Abstract

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

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