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Article

Transcriptome Analysis Reveals Differential Gene Expression between the Closing Ductus Arteriosus and the Patent Ductus Arteriosus in Humans

by
Junichi Saito
1,
Tomoyuki Kojima
1,2,
Shota Tanifuji
1,
Yuko Kato
1,
Sayuki Oka
1,
Yasuhiro Ichikawa
3,
Etsuko Miyagi
2,
Tsuyoshi Tachibana
3,
Toshihide Asou
3 and
Utako Yokoyama
1,*
1
Department of Physiology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan
2
Department of Obstetrics and Gynecology, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan
3
Department of Cardiovascular Surgery, Kanagawa Children’s Medical Center, 2-138-4 Mutsukawa, Minami-ku, Yokohama, Kanagawa 232-8555, Japan
*
Author to whom correspondence should be addressed.
J. Cardiovasc. Dev. Dis. 2021, 8(4), 45; https://0-doi-org.brum.beds.ac.uk/10.3390/jcdd8040045
Submission received: 8 March 2021 / Revised: 8 April 2021 / Accepted: 14 April 2021 / Published: 16 April 2021
(This article belongs to the Special Issue Leaders in Cardiovascular Research: A Special Issue Dedicated to Professor Adriana Gittenberger-De Groot)
Browse Figures
Versions Notes

Abstract

:
The ductus arteriosus (DA) immediately starts closing after birth. This dynamic process involves DA-specific properties, including highly differentiated smooth muscle, sparse elastic fibers, and intimal thickening (IT). Although several studies have demonstrated DA-specific gene expressions using animal tissues and human fetuses, the transcriptional profiles of the closing DA and the patent DA remain largely unknown. We performed transcriptome analysis using four human DA samples. The three closing DA samples exhibited typical DA morphology, but the patent DA exhibited aorta-like elastic lamellae and poorly formed IT. A cluster analysis revealed that samples were clearly divided into two major clusters, the closing DA and patent DA clusters, and showed distinct gene expression profiles in IT and the tunica media of the closing DA samples. Cardiac neural crest-related genes such as JAG1 were highly expressed in the tunica media and IT of the closing DA samples compared to the patent DA sample. Abundant protein expressions of jagged 1 and the differentiated smooth muscle marker calponin were observed in the closing DA samples but not in the patent DA sample. Second heart field-related genes such as ISL1 were enriched in the patent DA sample. These data indicate that the patent DA may have different cell lineages compared to the closing DA.

1. Introduction

The ductus arteriosus (DA) is a fetal vascular shunt that connects the pulmonary artery and the aorta, and it is essential for maintaining fetal circulation. The DA begins to close immediately after birth. This closing process is characterized by several DA-specific features including differentiated contractile smooth muscle cells (SMCs), fragmentation of internal elastic laminae, sparse elastic fiber formation in the tunica media, and intimal thickening (IT) formation [1,2,3,4,5,6]. Histological analyses of human DA samples and several animal models indicated that these DA-specific features gradually develop throughout the fetal and neonatal periods [7]. Patent DA is a condition where the DA does not close properly after birth. Patent DA occurs in approximately 1 in 2000 full-term infants and occurs more frequently in premature neonates [8]. PDA samples exhibit fewer DA-specific structural features [9]. Comprehensive analysis of gene expression comparing the closing DA and the patent DA is necessary in order to better understand DA-specific remodeling and to explore methods to regulate patency of the DA.
Several research groups have previously reported the gene expression profiles of mouse, rat, and ovine DAs [10,11,12,13,14,15,16,17]. However, transcriptome analyses of human DA tissues are limited [18,19]. Yarboro et al. performed RNA sequencing using human DA tissues of 21 weeks gestation and identified genes that were distinctly expressed in the DA tissue compared to the aorta [18]. Additionally, they compared their human RNA sequencing data with previously reported rodent microarray data and demonstrated transcriptional commonalities between human and rodent DAs [18]. A report by Mueller et al. [19] is presently the only published study that demonstrated the differences of transcriptional profiles using postnatal human DAs. They compared gene expression between stent-implanted open DAs on postnatal days 222 and 239, closed ligamentous DAs on day 147, and an open unstented DA on day 1. To the best of our knowledge, transcriptomic comparisons of the closing DA and the patent DA in human infants have not been previously reported.
Using four postnatal human DA tissues, we performed an unbiased transcriptome analysis using the IT and the tunica media of each DA tissue. We found differential gene expression between the tunica media of the closing DA and that of the patent DA. Additionally, we investigated genes enriched in the IT or the tunica media of closing DA tissues to identify genes that potentially contribute to DA-specific remodeling.

2. Materials and Methods

2.1. Study Subjects and Ethics Statements

The protocol for using human DA tissues was approved by the Research Ethics Committees at Tokyo Medical University and Kanagawa Children’s Medical Center (reference numbers: T2020-0238 and 1502-05, respectively). The protocol conformed to the principles outlined in the Declaration of Helsinki. After receiving written informed parental consent, human DA tissues were obtained from four patients with congenital heart diseases, during cardiac surgeries in Kanagawa Children’s Medical Center. The patient information for each of the four cases included in this study is summarized in Table 1.

2.2. Total RNA Preparation and Microarray Analysis

Four human DA tissues were subjected to tissue staining and transcriptome analysis. Each DA tissue was divided into two pieces. One piece was fixed with 10% buffered formalin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for tissue staining. The other piece of tissue was prepared for microarray analysis as follows. After the adventitia was removed, each DA tissue was divided into two parts: the inner part and the outer part, which mainly contained IT and the tunica media, respectively, as indicated by the yellow dotted lines in Figure 1A. The tissues were immediately frozen in liquid nitrogen and stored at −80 °C until all patient samples were collected. Total RNA preparation and microarray analysis were performed as described previously [10,11]. Briefly, the frozen tissues were disrupted by a multi-bead shocker instrument (Yasui Kikai, Osaka, Japan). After buffer RLT with β-mercaptoethanol was added to the tissues, they were sonicated to ensure the samples were uniformly homogeneous. Total RNA was isolated using a RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). Microarray experiments were carried out using a SurePrint G3 Human GE 8 × 60 K v2 Microarray (Agilent, Santa Clara, CA, USA) according to the manufacturer’s protocol.

2.3. Generation of a Dendrogram, Venn Diagrams, and a Heatmap

The dendrogram was generated with Ward’s method using the hclust and dendrogram functions in R. The packages gplots and genefilter in R were used to create a heatmap in which data was normalized into a z-score. The mapping grids were subsequently colored according to their z-score. Venn diagrams of the number of differentially expressed genes in each sample group were generated using the gplots package in R.

2.4. Gene Set Enrichment Analyses (GSEAs)

Gene set enrichment analyses (GSEAs) were conducted to investigate the functions of genes that significantly correlated with each sample group. GSEAs ranked the gene list by the correlation between genes and phenotype, and an enrichment score was calculated to assess the gene distribution. Each analysis was carried out with 1000 permutations. Gene sets were considered significantly enriched if the false discovery rate (FDR) q-value < 0.25 [20].

2.5. Tissue Staining and Immunohistochemistry

Paraffin-embedded blocks containing the human DA tissues were cut into 4 μm thick sections and placed on glass slides. Elastica van Gieson staining was performed for morphological analysis to evaluate IT and the tunica media, as described previously [21,22]. Immunohistochemistry was performed using primary antibodies for jagged 1 (sc-390177, Santa Cruz Biotechnology, Dallas, TX, USA) and calponin (M3556, DakoCytomation, Glostrup, Denmark). Biotinylated rabbit antibody (Vectastain Elite ABC IgG kit, Vector Labs, Burlingame, CA, USA) was used as a secondary antibody, and the presence of targeted proteins was determined by 3,3′-diaminobenzidine tetrahydrochloride (DAB) (DakoCytomation, Glostrup, Denmark). Negative staining of immunohistochemistry was confirmed by the omission of primary antibodies.

3. Results

3.1. DA-Related Clinical Course of Each Participant

Four patients with congenital heart diseases were analyzed in this study. Patient profiles are presented in Table 1. Case 1 was considered a patent DA case because the DA did not exhibit closing tendency throughout the clinical course. The DA tissue was isolated during an operation for an atrioventricular septal defect closure and repairs of the aortic arch and pulmonary venous returns. The other three cases (Cases 2–4) exhibited complex congenital heart diseases that required DA patency to maintain systemic circulation. Cases 2–4 were administered prostaglandin E1 (PGE1) because they exhibited closing tendency of the DA.
In Case 2, lipo-PGE1 (1 ng/kg/min) was administered 8 h after birth when an echocardiography indicated narrowing of the DA. Case 2 continued lipo-PGE1 treatment until the operation. The patient had an aortic repair and pulmonary artery banding (PAB) conducted on postnatal day 5.
Case 3 showed closing tendency of the DA soon after birth and was administrated lipo-PGE1 (2 ng/kg/min). The dose of lipo-PGE1 was increased (4 ng/kg/min) 8 h after birth due to further closing tendency. The patient had PAB conducted on postnatal day 3. The closing tendency of the DA remained and required PGE1-cyclodextrin (30 ng/kg/min) on postnatal day 4. The patient received PGE1-cyclodextrin until the Norwood operation was conducted on postnatal day 24.
Case 4 showed closing tendency of the DA at 9 h after birth and received a lipo-PGE1 infusion (1 ng/kg/min). The patient underwent PAB on postnatal day 3. The DA was gradually narrowed and required an increased dose of lipo-PGE1 (5 ng/kg/min) on postnatal day 70. The patient underwent the Norwood operation on postnatal day 98. On the basis of their clinical courses, Cases 2–4 were considered as closing DAs.

