Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: AOPP-BSA mediates endothelial senescence and apoptosis evasion. HUVECs were treated with 200 μg/ml AOPP-BSA for 12 h. Serum-free medium was considered as blank control and BSA was applied as negative control. (A) The expression of senescence-associated proteins p21 and p16 were detected by western blot. GAPDH was used as loading control. (B,C) Quantification of p21 and p16 protein expression ( n = 4). (D) The senescent cells was identified with SA-β-gal assay. Scale bar for upper panel = 20 μm, scale bar for lower panel = 50 μm. (E) The SA-β-gal positive rate was quantitated ( n = 4). (F) The expression of apoptosis-related proteins CASP3, Bax, and Bcl-2 were detected by western blot. β-actin was used as loading control. (G,H) The changes of cleaved CASP3/Pro-CASP3 ratio and Bax/Bcl-2 ratio were compared ( n = 5). (I) Apoptosis rate in HUVECs was detected by Annexin V-FITC/PI assays with flow cytometry. (J) Apoptosis rate was compared ( n = 6). HUVECs exposed to the temperature of 55°C for 10 min were used as apoptosis positive control. One-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons was used for statistical analysis. All data are shown as mean ± SEM. CASP3, caspase 3.

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Control, Negative Control, Expressing, Western Blot, Flow Cytometry, Positive Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: AOPP-BSA promotes endothelial senescence via RAGE. (A) HUVECs were transfected with RAGE siRNA for 48 h before exposure to AOPP-BSA (200 μg/ml, 12 h). Serum-free medium was used as blank control and control siRNA as negative control. The expression of RAGE, p21 and p16 were detected by western blot. GAPDH was used as loading control. (B–D) Quantification of RAGE ( n = 4), p21 ( n = 3), and p16 ( n = 3) protein expression. (E) HUVECs were transfected with RAGE siRNA or control siRNA before AOPP-BSA application. The senescent cells was identified with SA-β-gal assay. Scale bar for upper panel = 20 μm, scale bar for lower panel = 50 μm. (F) The SA-β-gal positive rate was quantitated ( n = 4). Data were analyzed by one-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons. All data are shown as mean ± SEM. ns, non-significant ( P > 0.05).

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Transfection, Control, Negative Control, Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: AOPP-BSA mediates endothelial hyperpermeability and impairs angiogenesis. (A,C) HUVECs were stimulated with 200 μg/ml AOPP-BSA for 0, 1, 3, 6, 12, 24 h, then transendothelial electrical resistance (TER) and permeability coefficient of FITC-dextran (Pa) were detected ( n = 4). (B,D) HUVECs were treated with AOPP-BSA at 0~400 μg/ml for 12 h, then TER ( n = 3) and Pa ( n = 4) were measured. (E,F) CCK-8 assay was used to evaluate the proliferation function of HUVECs with AOPP-BSA treatment for different time ( n = 4) or at different concentrations ( n = 5). (G,I) The migration function of HUVECs treated with AOPP-BSA was detected by trans-well migration assay. Scale bar = 50 μm. (H,J) The number of trans-well migration cells was counted ( n = 3–5). (K,N) Matrigel tube formation assay was used for tube formation evaluation in HUVECs incubated with AOPP-BSA. Scale bar = 30 μm. (L,M) Tube length and branching points of HUVECs treated with AOPP-BSA for different time were quantitated ( n = 5). (O,P) Tube length and branching points of HUVECs treated with AOPP-BSA at different concentrations were quantitated ( n = 3). Two-way ANOVA was used for statistical analysis. All data are shown as mean ± SEM. ns, non-significant ( P > 0.05).

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Permeability, CCK-8 Assay, Migration, Tube Formation Assay, Incubation

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: AOPP-BSA attenuates endothelial autophagy. (A) HUVECs were treated with AOPP-BSA at 100, 200, and 400 μg/ml for 24 h. Before AOPP-BSA treatment, HUVECs were incubated with Hanks balanced salt solution (HBSS) for 1 h to activate autophagy by starvation. The expression of autophagy-associated proteins LC3II, p62 and Beclin1 were detected by western blot. β-actin was used as loading control. (B-D) Quantification of LC3II ( n = 6), p62 ( n = 5) and Beclin1 ( n = 5) protein expression. (F) HUVECs were stimulated with 200 μg/ml AOPP-BSA for 0, 0.5, 1, 3, 6, 12 and 24 h. As mentioned above, HUVECs were starved with HBSS for 1 h before AOPP-BSA treatment. The expression of LC3II, p62 and Beclin1 were detected by western blot. β-actin and GAPDH were used as loading control. (G–I) Density analysis of LC3II ( n =6 ), Beclin1 ( n = 5) and p62 ( n = 5) protein levels. (E,J) The alteration of LC3 (red) level was also observed using immunostaining by a laser confocal microscope in HUVECs and nuclear was indicated with DAPI (blue). Scale bar = 10 μm. One-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons was used for statistical analysis. All data are shown as mean ± SEM.

