Article Figures & Data
Figures
- Figure 1 Effects of healthy and PE medium on mixed cortical cultures
Mixed cortical cultures were exposed to medium conditioned by healthy (n=6) or preeclamptic (n=6) placental explants that had been pre-treated with no NPs, blank-NPs or MitoQ-NPs. Following a 24-h exposure, cortical cultures were assessed for neuronal dendrite lengths (A), process lengths of TH+ cells (B), levels of glutamate receptor subunits GluN1 (C) and GluN3α (D), levels of GABA receptor subunits GABA Aα1 (E) and GABA B1 (F), along with astrocyte count (G) and astrocyte process lengths (H). Data are presented as means + S.E.M.; *P<0.05, **P<0.01, ***P<0.001.
- Figure 2 Role of glutamate inhibition in mediating effects of PE medium on mixed cortical cultures
Mixed cortical cultures were incubated with NMDA receptor antagonist MK801 prior to exposure to culture medium conditioned by healthy (n=6) or preeclamptic (n=6) placental explants that had been treated with no NPs or with MitoQ-NPs. Lengths of neuronal dendrites (A), lengths of TH+ cell processes (B) and levels of glutamate receptor subunit GluN1 (C) were measured in the cortical cultures (data are presented as means + S.E.M.; ***P<0.001).
- Figure 3 Role of astrocytes in mediating effects of PE medium on neurons
(A–C) Neuron-only cortical cultures were exposed directly to medium conditioned by healthy or preeclamptic placental explants, which had been pre-treated with no NPs, blank-NPs or MitoQ-NPs. (D–F) In a second experiment, neuron-only cultures were exposed to medium collected from astrocyte-only cortical cultures, which had previously been exposed to the placenta conditioned medium, and some neuronal cultures were additionally pre-treated with NMDA receptor antagonist MK801. Neuronal dendrite lengths (A,D), TH+ process lengths (B,E) and glutamate receptor subunit GluN1 levels (C,F) were measured (data are presented as means + S.E.M.; *P<0.05, **P<0.01, ***P<0.001).
- Figure 4 Role of neurons in mediating effects of PE medium on astrocytes
(A and B) Astrocyte-only cortical cultures were exposed directly to medium conditioned by healthy or preeclamptic placental explants, which had been pre-treated with no NPs, blank-NPs or MitoQ-NPs. (C–F) In a second experiment, astrocyte-only cultures were exposed to medium collected from neuron-only cortical cultures, which had previously been exposed to the placenta conditioned medium. Astrocyte cultures were assessed with regard to cell count (A and E), process lengths (B and F) along with levels of glutamate receptor subunits GluN1 (C) and GluN3α (D) (data are presented as means + S.E.M.; **P<0.01, ***P<0.001).
- Figure 5 Characterization of PE conditioned medium
(A) Total levels of proteinogenic amino acids (µmol/l ± S.E.M.) were measured in culture medium conditioned by healthy (n=3) or preeclamptic (n=3) placental explants, following pre-treatment with no NPs, blank-NPs or MitoQ-NPs. (B) Log 2 fold change values are shown for those microRNAs that were significantly altered in PE medium (n=4) compared with healthy medium (n=4) (blue). Log fold changes of those same microRNAs in medium conditioned by preeclamptic placental explants treated with MitoQ-NPs (n=3), compared with untreated PE medium, are shown in orange.
- Figure 6 Overview of potential mechanistic effects of PE medium on cortical cultures
Secreted molecules from the preeclamptic placenta, including potentially microRNAs, may have direct effects on neurons and astrocytes. Some of the effects of PE medium on cortical cultures may also be indirect via glutamate secretion from astrocytes or by secretion of unknown factors from neurons.