3.2. Histological Differences between the Patent DA and the Closing DA Tissues

The Elastica van Gieson stain demonstrated that Case 1 had well-organized layered elastic fibers in the tunica media and a poorly formed IT (Figure 1A, upper panel). In Case 1, there was no overt fragmentation of the internal elastic laminae (Figure 1B, upper panel). Case 2 and Case 3 showed prominent IT formation that protruded into the lumen (Figure 1A, middle panels). Circumferentially oriented layered elastic fibers in the tunica media were sparsely formed and the internal elastic laminae were highly fragmented (Figure 1B, middle panels). Similarly, Case 4, who received PGE1 administration for more than 3 months, had a prominent IT (Figure 1A, lower panel). However, the entire tunica media consisted of sparse elastic fibers radially oriented toward the internal lumen, and circumferentially oriented elastic fibers were not recognized (Figure 1B, lower panel). These findings indicated that the closing DA had well-recognized, DA-specific morphological features, including prominent IT formation, fragmented internal elastic laminae, and less elastic fibers in the tunica media, which seemed to reflect a normal closing process. On the other hand, the patent DA tissue (Case 1) was devoid of these structures and exhibited aortification of the vascular wall, which was consistent with previously reported morphological characteristics of the patent DA [9].

3.3. Microarray Analysis of the IT and the Tunica Media of Human DA Tissues

To elucidate a differential gene expression profile between the patent DA (Case 1) and the closing DA tissues (Cases 2–4), we performed an unbiased transcriptomic analysis using these human DA tissues. Each DA sample was divided into the IT and the tunica media in Cases 1–3. In Case 4, circumferentially oriented SMCs and layered elastic laminae could not be identified; therefore, the IT-like wall was divided into the inner part and the outer part (Figure 1A, lower). These samples were subjected to microarray analysis.
The dendrogram demonstrated that the human DA tissues were clearly divided into two major clusters, A and B (Figure 2). Cluster A consisted of both the IT and the tunica media from Case 1. Cluster B consisted of the samples from Cases 2–4, and this cluster was further divided into two subgroups, B1 and B2. Cluster B1 consisted of the IT tissues from Cases 2 and 3 and both the inner and outer IT-like parts from Case 4. Cluster B2 consisted of the tunica media samples from Cases 2 and 3. These data suggested that the patent DA tissue (Case 1) had a distinct gene expression pattern compared to the other closing DA samples (Cases 2–4). In Cases 2 and 3, the gene expression patterns of the tunica media samples were relatively similar. Additionally, the IT samples showed similar gene expression profiles, which were distant from the tunica media samples of Cases 2 and 3. In agreement with histological analysis showing that two parts of the DA tissue from Case 4 (inner and outer parts) exhibited an IT-like structure, these two samples of Case 4 showed similar gene patterns, which were close to that of the IT of Cases 2 and 3.

3.4. Transcriptomic Differences between the Tunica Media of Closing DA Tissues and the Patent DA Tissue

Both the histological assessment and the cluster analysis of DA tissues demonstrated that the gene expression profile of the tunica media of the patent DA tissue (Case 1) was markedly different from that of the closing DA tissues (Cases 2 and 3). We, thus, compared gene expressions between the tunica media of the patent DA and closing DA tissues.
The GSEAs between the tunica media of the closing DA and the patent DA tissues, using all gene sets related to biological processes in the Gene Ontology (GO) (size > 300), revealed that the closing DA tissues were significantly correlated to 87 biological processes (FDR < 0.25, Table 2). Notably, vascular development-related gene sets (GO_REGULATION_OF_VASCULATURE_DEVELOPMENT and GO_BLOOD_VESSEL_MORPHOGENESIS) were highly enriched in the tunica media of closing DA tissues (Figure 3). Kinase activation-related gene sets (GO_REGULATION_OF_MAP_KINASE_ACTIVITY, GO_ACTIVATION_OF_PROTEIN_KINASE_ACTIVITY, and GO_POSITIVE_REGULATION_OF_PROTEIN_SERINE_THREONINE_KINASE_ACTIVITY) and three catabolic process-related gene sets, including GO_REGULATION_OF_PROTEIN_CATABOLIC_PROCESS, were positively correlated with the closing DA tunica media tissue. This suggested that intracellular signaling was more actively regulated in the closing DA tissues compared to the patent DA tissue. Protein secretion-related gene sets (GO_POSITIVE_REGULATION_OF_SECRETION and GO_GOLGI_VESICLE_TRANSPORT) and adhesion-related gene sets (GO_POSITIVE_REGULATION_OF_CELL_ADHESION, GO_REGULATION_OF_CELL_CELL_ADHESION, and GO_CELL_SUBSTRATE_ADHESION) were also enriched in the closing DA tissues, which support previous reports which found that multiple extracellular matrices and cell–matrix interactions play roles in DA-specific physiological remodeling [5,21,22]. The gene set GO_RESPONSE_TO_OXYGEN_LEVELS was positively correlated to the closing DA tissues (Figure 3). In this gene set, EGR1, which was previously shown to increase immediately after birth in rat DA tissues [10], was upregulated in the tunica media of the closing DA tissues. Enrichment of the gene set GO_ACTIN_FILAMENT_ORGANIZATION in the closing DA tissues (Figure 3) contained the Rho GTPase RHOD, which regulates directed cell migration [23]. This may support the migratory feature of SMCs in the closing DA tissue.
Although we demonstrated several genes that were highly expressed in the tunica media of the closing DAs compared to that of the patent DA (Figure 3, and Table 2 and Table 3), postnatal PGE1 administration possibly affected these gene expressions of the tunica media. To address this issue, we compared gene expressions of the tunica media between shorter-term PGE1-treated DAs (less than one month of administration, Cases 2 and 3) and a longer-term PGE1-treated DA (more than three months of administration, Case 4). The GSEAs revealed that the outer part of longer-term PGE1-treated DA was significantly correlated to eight biological processes related to cell-cycle regulation (FDR < 0.25, Table S1 and Figure S1A,B, Supplementary Materials) compared to the tunica media of shorter-term PGE1-treated DAs. Among these gene sets, the gene sets GO_ORGANELLE_FISSION and GO_NEGATIVE_REGULATION_OF_CELL_CYCLE_PROCESS belonged to gene sets that were highly expressed in the tunica media of the closing DAs (Table 2). These two gene sets may be associated with PGE1 administration, but not with specific features of the closing DA. However, the remaining 85 gene sets in Table 2 seemed to be independent of duration of PGE1 administration.

3.5. Vascular Development-Related Genes in Human DA Tissues

The vascular development-related gene sets noted above (Table 2 and Figure 3) contain cardiovascular cell lineage-related genes. A heatmap composed of cardiovascular cell lineage-related genes demonstrated distinct gene expression patterns between the closing DA tissues (Cases 2 and 3) and the patent DA tissue (Case 1) (Figure 4A). The genes SEMA5A, SFRP1, NRG1, CTNNB1, PHACTR4, and JAG1 were highly expressed in the ITs of the closing DA tissues. Among these genes, the expression of PHACTR4 and JAG1, which are cardiac neural crest-related genes, was greater in the tunica media of the closing DA tissues than the patent DA tissue. The expression levels of CFL1, TWIST1, EDNRB, SMO, and MAPK1 were greater in the tunica media of the patent DA tissue compared to the closing DA tissues. Similarly, expressions of SEMA4F, NRP1, LTBP3, EDN3, and FGF8 were enriched in the tunica media of the patent DA tissue. SEMA3G, ALX1, SOX8, ALDH1A2, and SEMA7A were relatively highly expressed in the entire tissue of the patent DA. WNT8A, KLHL12, FBXL17, and ISL1, which is a second heart field-related gene, were relatively enriched in the patent DA tissue, and the expression levels of these genes were higher in the IT than in the tunica media.

3.6. The Closing or Patent DA Tissue-Specific Gene Expression

Figure 4B presents a Venn diagram that shows probe sets that were upregulated (>8-fold) in the tunica media of the closing DA tissues (Cases 2 and 3) compared to the patent DA tissue (Case 1). Twenty-one overlapped probe sets consisted of 16 genes (Table 3). APLN, CEMIP2, and GHRL are related to vascular development [24,25,26]. There were several genes related to adhesion and protein secretion such as APLN, CD83, FLCN, and NEDD9 [27,28,29,30]. GHRL and NEDD9 were reported to regulate actin filament organization [31,32]. APLN was reported to promote proliferation and migration of vascular SMCs, as well as promote SMC contraction [27]. NEDD9 is involved in embryonic neural crest cell development and promotes cell migration, cell adhesion, and actin fiber formation [31]. To examine the effect of PGE1 administration on the human DAs, we compared gene expressions of the tunica media between shorter-term PGE1-treated DAs (Cases 2 and 3) and a longer-term PGE1-treated DA (Case 4) using a Venn diagram (Figure S1C, Supplementary Materials). We identified 20 probe sets that overlapped and were enriched in the outer part of a longer-term PGE1-treated DA compared to the IT of shorter-term PGE1-treated DAs (Table S2, Supplementary Materials). These genes did not belong to the genes in Table 3, suggesting that the genes presented in Table 3 did not seem to have been strongly influenced by PGE1 administration.
In the Venn diagram in Figure 4C, 116 probe sets are presented, which were upregulated (>8-fold) in the tunica media of the patent DA tissue (Case 1) compared to the closing DA tissues (Cases 2 and 3). These probe sets contained 52 genes (Table 4). Latent transforming growth factor beta-binding protein 3 (LTBP3) was upregulated in the tunica media of the patent DA tissue. LTBP3 is related to extracellular matrix constituents [33] and second heart field-derived vascular SMCs [34]. Expression of PRSS55, identified as an aorta-dominant gene in rodent microarray data [11], was elevated in the patent DA tissue.

