus arteriosus (PDA) can cause morbidity and mortality in neonates. Vascular remodeling, characterized by proliferation and migration of smooth muscle cells (SMCs), is an essential process for postnatal DA closure. Notch signaling is an important mediator of vascular remodelling but its role in DA is unkonwn. We investigated the effects and underlying mechanisms of γ-secretase inhibitor DAPT, a Notch signaling inhibitor on angiotensin II (Ang II)-induced proliferation and migration of DASMCs. Proliferation and migration of DASMCs cultured from neonatal Wistar rats were induced by Ang II, with or without DAPT pre-treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca(2+) influx, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and Notch receptor with its target gene pathway were examined. We found that DAPT inhibited Ang II-induced DASMCs proliferation and migration dose dependently. DAPT also arrested the cell cycle progression in the G0/G1-phase, and attenuated calcium overload and ROS production caused by Ang II. Moreover, DAPT inhibited nuclear translocation of Notch3 receptor intracellular domain, with decreased expression of its down-stream genes including HES1, HES2 and HES5. Finally, Ang II-activated ERK1/2, JNK and Akt were also counteracted by DAPT. In conclusion, DAPT inhibits Ang II-induced DASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production, and down-regulation of ERK1/2, JNK and Akt, through the Notch3-HES1/2/5 pathway. Therefore, Notch signaling has a role in DA remodeling and may provide a target pathway for therapeutic intervention of PDA.

Circulatory changes associated with the closure of the ductus arteriosus in hatching emu (Dromaius novaehollandiae).

In developing avian embryos, the right and left ductus arteriosi (DA) allow for a shunt of systemic venous return away from the lungs to the body and chorioallantoic membrane (CAM). Unlike in mammals where the transition from placental respiration to lung respiration is instantaneous, in birds the transition from embryonic CAM respiration to lung respiration can take over 24h. To understand the physiological consequences of this long transition we examined circulatory changes and DA morphological changes during hatching in the emu (Dromaius novaehollandiae), a primitive ratite bird. By tracking microspheres injected into a CAM vein, we observed no change in DA blood flow between the pre-pipped to internally pipped stages. Two hours after external pipping, however, a significant decrease in DA blood flow occurred, evident from a decreased systemic blood flow and subsequent increased lung blood flow. Upon hatching, the right-to-left shunt disappeared. These physiological changes in DA blood flow correspond with a large decrease in DA lumen diameter from the pre-pipped stages to Day 1 hatchlings. Upon hatching, the right-to-left shunt disappeared and at the same time apoptosis of smooth muscle cells began remodeling the DA for permanent closure. After the initial smooth muscle contraction, the lumen disappeared as intimal cushioning formed, the internal elastic lamina degenerated, and numerous cells underwent regulated apoptosis. The DA closed rapidly between the initiation of external pipping and hatching, resulting in circulatory patterns similar to the adult. This response is most likely produced by increased DA constriction in response to increased arterial oxygen levels and the initiation of vessel remodeling.

Oxygenation decreases elastin secretion from rat ductus arteriosus smooth muscle cells.

BACKGROUND: The ductus arteriosus (DA), a fetal arterial connection between the main pulmonary artery and the descending aorta, normally closes immediately after birth. The oxygen concentration in the blood rises after birth, and in the DA this increase in oxygen concentration causes functional closure, which is induced by smooth muscle contraction. Previous studies have demonstrated that hypoxia and/or oxygenation affect vascular remodeling of various vessels. Therefore, we hypothesized that the rise in oxygen concentration would affect the vascular structure of the DA due to production of proteins secreted from DA smooth muscle cells (SMC). METHODS AND RESULTS: Liquid chromatography-tandem mass spectrometry was used to comprehensively investigate the secreted proteins in the supernatant of rat DA SMC harvested under hypoxic conditions (1% oxygen) or under normoxic conditions (21% oxygen). We found that the rise in oxygen concentration reduced the secretion of elastin from DA SMC. On reverse transcription-polymerase chain reaction, the expression of elastin mRNA was not significantly changed in DA SMC from hypoxic to normoxic conditions. CONCLUSIONS: Given that elastin forms internal elastic lamina and elastic fibers in the vascular muscle layers, and that a rise in oxygen concentration reduced the secretion of elastin, this suggests that the rise in blood oxygen concentration after birth reduces the secretion of elastin, and therefore may play a role in DA structural remodeling after birth.