- Figure 7 Overview of experimental setup
Culture medium conditioned by healthy or preeclamptic placental explants, following exposure to no NPs, blank-NPs or MitoQ-NPs, was applied to mixed cortical cultures, astrocyte-only cortical cultures or neuron-only cortical cultures (A). To establish the role of astrocytes in mediating the effects of placenta-conditioned medium on cortical cultures, culture medium from the exposed astrocyte-only cultures was applied to neuron-only cultures (B); in order to investigate the role of neurons in mediating the effects of placenta condition medium on cortical cultures, culture medium from neuron-only cultures that had been exposed to placenta-conditioned medium was applied to astrocyte-only cultures (C).
Tables
- Table 1 Amino acid concentration in μmol/l (±S.E.M.) in medium conditioned by healthy (n=3) or preeclamptic placentas (n=3), after exposure to no NPs, blank-NPs or MitoQ-NPs
Healthy PE Amino acid No NP Blank-NP MitoQ-NP No NP Blank-NP MitoQ-NP Ala 79.27 (45.02) 90.93 (64.43) 54.77 (24.70) 79.70 (52.22) 64.07 (42.47) 251.0 (228.84) Arg 357.0 (5.483) 335.7 (33.79) 331.8 (19.46) 336.6 (6.552) 332.7 (17.84) 371.1 (26.71) Asn 16.63 (16.63) 21.33 (21.33) 7.833 (7.833) 15.07 (15.07) 11.70 (11.70) 94.13 (94.13) Asp 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 67.17 (67.17) Cys 58.63 (7.636) 58.10 (2.001) 62.60 (7.614) 67.57 (4.927) 69.83 (4.190) 66.27 (4.983) Gln 1509 (396.8) 1411 (275.0) 1358 (318.2) 1293 (436.2) 1273 (442.3) 1464 (477.7) Glu 64.73 (53.55) 67.90 (67.90) 36.10 (30.62) 41.00 (24.97) 62.97 (50.80) 201.4 (188.5) Gly 409.4 (34.20) 384.3 (51.72) 356.8 (24.70) 398.9 (30.74) 393.3 (52.11) 585.4 (223.0) His 153.3 (12.70) 143.4 (21.24) 142.9 (15.69) 127.8 (7.503) 139.2 (10.12) 185.2 (59.89) Ile 702.4 (35.45) 629.6 (17.69) 647.5 (28.17) 683.4 (48.12) 682.9 (46.88) 770.0 (59.13) Leu 554.4 (192.9) 674.5 (23.68) 671.0 (24.70) 725.4 (30.02) 720.2 (53.58) 908.5 (177.8) Lys 736.7 (17.91) 674.5 (36.12) 675.6 (26.43) 726.3 (15.76) 721.9 (61.11) 890.9 (170.8) Met 171.6 (7.270) 154.1 (4.206) 152.5 (7.878) 162.5 (10.52) 175.0 (13.83) 208.4 (40.77) Phe 377.8 (14.26) 344.5 (15.59) 346.3 (14.85) 386.8 (9.103) 390.2 (33.93) 467.3 (79.52) Pro 86.00 (17.56) 87.20 (34.33) 72.50 (12.85) 146.1 (66.25) 100.4 (33.79) 252.7 (188.6) Ser 377.4 (9.481) 346.9 (23.40) 342.9 (13.37) 369.1 (13.16) 358.1 (31.58) 514.3 (151.0) Thr 740.6 (23.73) 671.67 (27.48) 679.6 (27.09) 711.6 27.48) 716.3 (53.71) 829.0 (106.1) Trp 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 23.73 (23.73) Tyr 366.2 (10.53) 330.2 (14.00) 336.4 (13.60) 364.8 (12.40) 355.7 (26.76) 433.0 (60.29) Val 733.8 (27.62) 673.4 (23.32) 671.8 (26.17) 734.27 (27.35) 741.7 (56.19) 893.6 (153.5) - Table 2 Enriched biological processes
Biological process Count Fold enrichment P value Sensory perception of chemical stimulus 3 <0.2 3.20E-23 Developmental process 468 1.53 1.99E-17 Sensory perception of smell 1 <0.2 1.17E-16 Intracellular signal transduction 262 1.67 4.44E-13 Sensory perception 40 0.38 3.47E-11 Signal transduction 518 1.37 4.18E-11 MAPK cascade 108 2.16 1.16E-10 Cell communication 565 1.34 1.88E-10 Cell cycle 226 1.59 5.55E-09 Nervous system development 173 1.64 1.42E-07 System development 248 1.47 5.14E-07 Cellular process 1461 1.13 8.56E-07 Death 118 1.74 3.21E-06 Cell death 118 1.74 3.21E-06 Results of gene ontology analysis of predicted targets of differentially abundant microRNAs in PE placenta conditioned medium compared with healthy placental conditioned medium are listed. P values have been corrected for multiple comparisons using FDR.