3.7. Jagged 1 Was Highly Expressed in the Closing DA Tissues

Previous reports using genetically modified mice clearly demonstrated that SMCs of the DA are derived from cardiac neural crest cells, and these cells contribute to SMC differentiation in the DA [35,36]. Since the transcriptome analysis revealed that the neural crest cell-related gene JAG1 was abundantly expressed in the closing DA tissues compared to the patent DA tissue (Figure 4A), we performed immunohistochemistry to examine protein expression of jagged 1. In agreement with the transcriptome data, jagged 1 was highly expressed in the closing DA tissues (Cases 2–4) (Figure 5A). Calponin is well recognized as a differentiated SMC marker [1], and it was decreased in the DA tissues of Jag1-deficient mice [37]. A strong immunoreaction for calponin was observed in the closing DA tissues but was not as strong in the patent DA tissue (Figure 5B).

3.8. Transcriptomic Characteristics of the IT and the Tunica Media in the Closing DA Tissues

Lastly, we investigated the difference in gene expression between the IT and the tunica media in the closing DA tissues. IT formation is partly attributed to migration and proliferation of the tunica media-derived SMCs [2,3,4,5,7]. Gene expression analysis indicated that there were different transcriptomic characteristics between the IT and the tunica media in the closing DA tissues (Clusters B1 and B2 in Figure 2). We, thus, compared the expression of genes between the IT and the tunica media of the closing DA tissues (Cases 2 and 3).
The GSEAs between the IT and the tunica media, using all gene sets which related to biological processes in the Gene Ontology (GO) (size > 300), were performed. The analyses revealed that the IT was significantly correlated to 89 biological processes (FDR < 0.25, Table 5), and that the tunica media correlated to 81 biological processes (FDR < 0.25, Table 6). The IT of the closing DAs was significantly correlated to more than 10 migration- and proliferation-related gene sets (GO_MICROTUBULE_CYTOSKELETON_ORGANIZATION and GO_CELL_DIVISION, etc.) (Figure 6A,B). Wnt signaling-related gene sets (GO_REGULATION_OF_WNT_SIGNALING_PATHWAY, GO_CANONICAL_WNT_SIGNALING_PATHWAY, and GO_CELL_CELL_SIGNALING_BY_WNT) were also enriched in the IT of the closing DA tissues. The tunica media of the closing DAs was significantly correlated to vascular development-related gene sets (GO_REGULATION_OF VASCULATURE_DEVELOPMENT, GO_BLOOD_VESSEL_MORPHOGENESIS, GO_VASCULAR _DEVELOPMENT, and GO_CIRCULATORY_SYSTEM_DEVELOPMENT) (Figure 6C,D). Five adhesion-related gene sets, including GO_BIOLOGICAL_ADHESION, were enriched in the tunica media compared to the IT in the closing DA tissues.
Figure 6E presents a Venn diagram that shows probe sets that were upregulated (>8-fold) in the IT of closing DA tissues compared to the tunica media of the same DA tissues (Cases 2 and 3). Eight overlapped probe sets consisted of eight genes (Figure 6E and Table 7). POU4F1, FGF1, and PROCR are related to cell division and cell cycle [38,39,40]. FGF1 is reportedly involved in proliferation and migration of vascular SMCs [39]. A Venn diagram in Figure 6F shows 12 probe sets that were commonly upregulated (>8-fold) in the tunica media of the closing DA tissues compared to the IT of the same DA tissues (Cases 2 and 3), which consisted of eight genes (Figure 6F and Table 8). There were several genes related to muscle structure development such as BDKRB2, MSC, and DCN [41,42,43]. DCN is also involved in extracellular constituents and stabilizes collagen and elastic fibers [44,45].

4. Discussion

The present study demonstrated that neonatal closing DAs exhibited prominent IT and sparse elastic fiber formation, which are typical human DA characteristics. Postnatal closing DA tissues had abundant expression of cardiac neural crest-related protein jagged 1 and the differentiated smooth muscle marker calponin compared to the patent DA tissue. On the other hand, the patent DA tissue had a distinct morphology (e.g., aorta-like elastic lamellae and a poorly formed IT) and gene profiles, such as second heart field-related genes, compared to the closing DA tissues.
The DA is originally derived from the sixth left aortic artery and has a unique cell-lineage [46]. SMCs of the DA are derived from cardiac neural crest cells [35,47,48] and the DA endothelial cells (ECs) are from second heart field [48,49], while both SMCs and ECs of the adjacent pulmonary artery are derived from second heart field [48,49]. In the ascending aorta, ECs are derived from second heart field, and SMCs of the inner medial layer and outer layer are derived from neural crest cells and second heart field, respectively [47,48]. The heatmap of transcriptome data indicated that these cell lineage-related genes were differentially expressed between the closing DA tissues and the patent DA tissue. Although it was difficult to clearly classify these genes into each lineage due to some overlap, cardiac neural crest-related genes such as JAG1 were highly expressed in the closing DA tissues. In contrast, second heart field-related genes, such as ISL1, were enriched in the patent DA tissue.
The DA has been reported to have differentiated SMCs compared to the adjacent great arteries [1,50]. Slomp et al. demonstrated high levels of calponin expression in the tunica media of the fetal human DA [1]. Similarly, Kim et al. reported the presence of highly differentiated SMCs in the fetal rabbit DA, according to SM2 expression [50]. These differentiated SMCs have a high contractile apparatus, which makes them compatible with the postnatal potent DA contraction [1,50]. Additionally, several mutant mice with the patent DA had less differentiated SMCs [35,36,37]. Ivey et al. reported that mice lacking Tfap2β, which is a neural crest-enriched transcription factor, had decreased expression of calponin on embryonic day 18.5 [36]. Huang et al. utilized mice that harbored a neural crest-restricted deletion of the myocardin gene and demonstrated that decreased SMC contractile proteins were present on embryonic day 16.5 [35]. In addition, SMC-specific Jag1-deficient mice had a limited expression of SMC contractile proteins, even at postnatal day 0 [37]. The transcriptome data of the human DA tissues in the present study exhibited decreased protein levels of Jagged 1 and calponin in the patent DA tissue compared to the closing DA tissues. These altered SMC differentiation markers may contribute to postnatal DA patency in humans.
Prematurity and several genetic syndromes are reported to increase the incidence of the patent DA [51], and the patent DA can be classified into three groups, i.e., (1) patent DA in preterm infants, (2) patent DA as a part of a clinical syndrome, and (3) non-syndromic patent DA. This study included only one case with patent DA who had heterotaxy syndrome (polysplenia), which is a major study limitation. Indeed, in the present study, the expression of Nodal, which plays a primary role in the determination of left–right asymmetry, was positively correlated to the closing DAs compared to the patent DA with heterotaxy (Figure 3B). Therefore, it was not able to conclude that differentially expressed genes between in the closing DAs and the patent DA were associated with DA patency, but not with heterotaxy.
There are several syndromes (mutated genes) associated with the patent DA, such as Cantú (ABCC9), Char (TFAP2B), DiGeorge (TBX1), Holt-Oram (TBX5), and Rubinstein–Taybi (CREBBP) syndromes [52,53,54]. In addition to these syndromes, heterotaxy syndrome was reported to have a higher incidence of patent DA [55]. Notch signaling pathways have been reported to play a role in the establishment of left–right asymmetry via regulating Nodal expression [56,57]. Mutant for the Notch ligand Dll1 or double mutants for Notch1 and Notch2 exhibited defects in left–right asymmetry [56,57]. Dll1-null mutants die at early embryonic days due to severe hemorrhages [58], and it is not yet elucidated whether this Dll1-mediated Notch signaling is involved in the pathogenesis of patent DA. It has been reported that combined SMC-specific deletion of Notch2 and heterozygous deletion of Notch3 in mice showed the patent DA, but not heterotaxy [59]. In this study, we delineated the low levels of JAG1, which is a Notch ligand, in the patent DA with heterotaxy syndrome. Mice with Jag1-null mutant are early embryonic lethal due to hemorrhage [60], and SMC-specific Jag1-deleted mice are postnatal lethal due to patent DA [37]. These Jag1 mutants were not reported to exhibit heterotaxy. In humans, there is no obvious relationship between Alagille syndrome (JAG1) and patent DA or heterotaxy [61,62]. In mice, phenotypes of patent DA or heterotaxy seem to depend on ligands and isoforms of receptors of Notch signaling. In addition, there are differences in phenotypes caused by genetic mutations between in mice and humans. Analysis of non-syndromic patent DA would provide further insights into molecular mechanisms of closing and patency of the human DA.
Yarboro et al. performed RNA sequencing to determine genes that were differentially expressed in the preterm human DA and aorta at 21 weeks gestation [18], which was a much earlier time point than we used in our study. They found that several previously recognized DA-dominant genes in rodent studies [11,14,15,17] (e.g., ABCC9, PTGER4, and TFAP2B) were also upregulated in the preterm human DA tissues compared to the aorta [18]. Some DA-dominant genes (e.g., ERG1 and SFRP1) in the preterm human DA [18] were upregulated in the closing DAs compared to the patent DA in the present study. In addition, some aorta-dominant genes, including ALX1 [18], were upregulated in the patent DA compared to the closing DA, which might partly support the aortification phenotype of the patent DA. Jag1 was reported to be the term DA dominant gene rather than the preterm DA dominant gene in rats [10], suggesting that Jag1 contributes to normal DA development. In addition to these previously reported genes, the gene profiles in our study potentially provide novel candidate genes (e.g., APLN and LTBP3) that may contribute to vascular SMC development and function [27,34]. Further study is needed to understand the roles of these genes in DA development.
Mueller et al. performed DNA microarray analysis using postnatal human DAs [19]. Their DA samples were composed of two stent-implanted DAs, one ligamentous DA, and one un-stented open DA [19]. We compared our data to their un-stented open DA dominant genes; however, we could not find obvious overlapped genes among them. One possible reason is that Mueller et al. compared the un-stented open DA on postnatal day 1 to the stented DAs on postnatal days 222 and 239. The stent implantation and time-course of sampling might affect the gene expressions.
This study elucidated the transcriptomic difference between the closing DA tissues and the patent DA tissue in humans. However, one of the limitations in this study pertains to the different durations of PGE1 administration. In utero, the DA is dilated by prostaglandin E2 (PGE2), which is mainly derived from the placenta [63]. After birth, the loss of the placenta and the increased flow of the lung, which is the major site of PGE catabolism, cause a decline in circulating PGE2 [64]. This decline in PGE2 contributes to a postnatal DA contraction [64]. We previously reported PGE2-induced structural DA remodeling via the prostaglandin receptor EP4 (e.g., IT formation [4,5,22] and attenuation of elastic laminae in the tunica media [6]). In humans, Mitani et al. reported that lipo-PGE1 administration increased IT formation in the DA [65]. Gittenberger-de Groot et al. reported that PGE1 treatment induced histopathologic changes (e.g., edema) in the human DA [66]. In this study, Case 4 who received the longest PGE1 administration, for 98 days, had prominent IT formation and less visible layered elastic fibers. This study demonstrated that the duration of PGE1-treatment affected gene expressions such as cell-cycle process-related genes. On the basis of these findings, postnatal PGE1 administration was thought to influence not only structural changes but also gene expression in the postnatal DA.
In 1977, Gittenberger-de Groot et al. performed histological analysis of 42 specimens of postnatal human DAs ranging in age from 12 h after premature delivery to 32 years [9]. An abnormal wall structure of the DA was found in all 14 patients that were over 4 months of age, and the most prominent feature was an aberrant distribution of elastic material, such as unfragmented subendothelial elastic lamina [9]. Three of the 14 patients also showed countable elastic laminae in the tunica media, namely, an aortification [9]. The histological finding of patent DA (Case 1) in the present study was consistent with this aortification type, showing aberrant distribution of elastic materials.
As mentioned above, a major limitation of this study is the use of only one sample of the patent DA, which could not represent the whole entity of patent DA. It is difficult to obtain large numbers of samples with a variety of different congenital heart diseases because isolation of the DA is possible only in the case of a limited number of surgical procedures (e.g., aortic arch repair). However, transcriptome comparisons of different types of patent DA tissues would be more informative to elucidate the pathogenesis of the human patent DA.

