Ductus Arteriosus Smooth Muscle Cells Research Articles

Electron Leak From the Mitochondrial Electron Transport Chain Complex I at Site IQ Is Crucial for Oxygen Sensing in Rabbit and Human Ductus Arteriosus.

Background As partial pressure of oxygen (pO2) rises with the first breath, the ductus arteriosus (DA) constricts, diverting blood flow to the pulmonary circulation. The DA's O2 sensor resides within smooth muscle cells. The DA smooth muscle cells' mitochondrial electron transport chain (ETC) produces reactive oxygen species (ROS) in proportion tooxygen tension, causing vasoconstriction by regulating redox-sensitive ion channels and enzymes. To identify which ETC complex contributes most to DA O2 sensing and determine whether ROS mediate O2 sensing independent of metabolism, we used electron leak suppressors, S1QEL (suppressor of site IQ electron leak) and S3QEL (suppressor of site IIIQo electron leak), which decrease ROS production by inhibiting electron leak from quinone sites IQ and IIIQo, respectively. Methods and Results The effects of S1QEL, S3QEL, and ETC inhibitors (rotenone and antimycin A) on DA tone, mitochondrial metabolism, O2-induced changes in intracellular calcium, and ROS were studied in rabbit DA rings, and human and rabbit DA smooth muscle cells. S1QEL's effects on DA patency were assessed in rabbit kits, using micro computed tomography. In DA rings, S1QEL, but not S3QEL, reversed O2-induced constriction (P=0.0034) without reducing phenylephrine-induced constriction. S1QEL did not inhibit mitochondrial metabolism or ETC-I activity. In human DA smooth muscle cells, S1QEL and rotenone inhibited O2-induced increases in intracellular calcium (P=0.02 and 0.001, respectively), a surrogate for DA constriction. S1QEL inhibited O2-induced ROS generation (P=0.02). In vivo, S1QEL prevented O2-induced DA closure (P<0.0001). Conclusions S1QEL, but not S3QEL, inhibited O2-induced rises in ROS and DA constriction ex vivo and in vivo. DA O2 sensing relies on pO2-dependent changes in electron leak at site IQ in ETC-I, independent of metabolism. S1QEL offers a therapeutic means to maintain DA patency.

Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility.

Based upon our demonstration that the smooth muscle cell-selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6's role in SMCs in vivo using cell type-specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest-derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation-sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.

Abstract 11961: Ductus Arteriosus Oxygen Response is Mediated by Mitochondrial Subunit NDUFS2

Introduction: The ductus arteriosus (DA) is a fetal vessel connecting the pulmonary artery (PA) and aorta, diverting oxygenated blood away from the developing lungs to the systemic circulation. The DA rapidly constricts in response to the increase in arterial oxygen (O 2 ) following the first breath. Failure of the DA to close in response to O 2 results in persistent ductus arteriosus, one of the most common complications of preterm birth. Mitochondria are known to play an important role in the O 2 response of DA smooth muscle cells (DASMC), however the mechanism is not fully understood. A subunit of mitochondrial Complex I, NDUFS2 (NADH:ubiquinone oxidoreductase core subunit 2) mediates the O 2 response of other specialized O 2 sensing tissues (PA and carotid body); we assess whether NDUFS2 also plays a role in DA O 2 sensing. Methods: DASMC were isolated from male term (29 days gestation) fetal New Zealand white rabbits. Cell lines were purified via fluorescence activated cell sorting, excluding CD90 + and CD31 + cells (fibroblasts and endothelial cells, respectively). Smooth muscle cell purity was confirmed using flow cytometry for α-smooth muscle actin (α-SMA). Primary cultures were 97.65% ± 0.65% CD90 - CD31 - , and were 100% α-SMA + post-sorting. We measured the O 2 responsiveness of DASMC using confocal microscopy, measuring changes in intracellular calcium with Cal-520AM. Cells were cultured in hypoxia (2.5% O 2 , pO 2 = 41 mmHg), with O 2 -induced increase in calcium relative to hypoxic baseline serving as a surrogate for constriction. DASMC were treated for 48 hours with 30 pmol siRNA and 90 pmol Lipofectamine® RNAiMAX to knockdown gene expression of NDUFS2 or a negative control. Knockdown was confirmed using qPCR. Results &amp; Conclusions: There was a 14.7% (±2.6%, n=33, p&lt;0.0001) increase in intracellular calcium relative to hypoxic baseline in siNC-treated cells. O 2 response was obliterated when treated with siNDUFS2 (0.7% ± 2.1%, n=29, p=0.798), with a 90.9% ± 1.89% knockdown of NDUFS2 mRNA expression. These data suggest a critical role for the mitochondrial electron transport chain, specifically NDUFS2, in DA oxygen responsiveness. While further functional studies will be required, our findings suggest a novel potential therapeutic target to modulate DA patency.

