Impact of excessive hypercholesterolemia in pregnancy on the placentas of the male and female offspring

Figures

• 

  Fig. 1. Lipid levels were increased in the male and female eHC placentas. Representative images and quantitative analysis of Sudan IV (lipids are stained in orange; i) and dihydroethidium (DHE, reactive oxygen species are stained in red; ii) staining in the placentas of the control diet (CD, closed symbols) and high cholesterol diet (eHC, open symbols) male (A) and female (B) offspring. Images were adjusted for brightness and contrast using the same parameters. Scale bar = 100 μm. Data are shown as means ± SEM; *p < 0.05 using a two-tailed unpaired t-test, n = 5/group. a.u.: Arbitrary units; MFI: Mean fluorescence intensity.

• 

  Fig. 2. LDLR levels were reduced in the male and female eHC placentas, VLDLR and LOX-1 levels were reduced in only the female eHC placentas, and ABCA1 levels were increased in only the male eHC placentas. Representative blots (i) and densitometry of scavenger receptor class B type I (SR-BI, normalized to total protein; ii), low-density lipoprotein receptor (LDLR, normalized to total protein; iii), very-low-density lipoprotein receptor (VLDLR, normalized to total protein; iv), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1, normalized to total protein, v), ATP-binding cassette transporter A1 (ABCA1, normalized to total protein; vi), and ATP-binding cassette sub-family G member 1 (ABCG1, normalized to total protein; vii) in the placentas of the control diet (CD, closed symbols) and high cholesterol diet (eHC, open symbols) male (A) and female (B) offspring. Data are shown as means ± SEM; *p < 0.05 and **p < 0.01 vs. CD using a two-tailed unpaired t-test, n = 4–5/group. Full uncropped blots are presented in Figure S1-S6.

• 

  Fig. 3. Total cholesterol levels were reduced in only the male eHC offspring. Total cholesterol levels in the plasma of the control diet (CD, closed symbols) and high cholesterol diet (eHC, open symbols) male (A) and female (B) offspring. Data are shown as means ± SEM; *p < 0.05 vs. CD using a two-tailed unpaired t-test, n = 5–6/group.

• 

  Fig. 4. Phosphorylated IRE1α levels were reduced in only the male eHC placentas while phosphorylated eIF2α were reduced in only the female eHC placentas. Representative blots (i) and densitometry of glucose-regulated protein 78 (GRP78, normalized to total protein; ii), cleaved activating transcription factor 6 (c-ATF6, normalized to full length ATF6; iii), phosphorylated inositol requiring-enzyme 1αSer724 (p-IRE1αSer724, normalized to IRE1α; iv), phosphorylated eukaryotic translation initiation factor 2αSer51 (p-eIF2αSer51, normalized to eIF2α; v), activating transcription factor 4 (ATF4, normalized to total protein; vi), and C/EBP homologous protein (CHOP, normalized to total protein; vii) in the placentas of the control diet (CD, closed symbols) and high cholesterol diet (eHC, open symbols) male (A) and female (B) offspring. Data are shown as means ± SEM; *p < 0.05 vs. CD using a two-tailed unpaired t-test, n = 5–8/group. Full uncropped blots are presented in Figure S7-S12.

• 

  Fig. 5. NLRP3 levels were increased and TLR4 levels were reduced in only the male eHC placentas while TXNIP were reduced in only the female eHC placentas. Representative blots (i) and densitometry of thioredoxin-interacting protein (TXNIP, normalized to total protein; ii), NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3, normalized to total protein; iii), and toll-like receptor 4 (TLR4, normalized to total protein; iv) in the placentas of the control diet (CD, closed symbols) and high cholesterol diet (eHC, open symbols) male (A) and female (B) offspring. Data are shown as means ± SEM; *p < 0.05 vs. CD using a two-tailed unpaired t-test, n = 5–8/group. Full uncropped blots are presented in Figure S13-S15.

• 

  Fig. 6. IL-1β levels were reduced and caspase 8 levels were increased in only the male eHC placentas while HMGB1 levels were reduced in only the female eHC placentas. Interleukin (IL)−1β (i) and IL-18 (ii) levels, as well as representative blots (iii) and densitometry of N-terminal gasdermin D (NT-GSDMD, normalized to total protein; iv) and high mobility group box 1 (HMGB1, normalized to total protein; v) and representative blots (vi) and densitometry of caspase 1 (normalized to pro-caspase 1; vii) and caspase 8 (normalized to pro-caspase 8; viii), in the placentas of the control diet (CD, closed symbols) and high cholesterol diet (eHC, open symbols) male (A) and female (B) offspring. Data are shown as means ± SEM; *p < 0.05 vs. CD using a two-tailed unpaired t-test, n = 4–7/group. Full uncropped blots are presented in Figure S16-S19.

• 

  Fig. 7. Schematic summary of the effects of eHC on the male and female placentas. ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette sub-family G member 1; ATF6: Activating transcription factor 6; eIF2α: Eukaryotic translation initiation factor 2α; ER: Endoplasmic reticulum; GRP78: Glucose-regulated protein 78; GSDMD: Gasdermin D; GSDMDNT: N-terminal gasdermin D; HMGB1: High mobility group box 1; IL: Interleukin; IRE1α: Inositol requiring-enzyme 1α; LDLR: Low-density lipoprotein receptor; LOX-1: Lectin-like oxidized low-density lipoprotein receptor-1; NLRP3: NOD-, LRR-, and pyrin domain-containing protein 3; ROS: Reactive oxygen species; SR-BI: Scavenger receptor class B type I; TLR4: Toll-like receptor 4; TXNIP: Thioredoxin-interacting protein; VLDLR: Very low-density lipoprotein receptor. Created in BioRender.com.