5. Conclusions

Transcriptome analysis using the IT and the tunica media of human DA tissue revealed different gene profiles between the patent DA and the closing DA tissues. Cardiac neural crest-related genes such as JAG1 were highly expressed in the tunica media and IT of the closing DA tissues compared to the patent DA. Second heart field-related genes, such as ISL1, were enriched in the patent DA. The data from this study indicate that patent DA tissue may have different cell lineages from closing DA tissue.

Supplementary Materials

The following are available online at https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/jcdd8040045/s1: Figure S1. Differential gene expression between the longer-term PGE1-treated human ductus arteriosus (DA) tissue (Case 4) and the shorter-term PGE1-treated DA tissues (Cases 2 and 3); Table S1. Gene Ontology biological process terms (size > 300) that were significantly upregulated (FDR < 0.25) in the outer part of long-term PGE1-treated human ductus arteriosus (DA) tissue (Case 4) compared to the tunica media of short-term PGE1-treated DA tissues (Case 2 and Case 3); Table S2. Twenty genes that overlapped and were enriched (>8-fold) in the outer part of a longer-term PGE1-treated DA tissue (Case 4) compared to the IT of shorter-term PGE1-treated DA tissues (Cases 2 and 3).

Author Contributions

Conceptualization, J.S. and U.Y.; methodology, U.Y.; validation, J.S., T.K., S.T., Y.K., and U.Y.; formal analysis, J.S., T.K., S.T., Y.K., and U.Y.; investigation, J.S., T.K., S.T., Y.K., S.O., Y.I., and U.Y.; resources, T.T. and T.A.; data curation, J.S., T.K., S.T., Y.K., and U.Y.; writing—original draft preparation, J.S. and U.Y.; writing—review and editing, J.S., T.K., S.T., Y.K., E.M., and U.Y.; visualization, J.S.; supervision, E.M.; project administration, U.Y.; funding acquisition, J.S., S.T., Y.K., and U.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (J.S., JP18K15681, JP16H07107; S.T., JP20K17730; Y.K., JP17K08976, JP21K07352; U.Y., JP20H03650, JP20K21638, JP18K08767), the Japan Agency for Medical Research and Development (AMED) (U.Y., 20ek0210117h0002), the Lydia O’Leary Memorial Pias Dermatological Foundation (Y.K.), and the Miyata Cardiac Research Promotion Foundation (U.Y.).

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Ethics Committee of Tokyo Medical University and Kanagawa Children’s Medical Center (protocol codes: T2020-0238 and 1502-05; date of approval: 16-November-2020 and 9-July-2015).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data presented in this study are available within the article.

Acknowledgments

The authors are grateful to Yuka Sawada for excellent technical assistance.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design, execution, interpretation, or writing of this study.

