| Abstract |
Preeclampsia (PE) affects 3-5% of all pregnancies worldwide and is a major cause of maternal mortality and morbidity. PE is a consequence of diverse pathological processes involving impaired implantation, endothelial dysfunction and systemic inflammation, and it is characterized by hypertension and proteinuria during pregnancy.
The Notch signaling pathway is an evolutionarily conserved intercellular signaling mechanism, which is involved in cell fate decisions and pattern formation during development. NOTCH1-4 receptors are single-pass transmembrane proteins that are activated by Delta (DLL1,-3,-4) and Jagged/Serrate (JAG1,-2) ligands. Each of these proteins shows cell-type- and tissue-specific expression during development. This ligand-receptor interaction leads to the proteolytic cleavage and release of the NOTCH intracellular domain (NICD), which translocates to the nucleus, where it interacts with the DNA with the help of transcription factor RBPJK, inducing the expression of numerous target genes. Members of the Notch signaling pathway have been detected in the developing placenta, where they play an important role in placentation.
NICD activates the transcription of several target genes, most notably the HEY1 and HEY2 genes, which are required for vascular development. These genes are expressed in the developing cardiovascular system, including the heart, endothelial cells and vascular smooth muscles, as well as in the placental labyrinth, while in mice Hey1 is additionally expressed in some trophoblast cells in the ectoplacental cone. Knockout of any of these genes leads to placental vascular deficiencies and to early embryonic lethality, due to major vascular and cardiac defects.
The aim of this study was to investigate the expression of receptors NOTCH1,-2,-3,-4, ligands DLL1,-3,-4, JAG1,-2 and target genes HEY1,-2 in placental tissue samples from 20 late preterm or term pregnancies complicated by PE versus 20 normal pregnancies. mRNA levels of the studied molecules were measured by quantitative Real-Time PCR (qRT-PCR), while the
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protein expression of the intracellular domain of NOTCH2 (NICD2) and NOTCH3 (NICD3) was measured by Western Blot (WB).
qRT-PCR analysis revealed that NOTCH1, NOTCH4 and DLL1 were not expressed in the placenta. Possible explanations regarding expression variations between our study and previous ones are the different experimental procedures used, since we measured expression with qRT-PCR while those studies used either Western Blot or immunohistochemistry, the different ethnic background of the study populations, and the temporal and spatial heterogeneity of the placental structures, since it is believed that CTBs alter the expression of Notch receptors and ligands as they differentiate.
On the contrary, NOTCH2, NOTCH3, DLL3, DLL4, JAG1, JAG2, HEY1 and HEY2 mRNA levels were downregulated in PE samples vs. controls (p<0.01). WB confirmed that NICD2 (p=0.014) and NICD3 (p<0.001) protein levels were also lower in PE specimens. Statistical analysis revealed several significant associations: of NOTCH3 mRNA expression with smoking during pregnancy (p=0.029), of NICD3 protein levels (p=0.028) and DLL3 mRNA levels (p=0.041) with birth weight centile, and of HEY2 transcript levels with parity (p=0.034) and mode of delivery (p=0.028).
The main strength of our research is that it is the first study to concurrently measure the mRNA levels of all key Notch signaling pathway molecules, and especially target genes HEY1 and HEY2, whose expression in human placenta have never been studied before, and to determine the associations between them. Study limitations include the fact that our research was conducted only on late preterm and term placentas, and therefore we were not able to measure the expression of the Notch pathway molecules during the earlier stages of pregnancy, and that our samples were whole tissue homogenates and not micro-dissected, which means that the measured expression of the Notch signaling pathway genes was their average expression among the various placenta cell types and not their expression in each cell type individually. Evidently, these limitations could also explain the differences between our results and those of some previously
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conducted studies. Another potential limitation would be the differences in gestation period (shorter in PE cases), mode of delivery (since elective caesarean sections were more common in PE samples than in controls) and smoking status (since more women with PE smoked) between the two groups, which could introduce a possible bias in the results. However, since univariate analysis, with the aforementioned parameters as co-factors, found that the differences in the expression of all studied genes between PE cases and controls retained their statistical significance, our findings cannot be attributed to sampling bias, but are linked to PE pathophysiology.
Our findings, that receptors NOTCH2,-3, ligands DLL3,-4 and JAG1,-2 and target genes HEY1,-2 are downregulated in PE-complicated late preterm and term placentas, provide evidence that the Notch signaling pathway is associated with this pregnancy complication (visual depiction of the pathway in Fig. 4). Further research regarding the function of Notch receptors, ligands and target genes in the placenta is required in order to elucidate their role in PE pathogenesis and to determine whether one or more of these genes could be used as biomarkers for the early detection of PE, or for new preventive and therapeutic strategies.
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