Prostaglandin E2 inhibits elastogenesis in the ductus arteriosus via EP4 signaling.

BACKGROUND: Elastic fiber formation begins in mid-gestation and increases dramatically during the last trimester in the great arteries, providing elasticity and thus preventing vascular wall structure collapse. However, the ductus arteriosus (DA), a fetal bypass artery between the aorta and pulmonary artery, exhibits lower levels of elastic fiber formation, which promotes vascular collapse and subsequent closure of the DA after birth. The molecular mechanisms for this inhibited elastogenesis in the DA, which is necessary for the establishment of adult circulation, remain largely unknown. METHODS AND RESULTS: Stimulation of the prostaglandin E2 (PGE2) receptor EP4 significantly inhibited elastogenesis and decreased lysyl oxidase (LOX) protein, which catalyzes elastin cross-links in DA smooth muscle cells (SMCs), but not in aortic SMCs. Aortic SMCs expressed much less EP4 than DASMCs. Adenovirus-mediated overexpression of LOX restored the EP4-mediated inhibition of elastogenesis in DASMCs. In EP4-knockout mice, electron microscopic examination showed that the DA acquired an elastic phenotype that was similar to the neighboring aorta. More importantly, human DA and aorta tissues from 7 patients showed a negative correlation between elastic fiber formation and EP4 expression, as well as between EP4 and LOX expression. The PGE2-EP4-c-Src-phospholipase C (PLC)γ-signaling pathway most likely promoted the lysosomal degradation of LOX. CONCLUSIONS: Our data suggest that PGE2 signaling inhibits elastogenesis in the DA, but not in the aorta, through degrading LOX protein. Elastogenesis is spatially regulated by PGE2-EP4 signaling in the DA.

Transcription profiles of endothelial cells in the rat ductus arteriosus during a perinatal period.

Endothelial cells (ECs) lining the blood vessels serve a variety of functions and play a central role in the homeostasis of the circulatory system. Since the ductus arteriosus (DA) has different arterial characteristics from its connecting vessels, we hypothesized that ECs of the DA exhibited a unique gene profile involved in the regulation of DA-specific morphology and function. Using a fluorescence-activated cell sorter, we isolated ECs from pooled tissues from the DA or the descending aorta of Wistar rat fetuses at full-term of gestation (F group) or neonates 30 minutes after birth (N group). Using anti-CD31 and anti-CD45 antibodies as cell surface markers for ECs and hematopoietic derived cells, respectively, cDNAs from the CD31-positive and CD45-negative cells were hybridized to the Affymetrix GeneChip® Rat Gene 1.0 ST Array. Among 26,469 gene-level probe sets, 82 genes in the F group and 81 genes in the N group were expressed at higher levels in DA ECs than in aortic ECs (p<0.05, fold change>2.0). In addition to well-known endothelium-enriched genes such as Tgfb2 and Vegfa, novel DA endothelium-dominant genes including Slc38a1, Capn6, and Lrat were discovered. Enrichment analysis using GeneGo MetaCore software showed that DA endothelium-related biological processes were involved in morphogenesis and development. We identified many overlapping genes in each process including neural crest-related genes (Hoxa1, Hoxa4, and Hand2, etc) and the second heart field-related genes (Tbx1, Isl1, and Fgf10, etc). Moreover, we found that regulation of epithelial-to-mesenchymal transition, cell adhesion, and retinol metabolism are the active pathways involved in the network via potential interactions with many of the identified genes to form DA-specific endothelia. In conclusion, the present study uncovered several significant differences of the transcriptional profile between the DA and aortic ECs. Newly identified DA endothelium-dominant genes may play an important role in DA-specific functional and morphologic characteristics.

