Identification and Functional Verification of CITED2 Gene Promoter Region in Patients with Patent Ductus Arteriosus.

Patent ductus arteriosus (PDA) is a common congenital heart disease. CITED2 plays an important role in the development of the heart, and genetic variants in its coding region are significantly associated with cardiac malformations. However, the role of variants in the promoter region of CITED2 in the development of PDA remains unclear. We extracted the peripheral blood of 646 subjects (including 353 PDA patients and 293 unrelated healthy controls) for sequencing. We identified 13 promoter variants of the CITED2 gene (including 2 novel heterozygous variants). Of the 13 variants, 10 were found only in PDA patients. In mouse cardiomyocytes (HL-1) and rat cardiac myocytes (RCM), the transcriptional activity of the CITED2 gene promoter was significantly changed by the variants (p < 0.05). The results of the experiments of electrophoretic mobility indicated that these variants may affect the transcription of the CITED2 gene by influencing the binding ability of transcription factors. These results, combined with the JASPAR database analysis, showed that the destruction/production of transcription factor binding sites due to the variants in the promoter region of the CITED2 gene may directly or indirectly affect the binding ability of transcription factors. Our results suggest for the first time that variants at the CITED2 promoter region may cause low expression of CITED2 protein related to the formation of PDA.

Vav2 promotes ductus arteriosus anatomic closure via the remodeling of smooth muscle cells by Rac1 activation.

The ductus arteriosus (DA), bridging the aorta and pulmonary artery, immediately starts closing after birth. Remodeling of DA leads to anatomic obstruction to prevent repatency. Several histological changes, especially extracellular matrices (ECMs) deposition and smooth muscle cells (SMCs) migration bring to anatomic closure. The genetic etiology and mechanism of DA closure remain elusive. We have previously reported a novel copy number variant containing Vav2 in patent ductus arteriosus (PDA) patients, but its specific role in DA closure remains unknown. The present study revealed that the expression of Vav2 was reduced in human patent DA, and it was less enrichment in the adjacent aorta. Matrigel experiments demonstrated that Vav2 could promote SMC migration from PDA patient explants. Smooth muscle cells with Vav2 overexpression also presented an increased capacity in migration and downregulated contractile-related proteins. Meanwhile, SMCs with Vav2 overexpression exhibited higher expression of collagen III and lessened protein abundance of lysyl oxidase, and both changes are beneficial to DA remodeling. Overexpression of Vav2 resulted in increased activity of Rac1, Cdc42, and RhoA in SMCs. Further investigation noteworthily found that the above alterations caused by Vav2 overexpression were particularly reversed by Rac1 inhibitor. A heterozygous, rare Vav2 variant was identified in PDA patients. Compared with the wild type, this variant attenuated Vav2 protein expression and weakened the activation of downstream Rac1, further impairing its functions in SMCs. In conclusion, Vav2 functions as an activator for Rac1 in SMCs to promote SMCs migration, dedifferentiation, and ECMs production. Deleterious variant potentially induces Vav2 loss of function, further providing possible molecular mechanisms about Vav2 in PDA pathogenesis. These findings enriched the current genetic etiology of PDA, which may provide a novel target for prenatal diagnosis and treatment. KEY MESSAGES: Although we have proposed the potential association between Vav2 and PDA incidence through whole exome sequencing, the molecular mechanisms underlying Vav2 in PDA have never been reported. This work, for the first time, demonstrated that Vav2 was exclusively expressed in closed DAs. Moreover, we found that Vav2 participated in the process of anatomic closure by mediating SMCs migration, dedifferentiation, and ECMs deposition through Rac1 activation. Our findings first identified a deleterious Vav2 c.701C>T variant that affected its function in SMCs by impairing Rac1 activation, which may lead to PDA defect. Vav2 may become an early diagnosis and an effective intervention target for PDA clinical therapy.

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.

Transcriptional Evaluation of the Ductus Arteriosus at the Single-Cell Level Uncovers a Requirement for Vim (Vimentin) for Complete Closure.