Contractile response of avian ductus arteriosus to chronic elevated oxygen levels

The ductus arteriosus (DA) is an oxygen sensitive embryonic blood vessel present in all developing amniotes. The DA connects the pulmonary artery to the aorta and shunts blood away from nonfunctioning lungs and towards the fetal respiratory organ. In birds at hatching, a rise in arterial PO2 initiates DA smooth muscle cell constriction to initiate closure. As the avian embryo approaches hatching, the DA contraction becomes more sensitive to oxygen. In this study we asked how 3‐hr chronic exposure to elevated oxygen levels of 5, 25, or 73 kPa oxygen influenced the oxygen stimulated contraction of the late term chicken DA. During the 3‐hr exposure, the greatest increase in contraction was seen in response to 73 kPa oxygen with smaller change occurring at 5 kPa oxygen exposure. Exposure to all three levels of oxygen resulted in resetting the baseline tension when oxygen was returned to 2.5 kPa. These changes in response to chronic elevated oxygen disappeared in the presence of cytochalasin B. These findings suggest that DA exposure to chronic elevated oxygen results in cellular changes in the contractile properties of the vessel allowing it to respond with greater contractile strength in response to a rise in oxygen.

Abstract 12927: The Human Ductus Arteriosus Smooth Muscle Cell Transcriptome Indicates a Central Role for Mitochondria in Postnatal Oxygen Sensing

Introduction: The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increased oxygen (O 2 ) immediately post-partum, resulting in functional DA closure, with anatomic closure ensuing within the first days of life. Failure of DA closure results in patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA response to O 2 , though modulated by the endothelium, is intrinsic to DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondria-derived reactive oxygen species, which increase in proportion to PaO 2 . The molecular signature of the DASMC response to O 2 is not well understood. Methods &amp; Results: DASMC were isolated from DA obtained from 10 infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorted to eliminate fibroblasts (CD90+) or endothelial cells (CD31+). DASMC purity was confirmed by α-smooth muscle actin staining. Cells were grown for 96 hours in hypoxia (2.5% O 2 ) or normoxia (19% O 2 ) and confocal calcium imaging confirmed O 2 -responsiveness of all cell lines: O 2 exposure increased intracellular calcium 18.1%±4.4% and decreased cell length 27%±1.5% relative to hypoxic baseline. RNA sequencing (Seq) of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p&lt;0.05). Gene Ontology (GO) analysis provided an unbiased assessment of genes differentially regulated by O 2 , revealing enrichment of functional groups including mitochondria, cellular respiration and transcription. Multiple subunits of electron transport chain complex I, NDUF (NADH:ubiquinone oxidoreductase) family genes, are upregulated in hDASMC following normoxia. Upregulated NDUF genes were validated with qPCR (t-test p&lt;0.05): NDUFS5 (Seq = 0.62-fold, PCR = 3.8-fold), NDUFA9 (Seq = 0.8-fold, Seq = 4.3-fold), NDUFA13 (Seq = 0.7-fold, PCR = 6.1-fold), and NDUFS8 (Seq = 0.6-fold, PCR = 7.1-fold). Conclusion: This first examination of the effects of O 2 on human DA transcriptomics supports a putative role for mitochondria as O 2 sensors, with mitochondrial subunits as potential future therapeutic targets for PDA.