Role of dynamin-related protein 1 (Drp1)-mediated mitochondrial fission in oxygen sensing and constriction of the ductus arteriosus.

RATIONALE: Closure of the ductus arteriosus (DA) is essential for the transition from fetal to neonatal patterns of circulation. Initial PO2-dependent vasoconstriction causes functional DA closure within minutes. Within days a fibrogenic, proliferative mechanism causes anatomic closure. Though modulated by endothelial-derived vasodilators and constrictors, O2 sensing is intrinsic to ductal smooth muscle cells and oxygen-induced DA constriction persists in the absence of endothelium, endothelin, and cyclooxygenase mediators. O2 increases mitochondrial-derived H2O2, which constricts ductal smooth muscle cells by raising intracellular calcium and activating rho kinase. However, the mechanism by which oxygen changes mitochondrial function is unknown. OBJECTIVE: The purpose of this study was to determine whether mitochondrial fission is crucial for O2-induced DA constriction and closure. METHODS AND RESULTS: Using DA harvested from 30 term infants during correction of congenital heart disease, as well as DA from term rabbits, we demonstrate that mitochondrial fission is crucial for O2-induced constriction and closure. O2 rapidly (<5 minutes) causes mitochondrial fission by a cyclin-dependent kinase- mediated phosphorylation of dynamin-related protein 1 (Drp1) at serine 616. Fission triggers a metabolic shift in the ductal smooth muscle cells that activates pyruvate dehydrogenase and increases mitochondrial H2O2 production. Subsequently, fission increases complex I activity. Mitochondrial-targeted catalase overexpression eliminates PO2-induced increases in mitochondrial-derived H2O2 and cytosolic calcium. The small molecule Drp1 inhibitor, Mdivi-1, and siDRP1 yield concordant results, inhibiting O2-induced constriction (without altering the response to phenylephrine or KCl) and preventing O2-induced increases in oxidative metabolism, cytosolic calcium, and ductal smooth muscle cells proliferation. Prolonged Drp1 inhibition reduces DA closure in a tissue culture model. CONCLUSIONS: Mitochondrial fission is an obligatory, early step in mammalian O2 sensing and offers a promising target for modulating DA patency.

Isolation and culture of fibroblasts, vascular smooth muscle, and endothelial cells from the fetal rat ductus arteriosus.

The ductus arteriosus (DA), a fetal arterial shunt vessel between the proximal descending aorta and the pulmonary artery, closes shortly after birth. Initial functional closure as a result of the DA's smooth muscle contraction is followed by definite anatomical closure. The latter involves several complex mechanisms like endothelial cushion formation and smooth muscle cell migration resulting in fibrosis and sealing of the vessel. These complex steps indicate highly specialized functions of the DA vascular smooth muscle cells (VSMCs), endothelial cells, and fibroblasts. Herein, we describe a new reproducible method for isolating VSMCs, endothelial cells, and fibroblasts of high viability from fetal rat DA using immunomagnetic cell sorting. Purity of the different cell cultures was assessed by immunohistochemistry and flow cytometry and ranged between 85 and 94%. The capability of the VSMCs to react to hypoxic stimuli was assessed by intracellular calcium and ATP measurements and by VEGF mRNA expression analysis. VSMCs respond to hypoxia with decreases in intracellular calcium concentrations and ATP levels, whereas VEGF mRNA expression increased 3.2-fold. The purified vessel-specific different cell types are suitable for subsequent gene expression profiling and functional studies and provide important tools for improving our understanding of the complex processes involved in the closure of the DA.

Differential regulation of vascular tone and remodeling via stimulation of type 2 and type 6 adenylyl cyclases in the ductus arteriosus.