OBJECTIVE: Failure to close the ductus arteriosus, patent ductus arteriosus, accounts for 10% of all congenital heart defects. Despite significant advances in patent ductus arteriosus management, including pharmacological treatment targeting the prostaglandin pathway, a proportion of patients fail to respond and must undergo surgical intervention. Thus, further refinement of the cellular and molecular mechanisms that govern vascular remodeling of this vessel is required. METHODS: We performed single-cell RNA-sequencing of the ductus arteriosus in mouse embryos at E18.5 (embryonic day 18.5), and P0.5 (postnatal day 0.5), and P5 to identify transcriptional alterations that might be associated with remodeling. We further confirmed our findings using transgenic mouse models coupled with immunohistochemistry analysis. RESULTS: The intermediate filament vimentin emerged as a candidate that might contribute to closure of the ductus arteriosus. Indeed, mice with genetic deletion of vimentin fail to complete vascular remodeling of the ductus arteriosus. To seek mechanisms, we turned to the RNA-sequencing data that indicated changes in Jagged1 with similar profile to vimentin and pointed to potential links with Notch. In fact, Notch3 signaling was impaired in vimentin null mice and vimentin null mice phenocopies patent ductus arteriosus in Jagged1 endothelial and smooth muscle deleted mice. CONCLUSIONS: Through single-cell RNA-sequencing and by tracking closure of the ductus arteriosus in mice, we uncovered the unexpected contribution of vimentin in driving complete closure of the ductus arteriosus through a mechanism that includes deregulation of the Notch signaling pathway.

Interactions between PDA-associated polymorphisms and genetic ancestry alter ductus arteriosus gene expression.

BACKGROUND: DNA polymorphisms in PTGIS and TFAP2B have been identified as risk factors for patent ductus arteriosus (PDA) in a population composed of preterm infants with European genetic ancestry but not in more genetically diverse populations. GOAL: To determine if the effects of TFAP2B and PTGIS polymorphisms on ductus arteriosus (DA) gene expression differ based on genetic ancestry. METHODS: DA from 273 human second trimester fetuses were genotyped for TFAP2B and PTGIS polymorphisms and for polymorphisms distributing along genetic ancestry lines. RT-PCR was used to measure the RNA expression of 49 candidate genes involved with DA closure. RESULTS: Seventeen percent of the DA analyzed were of European ancestry. In multivariable regression analyses we found consistent associations between four PDA-related TFAP2B polymorphisms (rs2817399(A), rs987237(G), rs760900(C), and rs2817416(C)) and expression of the following genes: EPAS1, CACNB2, ECE1, KCNA2, ATP2A3, EDNRA, EDNRB, BMP9, and BMP10, and between the PTGIS haplotype rs493694(G)/rs693649(A) and PTGIS and NOS3. These changes only occurred in DA with European ancestry. No consistent positive or negative associations were found among DA samples unless an interaction between the polymorphisms and genetic ancestry was taken into account. CONCLUSION: PTGIS and TFAP2B polymorphisms were associated with consistent changes in DA gene expression when present in fetuses with European ancestry. IMPACT: DNA polymorphisms in PTGIS and TFAP2B have been identified as risk factors for patent ductus arteriosus (PDA) in a population composed primarily of preterm infants with European genetic ancestry but not in more genetically diverse populations. The same PTGIS and TFAP2B polymorphisms are associated with changes in ductus gene expression when present in ductus from fetuses with European genetic ancestry. No consistent associations with gene expression can be found unless an interaction between the polymorphisms and genetic ancestry is taken into account.

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.

Molecular Mechanisms Underlying Remodeling of Ductus Arteriosus: Looking beyond the Prostaglandin Pathway.

The ductus arteriosus (DA) is a physiologic vessel crucial for fetal circulation. As a major regulating factor, the prostaglandin pathway has long been the target for DA patency maintenance or closure. However, the adverse effect of prostaglandins and their inhibitors has been a major unsolved clinical problem. Furthermore, a significant portion of patients with patent DA fail to respond to cyclooxygenase inhibitors that target the prostaglandin pathway. These unresponsive medical patients ultimately require surgical intervention and highlight the importance of exploring pathways independent from this well-recognized prostaglandin pathway. The clinical limitations of prostaglandin-targeting therapeutics prompted us to investigate molecules beyond the prostaglandin pathway. Thus, this article introduces molecules independent from the prostaglandin pathway based on their correlating mechanisms contributing to vascular remodeling. These molecules may serve as potential targets for future DA patency clinical management.

Transcription factor AP-2beta in development, differentiation and tumorigenesis.