Abstract 13389: Critical Role of Electron Leak From Mitochondrial Electron Transport Chain Complex I in Oxygen Sensing in the Rabbit Ductus Arteriosus

Introduction: The ductus arteriosus (DA) allows placentally oxygenated blood to bypass the non-ventilated fetal lungs and enter the systemic circulation. The fetus’ systemic pO 2 is ~40 mmHg, maintaining the DA in a state of hypoxic vasodilation. As pO 2 rises, with the first breath, the DA constricts and achieves functional closure within minutes. We have identified a mitochondrial O 2 -sensor controlling functional closure within DA smooth muscle cells (DASMC). Increased pO 2 leads to increased reactive oxygen species (ROS) production by complexes I and III of the electron transport chain (ETC). These ROS are diffusible signaling molecules and inhibit redox sensitive potassium channels causing DASMC depolarization and vasoconstriction. We propose this reflects electron leak from ETC complexes. Using the term rabbit model, we investigated the differential function of ETC I vs III complex-specific electron leak suppressors (S1QEL vs S3QEL) on vascular tone in DA rings. Methods: DA’s from term rabbits (n=11 rabbits, n=29 individual DA rings) were mounted on a force transducer at ~800 mg in Krebs at a PO 2 of ~40mmHg. DA’s were subject to varying pO 2 levels (from 35-155 mmHg) and at peak O 2 constriction were treated with ETC leak suppressors, ETC complex inhibitors or vehicle controls (DMSO). Results: Exposure to normoxia caused an average 1100±62 mg increase in DA tension. S1QEL (50 μM) caused an average 20.9±6.2% reduction in DA tension (n=8) whereas S3QEL (100 μM) only reduced DA tension by 4.4±2.2% (n=4). Rotenone (complex I inhibitor) and antimycin A (complex III inhibitor) also relaxed the DA while vehicle had minimal effect (-2.6±1.5%). Conclusions: Electron leak from complex 1, but not complex 3, is implicated in DA O 2 -sensing.

Cinaciguat Prevents Postnatal Closure of Ductus Arteriosus by Vasodilation and Anti-remodeling in Neonatal Rats.

Closure of the ductus arteriosus (DA) involves vasoconstriction and vascular remodeling. Cinaciguat, a soluble guanylyl cyclase (sGC) activator, was reported with vasodilatory and anti-remodeling effects on pulmonary hypertensive vessels. However, its effects on DA are not understood. Therefore, we investigated whether cinaciguat regulated DA patency and examined its underlying mechanisms. In vivo, we found that cinaciguat (10 mg/kg, i.p. at birth) prevented DA closure at 2 h after birth with luminal patency and attenuated intimal thickening. These anti-remodeling effects were associated with enhanced expression of cyclic guanosine monophosphate (cGMP) in DA. Ex vivo, cinaciguat dilated oxygen-induced DA constriction dose-dependently. Such vasodilatory effect was blunted by KT-5823, a PKG inhibitor. In DA smooth muscle cells (DASMCs), we further showed that cinaciguat inhibited angiotensin II (Ang II)-induced proliferation and migration of DASMCs. In addition, cinaciguat inhibited Ang II-induced mitochondrial reactive oxygen species (ROS) production. Finally, Ang II-activated MAPKs and Akt were also inhibited by cinaciguat. In conclusion, cinaciguat prevents postnatal DA closure by vasodilation and anti-remodeling through the cGMP/PKG pathway. The mechanisms underlying anti-remodeling effects include anti-proliferation and anti-migration, with attenuation of mitochondrial ROS production, MAPKs, and Akt signaling. Thus, this study implicates that sGC activation may be a promising novel strategy to regulate DA patency.

