God's Hobby

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

Color scale table imitating heat maps of the DA dominant genes categorized by GeneGo processes.

<p>DA dominant genes are identified using GO analysis (MetaCore). The whole expression data set was processed by importing it into the MetaCore system. The MetaCore system lined up the top 10 (based on <i>p</i>-value) sets of categorized genes according to their GO biological processes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-t005" target="_blank">Table 5</a>). The color scale table imitating heat maps was created manually based on the genes in GO biological processes. A) The genes in all the top processes except the development or morphogenesis processes that emerged in both F and N. B) The genes categorized in the processes related to the development and morphogenesis in both F and N. C) The genes categorized only in cardiovascular or circulatory specific development processes. The color scale is the same as that used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-g003" target="_blank">Figure 3</a>. All heat maps were created manually based on the genes in the GeneGo biological processes. a) The genes in all the processes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-t004" target="_blank">Table 4</a>, except the development or morphogenesis processes which emerged in both F and N. b) The genes categorized in the processes related to development and morphogenesis in both F and N. c) The genes categorized only in cardiovascular or circulatory specific development processes.</p

Obvious differences in gene expression between sorted ECs and SMCs.

<p>A) The expression levels of Tie2 mRNA were significantly higher in ECs than in SMCs. (*<i>p</i><0.05, n = 5) B) The expression levels of γ2-actin mRNA were significantly lower in ECs than in SMCs. (**<i>p</i><0.001, n = 5) C) The expression levels of Ednra mRNA were significantly lower in ECs than in SMCs. (**<i>p</i><0.001, n = 3).</p

Color scale table imitating heat maps of DA dominant genes and Ao dominant genes.

<p>The listed genes in A) and B) are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-t003" target="_blank">Table 3</a>, respectively. The color scale is based on their expression intensities. The green or red color indicates the lowest or the highest expression levels, respectively. The midpoint shown as a dark color represents 235 since it is the average of whole gene expression.</p

Aorta endothelium-dominant genes.

<p>Sixty five genes in the F group and 52 genes in the N group were expressed more than 2-fold in ECs of the aorta than in ECs of the DA (<i>p</i><0.05). Among these aorta dominant genes, 43 genes were expressed more than 2-fold in ECs of the aorta in both groups(above the thick line). F: fetuses before breathing; N: neonates obtained 30 minutes after breathing.</p

DA endothelium-dominant genes.

<p>Eighty two genes in the F group and 81 genes in the N group were expressed more than 2-fold in ECs of the DA than in ECs of the aorta (<i>p</i><0.05). Among these DA dominant genes, 71 genes were expressed more than 2-fold in ECs of the DA in both groups (above the thick line). F: fetuses before breathing; N: neonates obtained 30 minutes after breathing.</p

Top 10 regulatory biological processes worked in the DA ECs.

<p>The MetaCore systems defined the top 10 ranked regulatory biological processes that were dominantly worked in each stage of the DA ECs based upon their <i>p</i> values. F: fetuses before breathing; N: neonates obtained 30 minutes after breathing.</p

Genes showed a significant change (<i>p</i><0.01) between F and N in the DA.

<p>Twenty five genes shows a significant difference between F and N groups in the DA (<i>p</i> values were less than 0.01. Among these 25 genes, Ctgf and Tbc1d30 are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-t003" target="_blank">Table 3</a>, respectively. F: fetuses before breathing; N: neonates obtained 30 minutes after breathing.</p

Thirty overlapping genes that appeared in more than five processes of the top ten ranking as active genes.

<p>Thirty genes that frequently appeared in more than five processes of the top ten ranking in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073685#pone-0073685-t005" target="_blank">Table 5</a> are regarded as active genes. These genes are listed in accordance with their function. F: fetuses before breathing; N: neonates obtained 30 minutes after breathing.</p