To date, the AP-2 family of transcription factors comprises five members. Transcription factor AP-2beta (TFAP2B)/AP-2ß was first described in 1995. Several studies indicate a critical role of AP-2ß in the development of tissues and organs of ectodermal, neuroectodermal and also mesodermal origin. Germline mutation of TFAP2B is known to cause the Char syndrome, an autosomal dominant disorder characterized by facial dysmorphism, patent ductus arteriosus and anatomical abnormalities of the fifth digit. Furthermore, single-nucleotide polymorphisms in TFAP2B were linked to obesity and specific personality traits. In neoplasias, AP-2ß was first described in alveolar rhabdomyosarcoma. Immunohistochemical staining of AP-2ß is a recommended ancillary test for the histopathological diagnosis of this uncommon childhood malignancy. In neuroblastoma, AP-2ß supports noradrenergic differentiation. Recently, the function of AP-2ß in breast cancer (BC) has gained interest. AP-2ß is associated with the lobular BC subtype. Moreover, AP-2ß controls BC cell proliferation and has a prognostic impact in patients with BC. This review provides a comprehensive overview of the current knowledge about AP-2ß and its function in organ development, differentiation and tumorigenesis.

Effect of Beta 3 Adrenoreceptor Modulation on Patency of the Ductus Arteriosus.

ß3-adrenoreceptor (ß3-AR), a G-protein coupled receptor, has peculiar regulatory properties in response to oxygen and widespread localization. ß3-AR is expressed in the most frequent neoplasms, also occurring in pregnant women, and its blockade reduces tumor growth, indicating ß3-AR-blockers as a promising alternative to antineoplastic drugs during pregnancy. However, ß3-AR involvement in prenatal morphogenesis and the consequences of its blockade for the fetus remain unknown. In this study, after the demonstrated expression of ß3-AR in endothelial and smooth muscle cells of ductus arteriosus (DA), C57BL/6 pregnant mice were acutely treated at 18.5 of gestational day (GD) with indomethacin or with the selective ß3-AR antagonist SR59230A, or chronically exposed to SR59230A from 15.5 to 18.5 GD. Six hours after the last treatment, fetuses were collected. Furthermore, newborn mice were treated straight after birth with BRL37344, a ß3-AR agonist, and sacrificed after 7 h. SR59230A, at the doses demonstrated effective in reducing cancer progression (10 and 20 mg/kg) in acute and chronic mode, did not induce fetal DA constriction and did not impair the DA ability to close after birth, whereas at the highest dose (40 mg/kg), it was shown to cause DA constriction and preterm-delivery. BRL37344 administered immediately after birth did not alter the physiological DA closure.

KCTD1 mutants in scalp­ear­nipple syndrome and AP­2α P59A in Char syndrome reciprocally abrogate their interactions, but can regulate Wnt/ß­catenin signaling.

Potassium­channel tetramerization-domain-containing 1 (KCTD1) mutations are reported to result in scalp­ear­nipple syndrome. These mutations occur in the conserved broad­complex, tramtrack and bric a brac domain, which is associated with inhibited transcriptional activity. However, the mechanisms of KCTD1 mutants have not previously been elucidated; thus, the present study aimed to investigate whether KCTD1 mutants affect their interaction with transcription factor AP­2α and their regulation of the Wnt pathway. Results from the present study demonstrated that none of the ten KCTD1 mutants had an inhibitory effect on the transcriptional activity of AP­2α. Co­immunoprecipitation assays demonstrated that certain mutants exhibited changeable localization compared with the nuclear localization of wild­type KCTD1, but no KCTD1 mutant interacted with AP­2α. Almost all KCTD1 mutants, except KCTD1 A30E and H33Q, exhibited differential inhibitory effects on regulating TOPFLASH luciferase reporter activity. In addition, the interaction region of KCTD1 to the PY motif (amino acids 59­62) in AP­2α was identified. KCTD1 exhibited no suppressive effects on the transcriptional activity of the AP­2α P59A mutant, resulting in Char syndrome, a genetic disorder characterized by a distinctive facial appearance, heart defect and hand abnormalities, by altered protein cellular localization that abolished protein interactions. However, the P59A, P60A, P61R and 4A AP­2α mutants inhibited TOPFLASH reporter activity. Moreover, AP­2α and KCTD1 inhibited ß­catenin expression levels and SW480 cell viability. The present study thus identified a putative mechanism of disease­related KCTD1 mutants and AP­2α mutants by disrupting their interaction with the wildtype proteins AP­2α and KCTD1 and influencing the regulation of the Wnt/ß­catenin pathway.