RATIONALE: Prostaglandin (PG)E(2), which increases intracellular cAMP via activation of adenylyl cyclases (ACs), induces vasodilation and hyaluronan-mediated intimal thickening (IT) in the ductus arteriosus (DA) during late gestation. After birth, however, differential regulation of vasodilation and IT is preferable for treatment of patients with patent DA and DA-dependent congenital cardiac malformations. OBJECTIVE: Our objectives were to examine whether AC isoforms play differential roles in DA vasodilation and IT. METHODS AND RESULTS: AC2 and AC6 were more highly expressed in rat DA than in the aorta during the perinatal period. AC6-targeted siRNA counteracted PGE(1)-induced hyaluronan production in rat DA smooth muscle cells. Overexpression of AC6 enhanced PGE(1)-induced hyaluronan production and induced IT in DA explants. Furthermore, IT of the DA was less marked in mice lacking AC6 than in wild-type and AC5-deficient mice. Stimulation of AC2 attenuated AC6-induced hyaluronan production via inhibition of the p38 mitogen-activated protein kinase pathway and AC6-induced IT of the DA. An AC2/6 activator, 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] forskolin (FD1), did not induce hyaluronan-mediated IT in DA explants, although an AC5/6 activator, 6-[3-(dimethylamino)propionyl]-14,15-dihydroforskolin (FD6) did. Moreover, FD1 induced longer vasodilation of the DA than did PGE(1) without significant adverse effects in vivo. CONCLUSIONS: AC6 is responsible for hyaluronan-mediated IT of the DA and AC2 inhibited AC6-induced hyaluronan production. Stimulation of both AC2 and AC6 by FD1 induced longer vasodilation without hyaluronan-mediated IT in the DA in vivo. FD1 may be a novel alternative therapy to currently available PGE therapy for patients with DA-dependent congenital heart disease.

Effect of aerosolized PGE(1) on the ductus arteriosus of neonatal swine.

BACKGROUND: Inhaled PGE(1) (IPGE(1)) is a potential pulmonary vasodilator in neonatal respiratory failure. However, its effect on the patency of the ductus arteriosus (DA) has not been described. OBJECTIVE: To investigate the effect of IPGE(1) on the DA in healthy piglets. DESIGN/METHODS: IPGE(1) (1200ng/kg/min) [Study] or nebulized saline [Control] was administered using a jet nebulizer. Transthoracic echocardiography (TTE) was performed prior to (T0) and after 24h of aerosol therapy (T24). The DA was also evaluated histomorphologically at autopsy. RESULTS: Fifteen piglets, 1-9 days old (study=9; control=6), were evaluated for DA patency. Study piglets received IPGE(1) for 12-24h. TTE was performed on 12 piglets at T0. Nine animals showed no ductal flow and 3 (1 study, 2 control) had a small DA. TTE at T24 in 5 animals showed no change in DA. At autopsy, the ductal diameter and histologic maturity stage were comparable in study and control animals. CONCLUSIONS: High dose IPGE(1) given for 12-24h does not exert significant effect on the DA of healthy term piglets as evaluated by echocardiography and histomorphology. We conclude that ductal patency in neonates is influenced not only by prostaglandins but also by factors like hypoxemia, prematurity, and heart disease.

T-type Ca2+ channels promote oxygenation-induced closure of the rat ductus arteriosus not only by vasoconstriction but also by neointima formation.

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca(2+) influx through several membrane Ca(2+) channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca(2+) channel (VDCC) in DA closure remains unclear. Here we found that the expression of alpha1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that alpha1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by alpha1G overexpression. Moreover, it was decreased by adding alpha1G-specific small interfering RNAs or using R(-)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca(2+) concentration of DA SMCs was significantly decreased by adding alpha1G-specific siRNAs or using R(-)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(-)-efonidipine (10(-6) m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(-)-efonidipine (10(-6) m) significantly attenuated oxygenation-induced vasoconstriction by approximately 27% using a vascular ring of fetal DA at term. Finally, R(-)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially alpha1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.