- Table 3 Top 15 enriched pathways
KEGG pathway Count Fold enrichment P value MAPK signalling pathway 106 2.40 1.11E-17 Ras signalling pathway 89 2.27 5.58E-13 Pathways in cancer 127 1.86 1.51E-11 Neurotrophin signalling pathway 55 2.64 8.13E-11 Rap1 signalling pathway 79 2.17 1.58E-10 PI3K-Akt signalling pathway 112 1.87 1.89E-10 Melanoma 37 3.00 4.36E-09 Axon guidance 53 2.41 8.11E-09 ErbB signalling pathway 41 2.72 1.44E-08 FoxO signalling pathway 54 2.32 1.98E-08 Renal cell carcinoma 33 2.93 7.95E-08 T-cell receptor signalling pathway 44 2.46 9.83E-08 Signalling pathways regulating pluripotency of stem cells 54 2.22 9.61E-08 Regulation of actin cytoskeleton 71 1.94 2.24E-07 Insulin signalling pathway 52 2.17 4.19E-07 Results of KEGG pathway analysis of predicted targets of differentially abundant microRNAs in PE placenta conditioned medium compared with healthy placental conditioned medium are listed. P values have been corrected for multiple comparisons using Benjamini–Hochberg method.
- Table 4 microRNAs significantly affected in preeclampsia and by MitoQ-NP treatment
microRNA Known relevant functions Up-regulated in PE hsa-miR-561-5p Regulates 11β-HSD1, which is highly expressed in liver, adipose tissue and CNS [96] Associated with Parkinson’s disease [97] hsa-miR-548ai+hsa-miR-570-5p miR-570 associated with autism [98] and congenital heart disease [99] miR-570 regulates cytochrome P450 [100] hsa-miR-196b-5p Associated with ectopic pregnancy [101] Associated with endometriosis [102] hsa-miR-2117 None hsa-miR-3065-3p None Down-regulated in PE hsa-miR-596 None hsa-miR-451a Associated with type 2 diabetes [103] Listed are those microRNAs that were found to be up- or down-regulated in culture medium conditioned by preeclamptic placental explants, compared with healthy explants, and that were also affected in the opposite direction by treatment of the placenta with MitoQ-NPs but not blank-NPs.
- Table 5 Clinical data of collected preeclamptic placentae
Gestation Parity Severity PCR IUGR Co-morbidities 1* 35+5 0 Moderate 274 Unknown n/a 2* 37+3 0 Moderate 361 No n/a 3*†‡ 40+8 2 Mild 204 No n/a 4* 40+13 0 Moderate 331 No Hypothyroid 5*‡ 37 0 Severe 427 Unknown DM1 6*‡ 27+4 0 Severe 537 Yes APKD 7*†‡ 26+1 1 Severe 448 Yes SLE nephritis, previous DVT/pulmonary embolus, baby has metabolic disorder 8*† 38+4 4 Severe 32 No n/a APKD, adult polycystic kidney disease; DM1, type 1 diabetes; DVT, deep vein thrombosis; IUGR, intrauterine growth restriction; PCR, protein-creatinine ratio; SLE, systemic lupus erythematosus. Conditioned media from the highlighted placentas was *applied to cortical cultures; †analysed for amino acids; ‡analysed for microRNA.
December 2018
Volume: 2 Issue: 4
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