A novel homozygous TPM1 mutation in familial pediatric hypertrophic cardiomyopathy and in silico screening of potential targeting drugs.

OBJECTIVE: Familial hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease. While sarcomeric gene mutations explain many HCM cases, the genetic basis of about half of HCM cases remains elusive. Here we aimed to identify the gene causing HCM in a non-consanguineous Saudi Arabian family with affected family members and a history of sudden death. The impact of the identified mutation on protein structure and potential drug targets were evaluated in silico. MATERIALS AND METHODS: Triplets (two HCM subjects and one patent ductus arteriosus (PDA) case) and unaffected parents were screened by targeted next-generation sequencing (NGS) for 181 candidate cardiomyopathy genes. In silico structural and functional analyses, including protein modeling, structure prediction, drug screening, drug binding, and dynamic simulations were performed to explore the potential pathogenicity of the variant and to identify candidate drugs. RESULTS: A homozygous missense mutation in exon 1 of TMP1 (assembly GRCh37-chr15: 63340781; G>A) was identified in the triplets [two HCM and one patent ductus arteriosus (PDA)] that substituted glycine for arginine at codon 3 (p.Gly3Arg). The parents were heterozygous for the variant. The mutation was predicted to cause a significant and deleterious change in the TPM1 protein structure that slightly affected drug binding, stability, and conformation. In addition, we identified several putative TPM1-targeting drugs through structure-based in silico screening. CONCLUSIONS: TPM1 mutations are a common cause of HCM and other congenital heart defects. To date, TPM1 has not been associated with isolated PDA; to our knowledge, this is the first report of the homozygous missense variation p.Gly3Arg in TPM1 associated with familial autosomal recessive pediatric HCM and PDA. The identified candidate TPM1 inhibitors warrant further prospective investigation.

Fibulin-1 Integrates Subendothelial Extracellular Matrices and Contributes to Anatomical Closure of the Ductus Arteriosus.

OBJECTIVE: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1-deficient (Fbln1-/-) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4-/-) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. CONCLUSIONS: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.

Role of dopamine and selective dopamine receptor agonists on mouse ductus arteriosus tone and responsiveness.

BACKGROUND: Indomethacin treatment for patent ductus arteriosus (PDA) is associated with acute kidney injury (AKI). Fenoldopam, a dopamine (DA) DA1-like receptor agonist dilates the renal vasculature and may preserve renal function during indomethacin treatment. However, limited information exists on DA receptor-mediated signaling in the ductus and fenoldopam may prevent ductus closure given its vasodilatory nature. METHODS: DA receptor expression in CD-1 mouse vessels was analyzed by qPCR and immunohistochemistry. Concentration-response curves were established using pressure myography. Pretreatment with SCH23390 (DA1-like receptor antagonist), phentolamine (α -adrenergic receptor antagonist) or indomethacin addressed mechanisms for DA-induced changes. Fenoldopam's effects on postnatal ductus closure were evaluated in vivo. RESULTS: DA1 receptors were expressed equally in ductus and aorta. High-dose DA induced modest vasoconstriction under newborn O2 conditions. Phentolamine inhibited DA-induced constriction, while SCH23390 augmented constriction, consistent with a vasodilatory role for DA1 receptors. Despite this, fenoldopam had little effect on ductus tone nor indomethacin- or O2-induced constriction and did not impair postnatal closure in vivo. CONCLUSION(S): DA receptors are present in the ductus but have limited physiologic effects. DA-induced ductus vasoconstriction is mediated via α-adrenergic pathways. The absence of DA1-mediated impairment of ductus closure supports the study of potential role for fenoldopam during PDA treatment.

Base excess and hematocrit predict response to indomethacin in very low birth weight infants with patent ductus arteriosus.