The molecular mechanisms of oxygen-sensing in human ductus arteriosus smooth muscle cells: A comprehensive transcriptome profile reveals a central role for mitochondria

The ductus arteriosus (DA) connects the fetal pulmonary artery and aorta, diverting placentally oxygenated blood from the developing lungs to the systemic circulation. The DA constricts in response to increases in oxygen (O2) with the first breaths, resulting in functional DA closure, with anatomic closure occurring within the first days of life. Failure of DA closure results in persistent patent ductus arteriosus (PDA), a common complication of extreme preterm birth. The DA's response to O2, though modulated by the endothelium, is intrinsic to the DA smooth muscle cells (DASMC). DA constriction is mediated by mitochondrial-derived reactive oxygen species, which increase in proportion to arterial partial pressure of oxygen (PaO2). The resulting redox changes inhibit voltage-gated potassium channels (Kv) leading to cell depolarization, calcium influx and DASMC constriction. To date, there has not been an unbiased assessment of the human DA O2-sensors using transcriptomics, nor are there known molecular mechanisms which characterize DA closure. DASMCs were isolated from DAs obtained from 10 term infants at the time of congenital heart surgery. Cells were purified by flow cytometry, negatively sorting using CD90 and CD31 to eliminate fibroblasts or endothelial cells, respectively. The purity of the DASMC population was confirmed by positive staining for α-smooth muscle actin, smoothelin B and caldesmon. Cells were grown for 96 h in hypoxia (2.5% O2) or normoxia (19% O2) and confocal imaging with Cal-520 was used to determine oxygen responsiveness. An oxygen-induced increase in intracellular calcium of 18.1% ± 4.4% and SMC constriction (−27% ± 1.5% shortening) occurred in all cell lines within five minutes. RNA sequencing of the cells grown in hypoxia and normoxia revealed significant regulation of 1344 genes (corrected p < 0.05). We examined these genes using Gene Ontology (GO). This unbiased assessment of altered gene expression indicated significant enrichment of the following GOterms: mitochondria, cellular respiration and transcription. The top regulated biologic process was generation of precursor metabolites and energy. The top regulated cellular component was mitochondrial matrix. The top regulated molecular function was transcription coactivator activity. Multiple members of the NADH-ubiquinone oxidoreductase (NDUF) family are upregulated in human DASMC (hDASMC) following normoxia. Several of our differentially regulated transcripts are encoded by genes that have been associated with genetic syndromes that have an increased incidence of PDA (Crebb binding protein and Histone Acetyltransferase P300). This first examination of the effects of O2 on human DA transcriptomics supports a putative role for mitochondria as oxygen sensors.

Nr4a1 , Kcne3 and Dgkg Are Identified as Oxygen‐sensitive Genes in the Ductus Arteriosus

Background The ductus arteriosus (DA), a vessel that consists of many smooth muscle cells (SMCs) and is essential for fetal circulation, immediately closes after birth.Although oxygen plays a critical role in functional DA closure, the specific molecules regulating the oxygen-induced DA closure remain unknown. Considering the dilation of pulmonary arteriolae (PA) soon after birth, we hypothesized that genes which exhibit an opposite expression pattern in DA and PA before and after birth would be the candidate molecules to regulate the oxygen-induced DA closure. Aim The aim of the study is to find out the genes that regulate DA closure in respond to oxygen after birth. Materials We used the DA and PA of Wistar rats before (on embryonic day 19 and embryonic day 21: e19 and e21) and after birth (on 2 days after birth: d2). Methods and Results Using Agilent SurePrint G3 Rat GE 8X60K, V2 Microarrays (Agilent®), we compared the gene expression profiling in DA and PA tissues of rats before and after birth. We discovered that the expression levels of 19 genes were increased in DA and decreased in PA after birth (d2DA/e21DA>2.0 and d2PA/e21PA<-2.0), while those of 21 genes were vice versa. Among these 40 genes that showed those opposite expression patterns in DA and PA, we selected 5 candidate genes (Nr4a1, Dgkg, Scara5, Abcc8 and Kcne3) in terms of the database for their expressions in SMCs and for their expected oxygen sensitivity. RT-PCR confirmed that Nr4a1, Dgkg, Scara5 and Abcc8 wereincreased in DA and decreased in PA after birth, while Kcne3 showed the opposite response. In order to confirm the oxygen-sensitive mRNA expression levels of the candidate genes, we conducted RT-PCR of DA SMCs that were harvested in 4 different groups: under a hypoxic condition (4% oxygen), a normoxic condition (21% oxygen), 2-hour-normoxic after 48-hour-hypoxic condition, and 24-hour-normoxic after 48-hour-hypoxic condition. We found that the transcription factor Nr4a1 showed upregulated under both 2-hour and 24-hour normoxic stimulation, diacylglycerol kinase Dgkg upregulated under 24-hour normoxic stimulation and Kv channel subfamily Kcne3 downregulated under 2-hour-normoxic stimulation. Conclusion The transcription factor Nr4a1 and diacylglycerol kinase Dgkg is upregulated and Kv channel subfamily Kcne3 is downregulated in DA SMCs in respond to oxygen. Nr4a1, Dgkg and Kcne3 may be the key molecules to regulate the oxygen-induced DA closure.