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Figure 1. Histological analysis of the human ductus arteriosus (DA) tissues. (A) Lower magnification images of the Elastica van Gieson stain of the human DA tissues. The yellow dotted lines indicate the border between the intimal thickening (IT) and the tunica media and the border between the tunica media and the adventitia. Scale bars: 200 µm. (B) Magnified images of red boxes in (A). Scale bars: 100 µm.
Figure 1. Histological analysis of the human ductus arteriosus (DA) tissues. (A) Lower magnification images of the Elastica van Gieson stain of the human DA tissues. The yellow dotted lines indicate the border between the intimal thickening (IT) and the tunica media and the border between the tunica media and the adventitia. Scale bars: 200 µm. (B) Magnified images of red boxes in (A). Scale bars: 100 µm.
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Figure 2. A dendrogram of the gene expressions from human ductus arteriosus tissues. IT: intimal thickening.
Figure 2. A dendrogram of the gene expressions from human ductus arteriosus tissues. IT: intimal thickening.
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Figure 3. Gene set enrichment analyses (GSEAs) of the tunica media of the closing human ductus arteriosus (DA) and the patent DA tissues. (A) GSEAs revealed positive correlations between the tunica media of the closing human DA tissues and vascular development–related genes, oxygen level response-related genes, and actin filament organization-related genes. On the x-axis, the genes in each gene set are ranked from the left side (positively correlated) to the right side (negatively correlated). The vertical black lines that look like barcodes indicate each gene in the gene set. The y-axis displays the calculated enrichment score of each gene (green color). NES, normalized enrichment score; FDR, false discovery rate. (B) The top 25 genes that comprise the leading edge of the enrichment score in (A) are shown in each heatmap.
Figure 3. Gene set enrichment analyses (GSEAs) of the tunica media of the closing human ductus arteriosus (DA) and the patent DA tissues. (A) GSEAs revealed positive correlations between the tunica media of the closing human DA tissues and vascular development–related genes, oxygen level response-related genes, and actin filament organization-related genes. On the x-axis, the genes in each gene set are ranked from the left side (positively correlated) to the right side (negatively correlated). The vertical black lines that look like barcodes indicate each gene in the gene set. The y-axis displays the calculated enrichment score of each gene (green color). NES, normalized enrichment score; FDR, false discovery rate. (B) The top 25 genes that comprise the leading edge of the enrichment score in (A) are shown in each heatmap.
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Figure 4. Differential gene expression between the tunica media of the closing human ductus arteriosus (DA) tissues and that of the patent DA tissue. (A) A heatmap of vascular cell lineage-related genes is depicted. (B) A Venn diagram shows the number of probe sets that were highly expressed (>8-fold) in the tunica media of closing DA tissues (Cases 2 and 3) compared to that of the patent DA tissue (Case 1). (C) A Venn diagram shows the number of probe sets highly expressed (>8-fold) in the tunica media of the patent DA tissue (Case 1) compared to that of the closing DA tissues (Cases 2 and 3). IT, intimal thickening.
Figure 4. Differential gene expression between the tunica media of the closing human ductus arteriosus (DA) tissues and that of the patent DA tissue. (A) A heatmap of vascular cell lineage-related genes is depicted. (B) A Venn diagram shows the number of probe sets that were highly expressed (>8-fold) in the tunica media of closing DA tissues (Cases 2 and 3) compared to that of the patent DA tissue (Case 1). (C) A Venn diagram shows the number of probe sets highly expressed (>8-fold) in the tunica media of the patent DA tissue (Case 1) compared to that of the closing DA tissues (Cases 2 and 3). IT, intimal thickening.
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Figure 5. Immunohistochemistry for jagged 1 (A) and calponin (B) in the human ductus arteriosus tissues from Cases 1–4. A brown color indicates positive immunostaining. Scale bars: 100 µm.
Figure 5. Immunohistochemistry for jagged 1 (A) and calponin (B) in the human ductus arteriosus tissues from Cases 1–4. A brown color indicates positive immunostaining. Scale bars: 100 µm.
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Figure 6. Differential gene expression between the intimal thickening (IT) and the tunica media of the closing human ductus arteriosus (DA) tissues. (A) Gene set enrichment analyses (GSEAs) revealed positive correlations between the IT of closing DA tissues and migration- and proliferation–related genes. (B) The top 25 genes that comprise the leading edge of the enrichment score in (A) are shown as a heatmap. (C) GSEAs revealed positive correlations between the tunica media of the closing DA tissues and vascular morphogenesis-related genes. (D) The top 25 genes that comprise the leading edge of the enrichment score in (B) are shown as a heatmap. (E) A Venn diagram shows the number of probe sets that were highly expressed (>8-fold) in the IT compared to the tunica media of closing DA tissues (Cases 2 and 3). (F) A Venn diagram shows the numbers of probe sets that were highly expressed (>8-fold) in the tunica media compared to the IT of closing DA tissues (Cases 2 and 3). NES, normalized enrichment score; FDR, false discovery rate.
Figure 6. Differential gene expression between the intimal thickening (IT) and the tunica media of the closing human ductus arteriosus (DA) tissues. (A) Gene set enrichment analyses (GSEAs) revealed positive correlations between the IT of closing DA tissues and migration- and proliferation–related genes. (B) The top 25 genes that comprise the leading edge of the enrichment score in (A) are shown as a heatmap. (C) GSEAs revealed positive correlations between the tunica media of the closing DA tissues and vascular morphogenesis-related genes. (D) The top 25 genes that comprise the leading edge of the enrichment score in (B) are shown as a heatmap. (E) A Venn diagram shows the number of probe sets that were highly expressed (>8-fold) in the IT compared to the tunica media of closing DA tissues (Cases 2 and 3). (F) A Venn diagram shows the numbers of probe sets that were highly expressed (>8-fold) in the tunica media compared to the IT of closing DA tissues (Cases 2 and 3). NES, normalized enrichment score; FDR, false discovery rate.
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Table
Table 1. A summary of the patient profiles for the four cases included in this study.
Table 1. A summary of the patient profiles for the four cases included in this study.
Case NumberDiagnosisGestational Age (Weeks)Birth Weight (g)Age at Operation (Days)Duration of PGE1 Administration (Days)Closing Tendency of the DA
1Polysplenia, intermediate AVSD, CoA, TAPVC (cardiac type), PDA, IVC interruption (azygos connection)382714170No
2DORV (subpulmonary VSD), hypoplastic distal arch, CoA, PFO 41294855Yes
3HLHS, TR, cor triatriatum, PLSVC4033522424Yes
4HLHS (MA, AA), PLSVC3726549898Yes