BACKGROUND: The treatment of patent ductus arteriosus (PDA) in very low birth weight (VLBW) infants remains a challenge. The ability to predict which infants will respond to indomethacin could spare some from the risks of unnecessary medications. Our objective was to determine if indicators of acid-base homeostasis could predict response to indomethacin treatment for ductal closure, and thus help guide treatment decisions. METHODS: We performed a retrospective analysis of medical records of VLBW (< 1500 g) neonates with hemodynamically significant PDA born at our institution between January 2009 and December 2012; all infants included in the study were treated with indomethacin for ductal closure within the first 2 weeks of life. We extracted data for a number of clinical variables including gestational age, birth weight, blood chemistries, surfactant use, hematocrit, and blood gas parameters. Our primary outcome measure was successful closure of PDA following the first round of indomethacin. Using variables that were significant on initial testing, we created multivariable regression models to determine the independent association of selected variables with indomethacin response. RESULTS: Of the 91 infants included in the study, 62 (68%) responded to the first course of indomethacin with successful ductal closure. Multivariable regression modeling revealed that both base excess and hematocrit were independently associated with indomethacin response; odds of PDA closure increased with increasing base excess (OR [odds ratio]: 1.81; 95% confidence interval [CI]: 1.36-2.60) and increasing hematocrit (OR: 1.21; 95% CI: 1.01-1.45). The optimal cutoff value for base excess was - 4.56, with a sensitivity of 96.8% (95% CI: 89-100) and specificity of 79.3% (95% CI: 60-92); optimal cutoff value for hematocrit was 40, with 69.4% sensitivity (95% CI: 56-80) and 65.5% specificity (95% CI: 46-82). CONCLUSIONS: Base excess and hematocrit may be independent predictors of indomethacin response in VLBW infants with PDA. Low-cost and readily accessible, acid-base indicators such as base excess could help guide treatment decisions.

Molecular and mechanical factors contributing to ductus arteriosus patency and closure.

Regulation of the ductus arteriosus, an essential fetal vessel connecting the pulmonary artery and aorta, is complex. Failure of this vessel to close after birth may result in a persistent left-to-right shunt through the patent ductus arteriosus, a condition associated with significant morbidities. Numerous factors contribute to the shift from fetal ductus patency to postnatal closure, requiring precise coordination of molecular cues with biomechanical forces and underlying genetic influences. Despite significant advances, questions remain regarding signaling dynamics and the natural time course of ductus closure, particularly in preterm neonates. This review highlights the contributions of early investigators and more recent clinician scientists to our understanding of the molecular and mechanical factors that mediate ductus patency and closure.

Altered plasma proteins released from platelets and endothelial cells are associated with human patent ductus arteriosus.

Patent ductus arteriosus is the third most common congenital heart disease and resulted from the persistence of ductal patency after birth. Ductus arteriosus closure involves functional and structural remodeling, controlled by many factors. The changes in plasma protein levels associated with PDA closure are not known. Here we for the first time demonstrate six key differential plasma proteins in human patent ductus arteriosus patients using proteomic technology and present a model to illustrate the constriction and closure of ductus arteriosus. Differentially expressed proteins were analyzed by using isobaric tags for relative and absolute quantification and validated by enzyme-linked immunosorbent assay in new samples. The proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD008568. We found 74 upregulated and 98 downregulated proteins in the plasma of patients with PDA. Five decreased proteins (platelet factor 4, fibrinogen, von Willebrand factor, collagen, and mannose binding lectin-associated serine protease-2) and one increased protein (fibronectin) may increase the risk of patent ductus arteriosus. Those proteins are closely related to platelet activation and coagulation cascades, complement mannan-binding-lectin, and other systemic signaling pathways. Our findings for the first time indicate that the differential proteins involved in different pathways may play key roles in the nonclosure of the ductus arteriosus in humans and may be developed as biomarkers for diagnosis. All those findings may be served as the basis of understanding the etiology and pathogenesis of patent ductus arteriosus.

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.

Simulation-based suggestions to improve ibuprofen dosing for patent ductus arteriosus in preterm newborns.

