The Effect of Gestational Age on Angiogenic Gene Expression in the Rat Placenta

Abstract

The placenta plays a central role in determining the outcome of pregnancy. It undergoes changes during gestation as the fetus develops and as demands for energy substrate transfer and gas exchange increase. The molecular mechanisms that coordinate these changes have yet to be fully elucidated. The study performed a large scale screen of the transcriptome of the rat placenta throughout mid-late gestation (E14.25–E20) with emphasis on characterizing gestational age associated changes in the expression of genes invoved in angiogenic pathways. Sprague Dawley dams were sacrificed at E14.25, E15.25, E17.25 and E20 (n = 6 per group) and RNA was isolated from one placenta per dam. Changes in placental gene expression were identifed using Illumina Rat Ref-12 Expression BeadChip Microarrays. Differentially expressed genes (>2-fold change, <1% false discovery rate, FDR) were functionally categorised by gene ontology pathway analysis. A subset of differentially expressed genes identified by microarrays were confirmed using Real-Time qPCR. The expression of thirty one genes involved in the angiogenic pathway was shown to change over time, using microarray analysis (22 genes displayed increased and 9 gene decreased expression). Five genes (4 up regulated: Cd36, Mmp14, Rhob and Angpt4 and 1 down regulated: Foxm1) involved in angiogenesis and blood vessel morphogenesis were subjected to further validation. qPCR confirmed late gestational increased expression of Cd36, Mmp14, Rhob and Angpt4 and a decrease in expression of Foxm1 before labour onset (P<0.0001). The observed acute, pre-labour changes in the expression of the 31 genes during gestation warrant further investigation to elucidate their role in pregnancy.

Gestation Related Gene Expression of the Endocannabinoid Pathway in Rat Placenta

“…Ptgs1 and Ptgs2 have been widely studied in placentae of women and animals models for decades [ 18 ]. Here we observe that expression of Ptgs2 in whole rat placenta in both cohorts remained stable throughout E14.25, E15.25, and E17.25 but was significantly upregulated at E20, just before the onset of labor, consistent with previous studies [ 16 , 19 – 21 ]. Since Ptgs1 and Ptgs2 are directly involved in prostaglandin syntheses, any differential or aberrant expression of these two enzymes may have a dramatic effect in prostaglandin levels [ 19 ].…”

“…This control mechanism could be an important player in the process of labor and hence this study should be further expanded in preterm samples as there is a paucity of data with regard to the role of Plcd4 in pregnancy. Apart from identifying the presence of the gene transcripts in myometrium from pregnant versus nonpregnant rats, Plcd4 , unlike phospholipase A [ 16 , 33 ], has not been studied for its role in placenta especially with its link to labor and parturition. Nevertheless PLCD4 can be further investigated in human samples as a potential biomarker for preterm deliveries in perhaps other gestational tissues, including membranes and/or biological fluids, and a reduced level of the enzyme could be a useful prognostic for impending preterm birth.…”

“…Array experiment readout was deposited on ArrayExpress (ArrayExpress accession number E-MTAB-1987). Overall microarray results were previously published by our group [ 16 ]. For this study, expression of the 7 enzymes Faah, Mgll, Plcd4, Pld1, Nat1, Daglα, and Ptgs2 in the endocannabinoid system was analyzed.…”

The rat placental renin-angiotensin system - a gestational gene expression study

“…The microarray analysis (cohort 1) was performed as previously published [ 28 ]. In short, 500 ng of total RNA was converted to double stranded cDNA and this was used to generate biotinylated cRNA probes using the Illumina TotalPrep RNA Amplification Kit.…”

“…Four time points were chosen as they cover the time period from mid gestation to the day prior to labour onset (E14.25, E15.25, E17.25 and E20). These investigations are apart of a larger gene expression study [ 28 , 29 ]. The rat model allows the study of RAS compenents at time-points (mid to late gestation) difficult to be obtained from human and unlike in the mouse, placentation in the rat involves the deep invasion of trophoblast ( i.e.…”