Differential response of human fetal smooth muscle cells from arterial duct to retinoid acid.

AIM: The aim of the present study was to understand the role of retinoic acid (RA) in the development of isolated patent ductus arteriosus and the features of arterial duct-derived vascular smooth muscle cells (VSMC). METHODS: The VSMC were isolated, and the biological characteristics and the response to RA were investigated in the arterial duct, aorta, and pulmonary artery VSMC from 6 human embryonic samples. Western blotting, immunostaining, and cell-based ELISA were employed to analyze the proliferation regulation of VSMC. RESULTS: The VSMC from the arterial duct expressed proliferating cell nuclear antigen (PCNA) at a significantly lower rate than those from the aorta and pulmonary artery, but expressed a higher level of Bax and Bcl-2. The expression level of PCNA or Bcl-2 was associated with the embryonic age. The effects of RA on the VSMC from the arterial duct were quite different from those from the aorta and pulmonary artery. In arterial duct VSMC, RA stimulated PCNA expression, but such stimulation could be suppressed by CD2366, an antagonist of nuclear retinoid receptor activation. In aorta or pulmonary artery VSMC, the expression response of PCNA to RA was insignificant. The ratio of Bax/Bcl-2 decreased in arterial duct VSMC after RA treatment due to the significant inhibition of Bax expression. CONCLUSION: The VSMC from the arterial duct possessed distinct biological behaviors. RA might be important in the development of ductus arteriosus VSMC.

Chronic activation of the prostaglandin receptor EP4 promotes hyaluronan-mediated neointimal formation in the ductus arteriosus.

PGE, a potent vasodilator, plays a primary role in maintaining the patency of the ductus arteriosus (DA). Genetic disruption of the PGE-specific receptor EP4, however, paradoxically results in fatal patent DA (PDA) in mice. Here we demonstrate that EP4-mediated signals promote DA closure by hyaluronic acid-mediated (HA-mediated) intimal cushion formation (ICF). Chronic EP4 stimulation by ONO-AE1-329, a selective EP4 agonist, significantly enhanced migration and HA production in rat DA smooth muscle cells. When HA production was inhibited, EP4-mediated migration was negated. Activation of EP4, adenylyl cyclase, and PKA all increased HA production and the level of HA synthase 2 (HAS2) transcripts. In immature rat DA explants, ICF was promoted by EP4/PKA stimuli. Furthermore, adenovirus-mediated Has2 gene transfer was sufficient to induce ICF in EP4-disrupted DA explants in which the intimal cushion had not formed. Accordingly, signals through EP4 have 2 essential roles in DA development, namely, vascular dilation and ICF. The latter would lead to luminal narrowing, helping adhesive occlusion and permanent closure of the vascular lumen. Our results imply that HA induction serves as an alternative therapeutic strategy for the treatment of PDA to the current one, i.e., inhibition of PGE signaling by cyclooxygenase inhibitors, which might delay PGE-mediated ICF in immature infants.

ATP depletion and cell death in the neonatal lamb ductus arteriosus.

Postnatal constriction of the full-term ductus arteriosus produces cell death and remodeling of the ductus wall. Using a bioluminescence imaging technique, we found that after birth, the lamb ductus develops ATP, glucose, and glycogen depletion in addition to hypoxia. In vitro studies showed that cell death correlates best with ATP depletion and is most marked when both glucose and oxygen are severely depleted; in addition, the degree of ATP depletion found in vivo is sufficient to account for the extensive degree of cell death that occurs after birth. Under hypoxic conditions, the immature ductus is more capable of preserving its ATP supply than the mature ductus as a result of increased glucose availability, glycogen stores, and glucose utilization. However, the immature ductus is just as susceptible as the mature ductus to ATP depletion when glucose supplies are restricted. The extensive degree of cell death that occurs in the newborn ductus after birth is associated primarily with ATP depletion. The increased glycolytic capacity of the immature ductus may enable it to tolerate episodes of hypoxia and nutrient shortage, making it more resistant to developing postnatal cell death and permanent closure.

Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores.

In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca(2+)](i)) through inactivation of a K(+) channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca(2+)](i) by 11.7 +/- 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca(2+)](i) was mimicked by pharmacological blockade of the voltage-sensitive K(+) channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca(2+)](i). Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca(2+)](i). Endothelin increased [Ca(2+)](i) in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca(2+)](i) via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.

Apolipoprotein D and platelet-derived growth factor-BB synergism mediates vascular smooth muscle cell migration.

We identified apolipoprotein (apo)D in a search for proteins upregulated in a posttranscriptional manner similar to fibronectin in motile smooth muscle cells (SMCs). To address the function of apoD in SMCs, we cloned a partial apoD cDNA from ovine aortic (Ao) SMCs using RT-PCR. We documented a 2.5-fold increase in apoD protein but no increase in apoD mRNA in Ao SMCs 48 hours after a multiwound migration assay (P<0.01). Confocal microscopy revealed prominent perinuclear and trailing edge expression of apoD in migrating SMCs but not in the confluent monolayer. Stimulation of Ao SMCs with 10 ng/mL platelet-derived growth factor (PDGF)-BB increased apoD protein expression (P<0.05). Moreover, PDGF-BB-stimulated migration of human pulmonary artery SMCs was suppressed by knock-down of apoD using RNAi. Stable overexpression of apoD in Ao SMCs cultured in 10% fetal bovine serum promoted random migration by 62% compared with vector-transfected cells (P<0.01). Overexpression of apoD or addition of exogenous apoD to a rat aortic SMC line (A10) stimulated their migration in response to a subthreshold dose of PDGF-BB (P<0.05). This was unrelated to increased phosphorylation of ERK1/2 or of phospholipase C-gamma1, but correlated with enhanced Rac1 activation. This study shows that apoD can be expressed or taken up by SMCs and can regulate their motility in response to growth factors.

Effects of hypoxia, hypoglycemia, and muscle shortening on cell death in the sheep ductus arteriosus.

After birth, constriction of the full-term ductus arteriosus produces ischemic hypoxia, caspase activation, DNA fragmentation (>70% of cell nuclei are positive by the terminal deoxynucleotidyl transferase nick-end labeling [TUNEL] technique), and permanent ductus closure. In contrast, the preterm ductus frequently fails to develop these changes. We used the TUNEL technique to examine rings of fetal ductus arteriosus (incubated for 24 h at different oxygen and glucose concentrations) to determine the roles of 1) constriction and shortening, 2) hypoxia, and 3) hypoglycemia in producing cell death. Under controlled conditions, late-gestation ductus rings had a low rate of TUNEL-positive staining (0.6 +/- 0.9%) that did not change during muscle shortening. Although hypoxia (6.9 +/- 3.5%) and hypoglycemia (2.4 +/- 1.9%) increased the incidence of TUNEL-positive staining, only the combination of hypoxia-plus-hypoglycemia increased the incidence to the range found in vivo (83 +/- 9.5%). The combination of hypoxia-plus-hypoglycemia was associated with an oligonucleosomal pattern of DNA fragmentation. Under the same experimental conditions, the preterm ductus was capable of developing a similar degree of TUNEL-positive staining as found at term. Although caspase-3 and caspase-7 were activated in rings exposed to hypoxia-plus-hypoglycemia, a nonselective caspase inhibitor, Z-VAD.FMK (which inhibited caspase-3 and caspase-7 cleavage in the rings), did not diminish the degree of TUNEL-positive staining. We hypothesize that the preterm ductus is capable of developing an extensive degree of cell death, if it can develop the same degree of hypoxia and hypoglycemia found in the full-term newborn ductus. We also hypothesize that cell death in the ductus wall may involve pathways that are not dependent on caspase-3 or -7 activation.