PURPOSE: Ibuprofen is the drug of choice for treatment of patent ductus arteriosus (PDA). There is accumulating evidence that current ibuprofen-dosing regimens for PDA treatment are inadequate. We aimed to propose an improved dosing regimen, based on all current knowledge. METHODS: We performed a literature search on the clinical pharmacology and effectiveness of ibuprofen. (R)- and (S)-ibuprofen plasma concentration-time profiles of different dosing regimens were simulated using a population pharmacokinetic model and evaluated to obtain a safe, yet likely more efficacious ibuprofen exposure. RESULTS: The most effective intravenous ibuprofen dosing in previous clinical trials included a first dose of 20 mg kg-1 followed by 10 mg kg-1 every 24 h. Simulations of this dosing regimen show an (S)-ibuprofen trough concentration of 43 mg L-1 is reached at 48 h, which we assumed the target through concentration. We show that this target can be reached with a first dose of 18 mg kg-1, followed by 4 mg kg-1 every 12 h. After 96 h postnatal age, the dose should be increased to 5 mg kg-1 every 12 h due to maturation of clearance. This twice-daily dosing has the advantage over once-daily dosing that an effective trough level may be maintained, while peak concentrations are substantially (22%) lower. CONCLUSIONS: We propose to improve intermittent ibuprofen-dosing regimens by starting with a high first dose followed by a twice-daily maintenance dosing regimen that requires increase over time and should be continued until sufficient effect has been achieved.

Prospective observational study on assessing the hemodynamic relevance of patent ductus arteriosus with frequency domain near-infrared spectroscopy.

BACKGROUND: What constitutes a hemodynamically relevant patent ductus arteriosus (hrPDA) in preterm infants is unclear. Different clinical and echocardiographic parameters are used, but a gold standard definition is lacking. Our objective was to evaluate associations between regional cerebral tissue oxygen saturation (rcStO2), fraction of tissue oxygen extraction (rcFtO2E) measured by frequency domain near-infrared spectroscopy (fd-NIRS) and their correlation to echocardiographic, Doppler-ultrasound, and clinical parameters in preterm infants with and without a hrPDA. METHODS: In this prospective observational study, 22 infants < 1500 g (mean [± SD]: gestational age 28.6 [±1.8] weeks, birth weight 1076 [±284] g, median (interquartile range) postnatal age at measurement 7.6 (4.6-12.9) d) with a clinical suspicion of hrPDA were analysed. Twelve infants had left-to-right shunt through PDA, and in 6 of these the PDA was classified as hrPDA based on pre-defined clinical and echocardiographic criteria. fd-NIRS, echocardiographic and Doppler-ultrasound examinations were performed. After identification of blood hemoglobin (Hb) as confounding factor, rcStO2 and rcFtO2E were corrected for this effect. RESULTS: Overall mean ± standard deviation (normalised to a median Hb of 13.8 mg/dl) was 57 ±5% for rcStO2 and 0.39 ±0.05 for rcFtO2E. Comparing no-hrPDA with hrPDA infants, there were no significant differences in mean rcStO2 (58 ±5% vs. 54 ±5%; p = .102), but in mean rcFtO2E (0.38 ±0.05 vs. 0.43 ±0.05; p = .038). Echocardiographic parameter and Doppler indices did not correlate with cerebral oxygenation. CONCLUSION: Oxygen transport capacity of the blood may confound NIRS data interpretation. Cerebral oxygenation determined by fd-NIRS provided additional information for PDA treatment decisions not offered by routine investigations. Whether indicating PDA therapy based on echocardiography complemented by data on cerebral oxygenation results in better outcomes should be investigated in future studies.

Effectiveness of the critical congenital heart disease screening program for early diagnosis of cardiac abnormalities in newborn infants.

OBJECTIVES: To evaluate the effectiveness of critical congenital heart disease (CCHD) screening program for early diagnosis of cardiac anomalies in newborn infants.  Methods: This is a hospital-based prospective cross-sectional study conducted in the Pediatric and Neonatology Department, King Fahad Hospital at  Albaha, Saudi Arabia, between February 2016 and February 2017. Results: We screened 2961 (95.4%) of 3103 patients in a nursery unit; 142 (4.6%) patients were not screened. The test was positive in 114 (3.9%) patients and negative in 2847 (96.1%). There were 94 (3.2%) false positives and 20 (0.7%) true positives. Critical cardiac defects were diagnosed in 7 (0.2%) patients of all screened infants, and severe pulmonary hypertension was diagnosed in 13 (0.4%) patients. True negative results were found in 2841(96%) patients, and no cardiac defect was diagnosed, whereas false negative results were seen in 6 (0.2%) patients diagnosed with ventricular septal defect. The sensitivity was 77%, and the specificity was very high at 97%, with a positive predictive value of 18%, and a negative predictive value of 99.8% (95% confidence interval 13.78-19.18, p=0.0001). Conclusion: Pulse oximetry was found to be easy, safe, sensitive, and highly specific for diagnosis of CCHD.