Primary study on the mechanism of ductus arteriosus closure in rats: impact of aging pathway

Objective: To explore the role and mechanism of aging pathway in patent ductus arteriosus closure of rats. Methods: Thirty outbreeding Sprague Dawley rats(20 females, 10-15 weeks old, 270-330 g) underwent random mating and conception. The primary Ductus Arteriosus smooth muscle cells (DASMCs) of pregnant 19 days(E19 group), 21 days(E21 group) and newborn(Day0 group) fetus were extracted and cultured. mRNA expression of cell senescence related markers p16, 21 and 53 genes in each group were detected by real-time fluorescent quantitative PCR(RT-PCR) after 48 hours culture. After hypoxic culture on DASMCs for 3 days, the DASMCs were divided into 3 groups: hypoxic control group(G0 group), 3 hours normal oxygen concentration treatment group(G3 group) and 6 hours normal oxygen concentration treatment group(G6 group). After intervention, mRNA expression of p16, 21 and 53 RT-PCR was detected. The DASMCs of newborn rats(Day0 group) were extracted and divided into 3 groups:low-oxygen culture control group, low-oxygen+siRNA culture group and normal oxygen concentration culture group. The DASMCs migration ability was tested experimentally by Transwell method. Result: The mRNA levels of p16, 21 and 53 in DASMCs were higher in E19 group than in Day0 group(all P<0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were also higher in E21 group than those in Day0 group (all P<0.01). The mRNA levels of p16, 21 and 53 in DASMC were all higher in G0 group than those in G3 group (P<0.05 or 0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were all higher in G0 group than those in G6 group (all P<0.01), and the mRNA levels of p16, 21 and 53 in DASMCs were all higher in G3 group than those in G6 group (all P<0.05). DASMCs migration ability of newborn rats was higher in normal oxygen concentration culture group than that in low-oxygen culture group (P<0.01), and DASMCs migration ability of newborn rats was also higher in low-oxygen+siRNA culture group than that in low-oxygen culture group (P<0.01). Conclusion: The expression of senescence marker of DASMCs decreases with the birth in rats during the process of ductal closure, and the aging pathway may affect ductal closure by inhibiting DASMCs migration in rats.

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.

The Role of Na+/Ca2+ Exchanger 1 in Maintaining Ductus Arteriosus Patency

Patency of the ductus arteriosus (DA) is crucial for both fetal circulation and patients with DA-dependent congenital heart diseases (CHD). The Na+/Ca2+ exchanger 1 (NCX1) protein has been shown to play a key role in the regulation of vascular tone and is elevated in DA-dependent CHD. This current study was conducted to investigate the mechanisms underpinning the role of NCX1 in DA patency. Our data showed NCX1 expression was up-regulated in the DA of fetal mice. Up-regulation of NCX1 expression resulted in a concomitant decrease in cytosolic Ca2+ levels in human DA smooth muscle cells (DASMCs) and an inhibition of the proliferation and migration capacities of human DASMCs. Furthermore, treatment of DASMCs with KB-R7943, which can reduce Ca2+ influx, resulted in the inhibition of both cell proliferation and migration. These findings indicate that NCX1 may play a role in maintaining patent DA not only by preventing DA functional closure through reducing cytosolic Ca2+ level in DASMC but also by delaying the anatomical closure process. The latter delay is facilitated by the down-regulation of human DASMC proliferation and migration. It is also likely that a reduction in cytosolic Ca2+ levels inhibits the proliferation and migration capacities of human DASMCs in vitro.

