Syncytiotrophoblast vesicles show altered micro-RNA and haemoglobin content after ex-vivo perfusion of placentas with haemoglobin to mimic preeclampsia

Abstract

Background: Cell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content.

Methods: The dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR.

Results: All micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs.

Conclusion: This study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.

Figure 1. Transmission electron micrographs of isolated STBMs.

The TEM panel (A) shows a selection of the 150K control STBMs, prior to gold-labelling. The STBMs were mixed with gold-labelled antibodies, as described in the Materials and Methods section, in two preparations. In the first preparation, STBMs shown in panels (B, control) and (C, Hb perfusions), were treated with antibodies against TF, CD63 and hsa-mir-222 labelled with colloidal gold of different sizes; CD63 (30 nm colloidal gold), TF (15 nm) and mir-222 (5 nm). Panel (B) shows the 150K control STBMs which are similar to the 150K Hb STBMs (C) in regards to the number of vesicles and their size. The 150K control STBMs (B) contain more mir-222 than 150K Hb STBMs (C). For the second preparation, STBMs shown in panels (D, control) and (E, Hb perfusions), were treated only with antibodies against Hb, labelled with colloidal gold. Control STBMs (D) carried small amounts of Hb, whereas STBMs from the Hb perfused placentas (E) showed higher labelling for Hb on particles of all sizes.

Figure 2. NTA analysis of vesicle size distribution in the 10K and 150K fraction.

Nanoparticle tracking analysis (NTA) size distribution profiles for STBMs; comparing effect of Hb perfusion for 10K (A) and 150K (B) STBMs. In (C) the effect of centrifugation speed, 10K vs 150K, is compared. 10K STBMs had a significantly larger median size than 150K STBMs.

Figure 3. Bar charts showing miRNA fold change in STBMs from control and Hb perfusions.

Nine selected miRNAs were analysed using quantitative PCR, as described in the Materials and methods section. The miRNA expression was normalized against Rnu6b and given as fold change. The fold change values were calculated by normalizing against control samples from control perfused placentas. Results are presented as mean ±SD. Differences between the respective control and Hb perfusions were analysed using Mann-Whitney U-test. * p<0.05. In the 10K STBMs mir-517a, mir-141 and mir -517b were significantly down regulated in Hb perfusions compared to controls. No significant differences were detected in the 150K STBMs (data not shown).