Abbreviations: PGE1, prostaglandin E1; DA, ductus arteriosus; AVSD, atrioventricular septal defect; CoA, coarctation of the aorta; TAPVC, total anomalous pulmonary venous connection; PDA, patent ductus arteriosus; IVC, inferior vena cava; DORV, double outlet right ventricle; VSD, ventricular septal defect; PFO, patent foramen ovale; HLHS, hypoplastic left heart syndrome; TR, tricuspid regurgitation; PLSVC, persistent left superior vena cava; MA, mitral atresia; AA, aortic atresia.
Table
Table 2. Gene Ontology biological process terms (size > 300) that were significantly upregulated (FDR < 0.25) in the tunica media of the closing human DA tissues (Cases 2 and 3) compared to that of the patent DA tissue (Case 1).
Table 2. Gene Ontology biological process terms (size > 300) that were significantly upregulated (FDR < 0.25) in the tunica media of the closing human DA tissues (Cases 2 and 3) compared to that of the patent DA tissue (Case 1).
Gene Set NameSizeNESFDR q-ValueRank at Max
GO_REGULATION_OF_VASCULATURE_DEVELOPMENT3101.610.0006660
GO_BLOOD_VESSEL_MORPHOGENESIS5641.610.0006712
GO_GOLGI_VESICLE_TRANSPORT3501.610.0008448
GO_REGULATION_OF_HEMOPOIESIS4411.600.0006338
GO_NCRNA_PROCESSING3401.590.0005889
GO_VIRAL_LIFE_CYCLE3151.580.0007537
GO_REGULATION_OF_MAP_KINASE_ACTIVITY3321.550.0006500
GO_LEUKOCYTE_CELL_CELL_ADHESION3391.540.0005082
GO_NEGATIVE_REGULATION_OF_IMMUNE_SYSTEM_PROCESS4051.540.0004982
GO_NEGATIVE_REGULATION_OF_PHOSPHORYLATION4241.530.0005671
GO_POSITIVE_REGULATION_OF_CELL_ADHESION4101.530.0004942
GO_NEGATIVE_REGULATION_OF_CELL_CYCLE_PROCESS3121.530.0005276
GO_NUCLEAR_TRANSPORT3371.520.0007197
GO_T_CELL_ACTIVATION4581.510.0005293
GO_IN_UTERO_EMBRYONIC_DEVELOPMENT3721.470.0006660
GO_REGULATION_OF_INFLAMMATORY_RESPONSE3481.470.0005237
GO_PEPTIDYL_LYSINE_MODIFICATION3531.460.0008228
GO_REGULATION_OF_PROTEIN_CATABOLIC_PROCESS3721.450.0006333
GO_IMMUNE_RESPONSE_REGULATING_SIGNALING_PATHWAY3851.400.0007472
GO_CELLULAR_RESPONSE_TO_EXTERNAL_STIMULUS3051.400.0007120
GO_REGULATION_OF_CELL_CELL_ADHESION4061.390.0005082
GO_RNA_SPLICING4231.390.0007328
GO_ACTIVATION_OF_PROTEIN_KINASE_ACTIVITY3211.390.0005553
GO_REGULATION_OF_CELLULAR_RESPONSE_TO_STRESS6901.390.0006338
GO_RAS_PROTEIN_SIGNAL_TRANSDUCTION3301.390.0007040
GO_POSITIVE_REGULATION_OF_PROTEIN_SERINE_THREONINE_KINASE_ACTIVITY3291.380.0006543
GO_AMEBOIDAL_TYPE_CELL_MIGRATION3861.380.0007624
GO_REGULATION_OF_DNA_BINDING_TRANSCRIPTION_FACTOR_ACTIVITY4151.370.0006196
GO_REGULATION_OF_LYMPHOCYTE_ACTIVATION4201.350.0005268
GO_RNA_SPLICING_VIA_TRANSESTERIFICATION_REACTIONS3411.350.0007328
GO_REGULATION_OF_T_CELL_ACTIVATION3161.350.0005256
GO_LYMPHOCYTE_DIFFERENTIATION3541.340.0005436
GO_ORGANELLE_FISSION4141.340.0004640
GO_POSITIVE_REGULATION_OF_CATABOLIC_PROCESS4301.340.0004648
GO_EPITHELIAL_CELL_PROLIFERATION3791.330.0116543
GO_REGULATION_OF_PROTEIN_SERINE_THREONINE_KINASE_ACTIVITY4951.330.0106256
GO_MRNA_PROCESSING4771.330.0107113
GO_EMBRYO_DEVELOPMENT_ENDING_IN_BIRTH_OR_EGG_HATCHING6441.320.0106259
GO_NEGATIVE_REGULATION_OF_INTRACELLULAR_SIGNAL_TRANSDUCTION4751.320.0097981
GO_PROTEIN_POLYUBIQUITINATION3271.310.0146212
GO_RIBONUCLEOPROTEIN_COMPLEX_BIOGENESIS4051.310.0136510
GO_MAINTENANCE_OF_LOCATION3051.310.0135837
GO_LEUKOCYTE_DIFFERENTIATION5141.310.0137110
GO_POSITIVE_REGULATION_OF_CELL_CYCLE3571.310.0135009
GO_POSTTRANSCRIPTIONAL_REGULATION_OF_GENE_EXPRESSION5541.290.0126212
GO_RESPONSE_TO_OXYGEN_LEVELS3701.290.0126459
GO_POSITIVE_REGULATION_OF_ESTABLISHMENT_OF_PROTEIN_LOCALIZATION3711.280.0126982
GO_REGULATION_OF_METAL_ION_TRANSPORT3631.280.0124661
GO_PROTEASOMAL_PROTEIN_CATABOLIC_PROCESS4551.280.0115596
GO_NEGATIVE_REGULATION_OF_PHOSPHORUS_METABOLIC_PROCESS5301.260.0115671
GO_POSITIVE_REGULATION_OF_GTPASE_ACTIVITY3751.260.0117037
GO_REGULATION_OF_GTPASE_ACTIVITY4471.260.0117095
GO_POSITIVE_REGULATION_OF_RESPONSE_TO_EXTERNAL_STIMULUS4961.250.0106529
GO_COVALENT_CHROMATIN_MODIFICATION4361.250.0106473
GO_NEURON_DEATH3381.250.0107040
GO_POSITIVE_REGULATION_OF_PROTEOLYSIS3401.240.0106359
GO_POSITIVE_REGULATION_OF_CELLULAR_PROTEIN_LOCALIZATION3071.240.0106982
GO_POSITIVE_REGULATION_OF_CYTOKINE_PRODUCTION4321.240.0105082
GO_PROCESS_UTILIZING_AUTOPHAGIC_MECHANISM4951.230.0095327
GO_VESICLE_ORGANIZATION3151.230.0097253
GO_POSITIVE_REGULATION_OF_CELL_ACTIVATION3241.230.0097094
GO_POST_TRANSLATIONAL_PROTEIN_MODIFICATION3521.230.0097885
GO_LEUKOCYTE_MIGRATION4281.220.0097094
GO_ESTABLISHMENT_OF_ORGANELLE_LOCALIZATION3971.220.0096914
GO_CANONICAL_WNT_SIGNALING_PATHWAY3151.200.0095210
GO_REGULATION_OF_CELLULAR_AMIDE_METABOLIC_PROCESS3851.190.0086376
GO_REGULATION_OF_AUTOPHAGY3271.180.0084699
GO_REGULATION_OF_CHROMOSOME_ORGANIZATION3211.170.0146695
GO_REGULATION_OF_SMALL_GTPASE_MEDIATED_SIGNAL_TRANSDUCTION3121.160.0247040
GO_REPRODUCTIVE_SYSTEM_DEVELOPMENT4281.160.0263453
GO_REGULATION_OF_SUPRAMOLECULAR_FIBER_ORGANIZATION3391.150.0286891
GO_POSITIVE_REGULATION_OF_DEFENSE_RESPONSE3601.140.0485237
GO_POSITIVE_REGULATION_OF_NERVOUS_SYSTEM_DEVELOPMENT5131.140.0474697
GO_REGULATION_OF_APOPTOTIC_SIGNALING_PATHWAY3831.130.0475796
GO_MYELOID_CELL_DIFFERENTIATION3751.120.0616268
GO_RESPONSE_TO_VIRUS3151.120.0605563
GO_ACTIN_FILAMENT_ORGANIZATION4001.110.0776891
GO_RESPONSE_TO_MOLECULE_OF_BACTERIAL_ORIGIN3261.100.0976178
GO_REGULATION_OF_PROTEIN_CONTAINING_COMPLEX_ASSEMBLY4081.100.0986574
GO_OSSIFICATION3781.100.1066790
GO_POSITIVE_REGULATION_OF_SECRETION3581.090.1096953
GO_EPITHELIAL_TUBE_MORPHOGENESIS3261.090.1266477
GO_ACTIVATION_OF_IMMUNE_RESPONSE4331.090.1387472
GO_CELL_SUBSTRATE_ADHESION3421.080.1728285
GO_REGULATION_OF_BINDING3491.080.1786143
GO_REGULATION_OF_DEVELOPMENTAL_GROWTH3241.080.1784701
GO_CELLULAR_RESPONSE_TO_CHEMICAL_STRESS3291.070.2294800
Abbreviations: NES, normalized enrichment score; FDR, false discovery rate.
Table
Table 3. Sixteen genes that overlapped and were enriched (>8-fold) in the tunica media of the closing DA tissues (Cases 2 and 3) compared to that of the patent DA tissue (Case 1).
Table 3. Sixteen genes that overlapped and were enriched (>8-fold) in the tunica media of the closing DA tissues (Cases 2 and 3) compared to that of the patent DA tissue (Case 1).
Gene NameDescriptionFold Change
Case 2 vs. Case 1Case 3 vs. Case 1
CD83CD83 molecule29.929.9
AP1S3adaptor-related protein complex 1 subunit sigma 326.518.0
GSTT1glutathione S-transferase theta 121.610.7
BCL2L13BCL2-like 1312.913.1
NEDD9neural precursor cell expressed, developmentally downregulated 912.113.6
HLA-DMAmajor histocompatibility complex, class II, DM alpha9.116.3
GHRLghrelin and obestatin prepropeptide13.112.2
FLCNfolliculin12.29.1
TCF7transcription factor 7, T-cell-specific11.09.0
ELOVL5ELOVL fatty-acid elongase 59.09.9
APLNapelin9.29.6
MAFFMAF bZIP transcription factor F9.58.8
AURKAPS1aurora kinase A pseudogene 19.78.4
MIS12MIS12 kinetochore complex component 9.58.3
CEMIP2cell migration inducing hyaluronidase 28.49.1
GMCL1germ cell-less 1, spermatogenesis associated8.58.5
Table
Table 4. Fifty-two genes that overlapped and were enriched (>8-fold) in the tunica media of the patent DA tissue (Case 1) compared to that of the closing DA tissues (Cases 2 and 3).
Table 4. Fifty-two genes that overlapped and were enriched (>8-fold) in the tunica media of the patent DA tissue (Case 1) compared to that of the closing DA tissues (Cases 2 and 3).
Gene NameDescriptionFold Change
Case 1 vs. Case 2Case 1 vs. Case 3
MYH16myosin heavy chain 16 pseudogene70.168.9
PRDM12PR/SET domain 1269.868.5