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, Ca2+ 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.

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

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). 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. 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.

PO-0510 Inhibition Of Pentose Phosphate Pathway And Nadph Oxidase Impaired The Response Of Chicken Ductus Arteriosus To Oxygen

Introduction NADPH derived from the pentose phosphate pathway (PPP) is a key system involved in maintaining the function of several important redox and antioxidant defense mechanisms. NADPH oxidases contain a catalytic NOX subunit that transfers electrons from NADPH to oxygen, thereby forming reactive oxygen species (ROS). Normoxic contraction of the ductus arteriosus (DA), such as occurs at birth, appears to be dependent upon the increase of ROS in DA smooth muscle cells. We hypothesised a role for NOX-derived ROS in the signalling pathway of oxygen-induced contraction of the DA. Methods We investigated the effects of the inhibition of PPP and NOX in the ex vivo response of chicken DA to oxygen. Experiments were performed in myograph-mounted DA rings (pulmonary and aortic sides) isolated from chicken embryos incubated for 19 days (total incubation: 21-d). Results Exposure to oxygen (21%) induced a sustained contractile response in the pulmonary but relaxation in the aortic side of 19-d DA. Incubation with the PPP inhibitor epiandrosterone or with the NOX inhibitors GKT-136901, VAS2870 and VAS3947 elicited a partial or complete impairment of oxygen-induced contraction. Phenylephrine- and KCl-induced contraction of chicken DA were impaired by epiandrosterone and VAS3947 but not by the other NOX inhibitors. Moreover, VAS3947 evoked an irreversible impairment of the contractility of the vessel. Oxygen-induced relaxation in the aortic part of the DA was not affected by NOX inhibitors. Conclusions Our data indicate that PPP and NADPH oxidase activation are events involved in the signalling cascade ofnormoxic contraction of chicken DA.

Decreased serum osmolality promotes ductus arteriosus constriction.

At birth, dynamic changes occur in serum components and haemodynamics, such as closure of the ductus arteriosus (DA). A previous study demonstrated that, in full-term human neonates, serum osmolality decreased transiently after birth, but recovered over the next few days. However, the significance of this transient decrease in osmolality has never been addressed. The objective of the present study was to examine the role of changes in serum osmolality after birth in DA closure. We found that rats exhibited a similar transient hypoosmolality after birth. Hypotonic stimulation induced constriction of DA rings and increased Ca(2+) transient in DA smooth muscle cells, but not in the aorta. The hypoosmotic sensor transient receptor potential melastatin 3 (TRPM3) was highly expressed in the rat DA, and TRPM3 silencing abolished the Ca(2+) response to hypoosmolality. Pregnenolone sulfate stimulation of TRPM3 induced rat DA constriction ex vivo and in vivo. Furthermore, hypertonic fluid injection impaired rat DA closure. In humans, neonatal serum hypoosmolality was observed in relatively mature preterm infants (≥28 weeks). In extremely preterm infants (<28 weeks), however, this hypoosmolality was absent. Instead, a rapid increase in osmolality occurred thereafter. Such an increase was greater, in particular, among patent DA (PDA) patients. A transient decrease in serum osmolality may promote DA closure during the first few days of life. Adjusting serum osmolality to proper levels might help to prevent the onset of PDA, improving the therapeutic outcome in extremely preterm infants.

Activation of the EGFR/p38/JNK pathway by mitochondrial-derived hydrogen peroxide contributes to oxygen-induced contraction of ductus arteriosus.

