Transcription Profiles of Endothelial Cells in the Rat Ductus Arteriosus during a Perinatal Period

Abstract

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 (p<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.

Unique Phenotypes of Endothelial Cells in Developing Arteries: A Lesson from the Ductus Arteriosus

“…previous study, we used a lorescence-activated cell sorter (FACS) to purify ECs from collagenase-digested DA tissues [21]. In this experiment, we incubated pooled fresh cells with luorescein isothiocyanate (FITC)-conjugated anti-CD31 and APC/Cy7-conjugated anti-CD45 antibodies to separate EC and hematopoietic derivation cells, respectively.…”

“…Because the analysis of the expression proiles of the vascular ECs of the DA is challenging, only two studies have been published to date, including ours [21,22]. It is diicult to compare these two studies because we used pooled DA ECs puriied by FACS, whereas Bokenkamp et al used laser-capture microdissection to isolate DA ECs from a frozen sample.…”

“…The comprehensive gene analysis study identiied signiicantly high expressions of Ndeacetylase/N-sulfotransferase (Ndst3), Glipican 3(Gpc3), and heparan-sulfate 6-O-sulfotransferase 2 (Hs6st2), all of which are involved in heparasulfate synthesis, in DA ECs in both full-term fetal and neonatal periods [21]. Ndst3 is the most important heparin-sulfate synthase among the three members of the NDST family [33].…”

“…Other genes that are known to relate to ECM, especially collagen synthesis, were also found to show higher expression levels in DA ECs than aortic ECs: the glycosyltransferase25 domain containing 2 (Glt25d2), which is known to strengthen collagen activity [34]; growth diferentiation factor (Gdf6), and microibrillar-associated protein 5 (Mfap5), which promotes collagen production [35,36]; Mfap4, which stabilizes collagen activity [37]; anthrax toxin receptor 1 (Antxr1), which provides a link between collagen I and actin cytoskeleton [38]; and prolyl 4-hydroxylase-alpha polypeptide (P4ha1), which is related to the procollagen process [39]. ADAM metallopeptidase with thrombospondin type 1 motif-17 preproprotein (Adamts17), plasminogen activator tissue (Plat), and ibrillin 1 (Fbn1), which are also categorized as related to ECM formation, were upregulated in DA ECs [21]. Interestingly, connective tissue growth factor (CTGF) was found to show higher expression in DA ECs than in aortic ECs in the postnatal period, whereas there was no diference in the fetal period [21].…”

“…ADAM metallopeptidase with thrombospondin type 1 motif-17 preproprotein (Adamts17), plasminogen activator tissue (Plat), and ibrillin 1 (Fbn1), which are also categorized as related to ECM formation, were upregulated in DA ECs [21]. Interestingly, connective tissue growth factor (CTGF) was found to show higher expression in DA ECs than in aortic ECs in the postnatal period, whereas there was no diference in the fetal period [21]. CTGF is a well-known downstream mediator of TGF-β1 in various cells and it exhibits diverse functions, such as cell proliferation, apoptosis, cell adhesion, ECM or collagen production, and angiogenesis [40,41].…”