CXXC4CXXC finger protein 462.861.5
VENTXP1VENT homeobox pseudogene 160.457.3
MKRN3makorin ring finger protein 370.943.7
CLEC3AC-type lectin domain family 3 member A26.1251.3
TEX43testis expressed 4351.326.7
SCN11Asodium channel, voltage-gated, type XI, alpha subunit 1138.026.5
CYP3A43cytochrome P450 family 3 subfamily A member 4324.522.3
MROH2Amaestro heat-like repeat family member 2A21.121.4
MUC12mucin 12, cell-surface-associated30.515.3
MS4A6Amembrane-spanning 4-domains subfamily A member 6A46.811.7
RBFOX3RNA-binding fox-1 homolog 315.520.4
SLC2A1solute carrier family 2 member 130.410.7
CFAP299cilia- and flagella-associated protein 29915.415.8
PSG5pregnancy-specific beta-1-glycoprotein 516.314.6
LTBP3latent transforming growth factor beta-binding protein 313.716.9
ZFP57zinc finger protein 5721.811.0
MFSD4major facilitator superfamily domain-containing 4A13.813.8
HOXA11homeobox A1110.219.1
ALLCallantoicase24.88.8
SLC6A14solute carrier family 6 member 1413.112.1
SLC44A4solute carrier family 44 member 412.711.7
MAS1MAS1 proto-oncogene, G-protein-coupled receptor12.212.2
CARD18caspase recruitment domain family member 1812.911.5
LCE1Clate cornified envelope protein 1C12.210.6
PAMR1peptidase domain-containing associated with muscle regeneration 18.118.7
PRSS55serine protease 5511.410.5
RUNDC3BRUN domain-containing 3B10.011.8
LINC00114long intergenic non-protein-coding RNA 114 8.913.8
TFAP4transcription factor AP-411.210.1
PLIN2perilipin 29.811.1
CCR2C–C motif chemokine receptor 211.79.3
CDKN2B-ASCDKN2B antisense RNA 110.09.8
MYO7Amyosin VIIA10.49.3
PDILTprotein disulfide isomerase like, testis expressed 9.69.6
SPA17sperm autoantigenic protein 1710.88.5
SLITRK2SLIT and NTRK-like family member 29.59.5
SLC9B1solute carrier family 9 member B19.39.7
PANX2pannexin 211.48.0
PLPPR1phospholipid phosphatase-related 110.18.8
ASTE1asteroid homolog 19.29.4
MUC16mucin 16, cell-surface-associated9.58.7
OR51B2olfactory receptor family 51 subfamily B member 29.28.6
ARHGAP36Rho GTPase-activating protein 368.88.7
KRTAP4-8keratin-associated protein 4-88.88.5
METTL21CP1methyltransferase-like 21E, pseudogene9.18.3
BPIFB6BPI fold-containing family B member 6 8.98.4
HABP2hyaluronan-binding protein 29.08.2
DUSP13dual-specificity phosphatase 138.98.2
CXorf51Achromosome X open reading frame 51A8.28.0
MTM1myotubularin 18.28.1
Table
Table 5. Gene Ontology biological process terms (size > 300) that were significantly upregulated (FDR < 0.25) in the intimal thickening compared to the tunica media of the closing DA tissues (Cases 2 and 3).
Table 5. Gene Ontology biological process terms (size > 300) that were significantly upregulated (FDR < 0.25) in the intimal thickening compared to the tunica media of the closing DA tissues (Cases 2 and 3).
Gene Set NameSizeNESFDR q-ValueRank at Max
GO_MICROTUBULE_CYTOSKELETON_ORGANIZATION5261.670.0006797
GO_MICROTUBULE_BASED_PROCESS7571.590.0006797
GO_CELL_DIVISION5491.580.0006799
GO_PROTEIN_POLYUBIQUITINATION3271.560.0007778
GO_MITOTIC_CELL_CYCLE9541.540.0006055
GO_MODIFICATION_DEPENDENT_MACROMOLECULE_CATABOLIC_PROCESS6041.510.0006933
GO_NEGATIVE_REGULATION_OF_CELL_CYCLE5661.510.0006548
GO_NEGATIVE_REGULATION_OF_CELL_CYCLE_PROCESS3121.500.0016055
GO_MRNA_PROCESSING4771.500.0016995
GO_RNA_SPLICING_VIA_TRANSESTERIFICATION_REACTIONS3411.480.0016758
GO_ESTABLISHMENT_OF_ORGANELLE_LOCALIZATION3971.470.0016823
GO_REGULATION_OF_MRNA_METABOLIC_PROCESS3261.470.0019158
GO_ORGANELLE_LOCALIZATION6021.460.0016823
GO_RNA_SPLICING4231.450.0026007
GO_CELL_CYCLE16811.440.0026059
GO_ORGANELLE_FISSION4141.440.0025836
GO_CELL_CYCLE_PROCESS12511.430.0026470
GO_REGULATION_OF_MITOTIC_CELL_CYCLE6001.430.0026548
GO_MUSCLE_TISSUE_DEVELOPMENT3681.420.0034069
GO_CELLULAR_PROTEIN_CATABOLIC_PROCESS7331.410.0056933
GO_REGULATION_OF_CELL_CYCLE_PROCESS7061.410.0056450
GO_CELL_CYCLE_PHASE_TRANSITION5781.400.0056055
GO_REGULATION_OF_CELL_CYCLE11101.400.0046548
GO_MICROTUBULE_BASED_MOVEMENT3211.370.0075942
GO_PROTEIN_MODIFICATION_BY_SMALL_PROTEIN_CONJUGATION_OR_REMOVAL10331.360.0086585
GO_PROTEIN_CATABOLIC_PROCESS8761.360.0087607
GO_PROTEASOMAL_PROTEIN_CATABOLIC_PROCESS4551.340.0117536
GO_POST_TRANSLATIONAL_PROTEIN_MODIFICATION3521.330.0134520
GO_VESICLE_ORGANIZATION3151.330.0136530
GO_PROTEIN_MODIFICATION_BY_SMALL_PROTEIN_CONJUGATION8641.320.0147634
GO_PROTEIN_CONTAINING_COMPLEX_DISASSEMBLY3101.320.0146434
GO_CHROMOSOME_ORGANIZATION10591.320.0156004
GO_RNA_PROCESSING11491.310.0167021
GO_REGULATION_OF_CHROMOSOME_ORGANIZATION3211.290.0247299
GO_REGULATION_OF_CELL_CYCLE_PHASE_TRANSITION4241.290.0246055
GO_MRNA_METABOLIC_PROCESS7891.280.0267021
GO_REGULATION_OF_INTRACELLULAR_TRANSPORT3251.280.0266449
GO_MUSCLE_SYSTEM_PROCESS4231.280.0283948
GO_CELLULAR_MACROMOLECULE_CATABOLIC_PROCESS11001.260.0347104
GO_REGULATION_OF_AUTOPHAGY3271.250.0416901
GO_CELLULAR_PROTEIN_CONTAINING_COMPLEX_ASSEMBLY9091.250.0416799
GO_REGULATION_OF_WNT_SIGNALING_PATHWAY3471.250.0423438
GO_RIBONUCLEOPROTEIN_COMPLEX_BIOGENESIS4051.240.0428496
GO_PROCESS_UTILIZING_AUTOPHAGIC_MECHANISM4951.230.0496585
GO_ANATOMICAL_STRUCTURE_HOMEOSTASIS4261.230.0486332
GO_ORGANOPHOSPHATE_BIOSYNTHETIC_PROCESS5261.230.0493613
GO_REGULATION_OF_CELLULAR_CATABOLIC_PROCESS8121.230.0516913
GO_PROTEIN_CONTAINING_COMPLEX_SUBUNIT_ORGANIZATION17161.230.0525624
GO_REGULATION_OF_SYSTEM_PROCESS5711.220.0573069
GO_GLYCEROPHOSPHOLIPID_METABOLIC_PROCESS3251.220.0552842
GO_ORGANELLE_ASSEMBLY7801.220.0555055
GO_MUSCLE_CONTRACTION3391.200.0703948
GO_REGULATION_OF_CELLULAR_LOCALIZATION9391.200.0736537
GO_CYTOSKELETON_ORGANIZATION12781.200.0725064
GO_REGULATION_OF_CATABOLIC_PROCESS9601.200.0757638
GO_MACROMOLECULE_CATABOLIC_PROCESS13191.190.0777614
GO_ORGANONITROGEN_COMPOUND_CATABOLIC_PROCESS12331.190.0806337
GO_CANONICAL_WNT_SIGNALING_PATHWAY3151.180.0843438
GO_DIVALENT_INORGANIC_CATION_TRANSPORT4431.180.0872865
GO_MUSCLE_STRUCTURE_DEVELOPMENT6061.180.0944139
GO_POSITIVE_REGULATION_OF_ESTABLISHMENT_OF_PROTEIN_LOCALIZATION3711.180.0925436
GO_DNA_METABOLIC_PROCESS8221.180.0917381
GO_CELL_CELL_SIGNALING_BY_WNT4881.170.1023490
GO_INTRACELLULAR_TRANSPORT15991.160.1086629
GO_MUSCLE_ORGAN_DEVELOPMENT3601.160.1084772
GO_REGULATION_OF_PEPTIDE_TRANSPORT6411.160.1095020
GO_SECOND_MESSENGER_MEDIATED_SIGNALING4121.150.1253308
GO_PEPTIDE_SECRETION4951.150.1253880
GO_REGULATION_OF_CYTOSKELETON_ORGANIZATION5131.150.1283944
GO_SIGNAL_RELEASE5771.140.1303898
GO_DNA_REPAIR4801.140.1326578
GO_MUSCLE_CELL_DIFFERENTIATION3381.140.1314061
GO_NEGATIVE_REGULATION_OF_PROTEIN_MODIFICATION_PROCESS5651.140.1325343
GO_NEGATIVE_REGULATION_OF_PHOSPHORYLATION4241.140.1385364
GO_MITOCHONDRION_ORGANIZATION4591.140.1387936
GO_NEGATIVE_REGULATION_OF_PHOSPHORUS_METABOLIC_PROCESS5301.130.1445394
GO_HORMONE_TRANSPORT3091.120.1653880
GO_REGULATION_OF_PROTEIN_CATABOLIC_PROCESS3721.120.1767675
GO_REGULATION_OF_PROTEIN_LOCALIZATION9051.110.1846577
GO_RIBOSE_PHOSPHATE_METABOLIC_PROCESS3731.110.1834526
GO_OSSIFICATION3781.110.1805020
GO_REGULATION_OF_DNA_METABOLIC_PROCESS3141.110.1927720
GO_NUCLEOBASE_CONTAINING_SMALL_MOLECULE_METABOLIC_PROCESS5451.110.1963618
GO_PHOSPHOLIPID_METABOLIC_PROCESS4201.100.2155395
GO_REGULATION_OF_ORGANELLE_ORGANIZATION12091.100.2205212
GO_ORGANOPHOSPHATE_METABOLIC_PROCESS9501.090.2383618
GO_REGULATION_OF_CELLULAR_RESPONSE_TO_STRESS6901.090.2356943
GO_CELLULAR_RESPONSE_TO_DNA_DAMAGE_STIMULUS7611.090.2466578
GO_GLYCEROLIPID_METABOLIC_PROCESS4091.090.2465591
Abbreviations: NES, normalized enrichment score; FDR, false discovery rate.