Oxygen-induced contraction of the ductus arteriosus (DA) involves a mitochondrial oxygen sensor, which signals pO2 in the DA smooth muscle cell (DASMC) by increasing production of diffusible hydrogen peroxide (H2O2). H2O2 stimulates vasoconstriction by regulating ion channels and Rho kinase, leading to calcium influx and calcium sensitization. Because epidermal growth factor receptor (EGFR) signaling is also redox regulated and participates in oxygen sensing and vasoconstriction in other systems, we explored the role of the EGFR and its signaling cascade (p38 and c-Jun N-amino-terminal kinase (JNK)) in DA contraction. Experiments were performed in DA rings isolated from full-term New Zealand white rabbits and human DASMC. In human DASMCs, increasing pO2 from hypoxia to normoxia (40 to 100mmHg) significantly increased cytosolic calcium, p < 0.01. This normoxic rise in intracellular calcium was mimicked by EGF and inhibited by EGFR siRNA. In DA rings, EGF caused contraction while the specific EGFR inhibitor (AG1478) and the tyrosine kinase inhibitors (genistein or tyrphostin A23) selectively attenuated oxygen-induced contraction (p < 0.01). Conversely, orthovanadate, a tyrosine phosphatase inhibitor known to activate EGFR signaling, caused dose-dependent contraction of hypoxic DA and superimposed increases in oxygen caused minimal additional contraction. Anisomycin, an activator of EGFR's downstream kinases, p38 and JNK, caused DA contraction; conversely, oxygen-induced DA contraction was blocked by inhibitors of p38 mitogen-activated protein kinases (MAPK) (SB203580) or JNK (JNK inhibitor II). O2-induced phosphorylation of EGFR occurred within 5min of increasing pO2 and was inhibited by mitochondrial-targeted overexpression of catalase. AG1478 prevented the oxygen-induced p38 and JNK phosphorylation. In conclusion, O2-induced EGFR transactivation initiates p38/JNK-mediated increases in cytosolic calcium and contributes to DA contraction. The EGFR/p38/JNK pathway is regulated by mitochondrial redox signaling and is a promising therapeutic target for modulation of the patent ductus arteriosus. Oxygen activates epidermal growth factor receptor (EGFR) in ductus arteriosus (DA) smooth muscle cells. EGFR inhibition selectively attenuates O2-induced DA constriction. pO2-induced EGFR activation is mediated by mitochondrial-derived hydrogen peroxide. p38 MAPK and JNK mediated EGFR's effects on oxygen-induced DA contraction. Tyrosine kinases and phosphatases participate in oxygen sensing in the DA. The EGFR pathway offers new therapeutic targets to modulate patency of the ductus arteriosus.

Thromboxane A(2) receptor stimulation promotes closure of the rat ductus arteriosus through enhancing neointima formation.

Ductus arteriosus (DA) closure follows constriction and remodeling of the entire vessel wall. Patent ductus arteriosus occurs when the DA does not close after birth, and this condition is currently treated using cyclooxygenase inhibitors. However, the efficacy of cyclooxygenase inhibitors is often limited. Our previous study demonstrated that low-dose thromboxane A2 receptor (TP) stimulation constricted the DA with minimal adverse effects in rat neonates. However, its effect on DA remodeling remains unknown. In this study, we focused on the impact of the exogenous TP stimulation on the DA remodeling, especially intimal thickening. Using DA explants from rat fetuses at embryonic day 19 as a ex vivo model and primary cultured rat DA smooth muscle cells from embryonic day 21 as a in vitro model, we evaluated the effect of TP stimulation on the DA remodeling. The selective TP agonists U46619 and I-BOP promoted neointima formation in the ex vivo DA explants, and TP stimulation increased DA SMC migration in a dose-dependent manner. Both effects were inhibited by the selective TP antagonist SQ29548 or the siRNA against TP. TP stimulation also increased DA SMC proliferation in the presence of 10% fetal bovine serum. LC/MS/MS analysis revealed that TP stimulation increased secretion of several extracellular matrix proteins that may contribute to an increase in neointima formation. In conclusion, we uncovered that exogenous administration of TP agonist promotes neointima formation through the induction of migration and proliferation of DA SMC, which could contribute to DA closure and also to its vasoconstrictive action.