Table
Table 6. Gene Ontology biological process terms (size > 300) that were significantly upregulated (FDR < 0.25) in the tunica media compared to the intimal thickening of the closing DA tissues (Cases 2 and 3).
Table 6. Gene Ontology biological process terms (size > 300) that were significantly upregulated (FDR < 0.25) in the tunica media compared to the intimal thickening of the closing DA tissues (Cases 2 and 3).
Gene Set NameSizeNESFDR q-ValueRank at Max
GO_EXTRACELLULAR_STRUCTURE_ORGANIZATION3761.670.0025349
GO_SKELETAL_SYSTEM_DEVELOPMENT4941.550.0125310
GO_REGULATION_OF_VASCULATURE_DEVELOPMENT3101.490.0307236
GO_EMBRYONIC_ORGAN_DEVELOPMENT4431.490.0236270
GO_PATTERN_SPECIFICATION_PROCESS4421.480.0216393
GO_INFLAMMATORY_RESPONSE7061.450.0297279
GO_TAXIS6121.450.0277278
GO_NEGATIVE_REGULATION_OF_CELL_DEVELOPMENT3111.440.0306296
GO_BLOOD_VESSEL_MORPHOGENESIS5641.430.0307164
GO_NEGATIVE_REGULATION_OF_CELL_DIFFERENTIATION6681.420.0326377
GO_POSITIVE_REGULATION_OF_NERVOUS_SYSTEM_DEVELOPMENT5131.420.0366270
GO_REGIONALIZATION3471.400.0436393
GO_VASCULATURE_DEVELOPMENT6761.390.0506711
GO_EMBRYONIC_MORPHOGENESIS5781.390.0505668
GO_POSITIVE_REGULATION_OF_CELL_DEVELOPMENT5281.380.0525938
GO_REGULATION_OF_NERVOUS_SYSTEM_DEVELOPMENT8881.370.0586409
GO_BIOLOGICAL_ADHESION13791.370.0627362
GO_EPITHELIAL_TUBE_MORPHOGENESIS3261.360.0696392
GO_TUBE_MORPHOGENESIS8081.350.0756726
GO_REGULATION_OF_NEURON_DIFFERENTIATION6311.350.0734480
GO_POSITIVE_REGULATION_OF_DEVELOPMENTAL_PROCESS12981.350.0716726
GO_REGULATION_OF_INFLAMMATORY_RESPONSE3481.340.0746671
GO_POSITIVE_REGULATION_OF_CELL_DIFFERENTIATION9391.340.0726708
GO_AMEBOIDAL_TYPE_CELL_MIGRATION3861.340.0703718
GO_POSITIVE_REGULATION_OF_MULTICELLULAR_ORGANISMAL_PROCESS16621.340.0676427
GO_REGULATION_OF_CELL_ADHESION6821.340.0717361
GO_CELL_MORPHOGENESIS_INVOLVED_IN_NEURON_DIFFERENTIATION5851.330.0756775
GO_NEGATIVE_REGULATION_OF_DEVELOPMENTAL_PROCESS9051.330.0756377
GO_AXON_DEVELOPMENT5121.320.0797161
GO_TUBE_DEVELOPMENT9981.320.0806726
GO_NEUROGENESIS15711.320.0796708
GO_REGULATION_OF_ANATOMICAL_STRUCTURE_MORPHOGENESIS10321.320.0796334
GO_REGULATION_OF_CELL_DIFFERENTIATION17291.310.0866377
GO_REGULATION_OF_T_CELL_ACTIVATION3161.310.0845912
GO_POSITIVE_REGULATION_OF_CELL_ADHESION4101.310.0836893
GO_REGULATION_OF_CELL_DEVELOPMENT9041.310.0866400
GO_REGULATION_OF_CELL_MORPHOGENESIS4741.300.1026663
GO_REGULATION_OF_CELL_CELL_ADHESION4061.290.1056811
GO_ANATOMICAL_STRUCTURE_FORMATION_INVOLVED_IN_MORPHOGENESIS10441.290.1067374
GO_EPITHELIAL_CELL_PROLIFERATION3791.280.1196725
GO_POSITIVE_REGULATION_OF_NEURON_DIFFERENTIATION3561.280.1206270
GO_REGULATION_OF_PEPTIDASE_ACTIVITY4191.280.1186411
GO_LEUKOCYTE_CELL_CELL_ADHESION3391.280.1166889
GO_CELL_CELL_ADHESION8261.280.1197362
GO_CELL_MORPHOGENESIS9961.270.1186775
GO_MORPHOGENESIS_OF_AN_EPITHELIUM5391.270.1256433
GO_CIRCULATORY_SYSTEM_DEVELOPMENT10181.270.1276433
GO_POSITIVE_REGULATION_OF_HYDROLASE_ACTIVITY7191.260.1326562
GO_CELLULAR_PROCESS_INVOLVED_IN_REPRODUCTION_IN_MULTICELLULAR_ORGANISM3301.260.1316092
GO_GLAND_DEVELOPMENT4361.260.1436386
GO_NEGATIVE_REGULATION_OF_MULTICELLULAR_ORGANISMAL_PROCESS11451.250.1476749
GO_REGULATION_OF_CELLULAR_COMPONENT_SIZE3601.250.1523939
GO_SENSORY_ORGAN_DEVELOPMENT5601.250.1555533
GO_NEURON_DIFFERENTIATION13271.250.1556400
GO_REPRODUCTIVE_SYSTEM_DEVELOPMENT4281.250.1544925
GO_CELL_PART_MORPHOGENESIS6801.240.1556775
GO_NEURON_DEVELOPMENT10801.240.1556705
GO_T_CELL_ACTIVATION4581.240.1547224
GO_POSITIVE_REGULATION_OF_CELL_PROJECTION_ORGANIZATION3661.240.1696270
GO_ANIMAL_ORGAN_MORPHOGENESIS10481.240.1686373
GO_ORGANIC_ANION_TRANSPORT4911.220.1686889
GO_CELL_MORPHOGENESIS_INVOLVED_IN_DIFFERENTIATION7281.220.1686775
GO_ANION_TRANSMEMBRANE_TRANSPORT3031.220.1918857
GO_ALCOHOL_METABOLIC_PROCESS3621.220.1927136
GO_G_PROTEIN_COUPLED_RECEPTOR_SIGNALING_PATHWAY12351.220.1959098
GO_EMBRYO_DEVELOPMENT10181.220.1946690
GO_ANION_TRANSPORT6281.210.2046889
GO_REGULATION_OF_NEURON_PROJECTION_DEVELOPMENT4861.210.2024480
GO_REGULATION_OF_CELL_PROJECTION_ORGANIZATION6521.210.2036334
GO_LIPID_CATABOLIC_PROCESS3271.210.2036013
GO_UROGENITAL_SYSTEM_DEVELOPMENT3251.200.2336411
GO_TISSUE_MORPHOGENESIS6391.200.2336433
GO_REGULATION_OF_HEMOPOIESIS4411.200.2367164
GO_REGULATION_OF_HYDROLASE_ACTIVITY12051.200.2366564
GO_REGULATION_OF_IMMUNE_SYSTEM_PROCESS13871.200.2357164
GO_LYMPHOCYTE_DIFFERENTIATION3541.190.2438193
GO_EPITHELIUM_DEVELOPMENT12611.190.2406313
GO_HEAD_DEVELOPMENT7651.190.2387239
GO_ENDOCYTOSIS5411.190.2385944
GO_TRANSMEMBRANE_RECEPTOR_PROTEIN_TYROSINE_KINASE_SIGNALING_PATHWAY6951.190.2457173
GO_REGULATION_OF_CELL_ACTIVATION5381.190.2477342
Abbreviations: NES, normalized enrichment score; FDR, false discovery rate.
Table
Table 7. Eight genes that overlapped and were enriched (>8-fold) in the intimal thickening (IT) compared to the tunica media of the closing DA tissues (Cases 2 and 3).
Table 7. Eight genes that overlapped and were enriched (>8-fold) in the intimal thickening (IT) compared to the tunica media of the closing DA tissues (Cases 2 and 3).
Gene NameDescriptionFold Change IT vs. the Tunica Media
Case 2Case 3
POU4F1POU class 4 homeobox 122.116.4
BMXBMX non-receptor tyrosine kinase10.629.2
FGF1fibroblast growth factor 115.99.0
MPZL2myelin protein zero-like 218.810.2
FMO3flavin-containing dimethylaniline monooxygenase 312.810.0
PROCRprotein C receptor13.08.6
DSPdesmoplakin9.311.7
NR1I2nuclear receptor subfamily 1 group I member 29.710.7
Table
Table 8. Eight genes that overlapped and were enriched (>8-fold) in the tunica media compared to the intimal thickening (IT) of the closing DA tissues (Cases 2 and 3).
Table 8. Eight genes that overlapped and were enriched (>8-fold) in the tunica media compared to the intimal thickening (IT) of the closing DA tissues (Cases 2 and 3).
Gene NameDescriptionFold Change the Tunica Media vs. IT
Case 2Case 3
GAS7growth arrest-specific 752.49.8
H19H19 imprinted maternally expressed transcript14.218.5
BTNL9butyrophilin-like 98.417.9
SELENOPselenoprotein P14.813.4
BDKRB2bradykinin receptor B211.311.6
CHRDL1chordin-like 19.223.6
MSCmusculin11.78.4
DCNdecorin9.09.4
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Saito, J.; Kojima, T.; Tanifuji, S.; Kato, Y.; Oka, S.; Ichikawa, Y.; Miyagi, E.; Tachibana, T.; Asou, T.; Yokoyama, U. Transcriptome Analysis Reveals Differential Gene Expression between the Closing Ductus Arteriosus and the Patent Ductus Arteriosus in Humans. J. Cardiovasc. Dev. Dis. 2021, 8, 45. https://0-doi-org.brum.beds.ac.uk/10.3390/jcdd8040045

AMA Style

Saito J, Kojima T, Tanifuji S, Kato Y, Oka S, Ichikawa Y, Miyagi E, Tachibana T, Asou T, Yokoyama U. Transcriptome Analysis Reveals Differential Gene Expression between the Closing Ductus Arteriosus and the Patent Ductus Arteriosus in Humans. Journal of Cardiovascular Development and Disease. 2021; 8(4):45. https://0-doi-org.brum.beds.ac.uk/10.3390/jcdd8040045

Chicago/Turabian Style

Saito, Junichi, Tomoyuki Kojima, Shota Tanifuji, Yuko Kato, Sayuki Oka, Yasuhiro Ichikawa, Etsuko Miyagi, Tsuyoshi Tachibana, Toshihide Asou, and Utako Yokoyama. 2021. "Transcriptome Analysis Reveals Differential Gene Expression between the Closing Ductus Arteriosus and the Patent Ductus Arteriosus in Humans" Journal of Cardiovascular Development and Disease 8, no. 4: 45. https://0-doi-org.brum.beds.ac.uk/10.3390/jcdd8040045

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