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Incubation, Expressing, Western Blot, Control, Immunostaining, Microscopy

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: Down-regulation of RAGE alleviates AOPP-BSA-induced autophagy inhibition. (A) HUVECs were pretreated with RAGE siRNA and control siRNA for 48 h before AOPP-BSA (200 μg/ml, 12 h) application. The expression of RAGE was detected by western blot and was quantitated ( n = 4). (B) HUVECs were treated as mentioned above, and immunostained with LC3 (red) and DAPI (nuclear, blue). Scale bar = 10 μm. (C) HUVECs were treated as mentioned above, then the expression of LC3II and p62 were detected by western blot. GAPDH was used as loading control. (D,E) Quantification of LC3II and p62 protein expression ( n = 3). (F) HUVECs were incubated with RAGE neutralizing antibody (RAGE Ab, 10 μg/ml) for 1 h before AOPP-BSA application, then protein levels of LC3II and p62 were detected by western blot. GAPDH was used as loading control. (G,H) Quantification of LC3II ( n = 4) and p62 ( n = 3) protein expression. One-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons was used for statistical analysis. All data are shown as mean ± SEM. RAGE Ab, RAGE neutralizing antibody.

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Inhibition, Control, Expressing, Western Blot, Incubation

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: Autophagy inhibition leads to AOPP-BSA-induced endothelial senescence. (A) Effects of autophagy activation with Rapa and inhibition with 3-MA on AOPP-BSA-induced expression of senescence-associated protein p21 and p16 were measured by western blot. HUVECs were pretreated with Rapa (200 nM) or 3-MA (5 mM) for 2 h before AOPP-BSA stimulation. GAPDH was used as loading control. (B,C) Quantification of p21 and p16 protein expression ( n = 4). (D) HUVECs were pretreated as mentioned above. Effects of Rapa and 3-MA on HUVEC senescence were also detected by SA-β-gal assay. Scale bar for upper panel = 20 μm, scale bar for lower panel = 50 μm. (E) The SA-β-gal positive rate was quantitated ( n = 4). All data analyzed by one-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons are shown as mean ± SEM. Rapa, rapamycin; 3-MA, 3-methyladenine.

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: Autophagy inhibition enhances p53 SUMOylation induced by AOPP-BSA. (A,C) HUVECs were incubated with 200 μg/ml AOPP-BSA for 12 h. Effects of AOPP-BSA on p53 and p53 SUMOylation were detected by western blot and immunoprecipitation. (B) Quantification of p53 protein expression ( n = 4). (D) The ratio of SUMOylated p53 and total p53 was calculated ( n = 4). (E,G) HUVECs were pretreated with Rapa (200 nM) or 3-MA (5 mM) for 2 h before AOPP-BSA application, and the levels of p53 and p53 SUMOylation were detected by western blot and immunoprecipitation. (F) Quantification of p53 protein expression ( n = 4). (H) The ratio of SUMOylated p53 and total p53 was calculated ( n = 3). Two-tailed unpaired Student's t -test for (B) . One-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons for (D,F,H) . Data are shown as mean ± SEM. ns, non-significant ( P > 0.05). SUMO1, small ubiquitin related modifier 1; SUMO1-p53, SUMOylated p53; IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Inhibition, Incubation, Western Blot, Immunoprecipitation, Expressing, Two Tailed Test, Ubiquitin Proteomics

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: p53 SUMOylation at K386 mediates AOPP-BSA-induced endothelial senescence. (A) HUVECs were infected with adenovirus of K386R, WT-TP53, or Ad-GFP for 48 h. The effects of K386R, WT-TP53, and Ad-GFP on the levels of p53 and flag were detected by western blot. GAPDH was used as loading control. (B,C) Quantification of p53 and flag protein expression ( n = 4). (D,E) At 48 h post-infection, HUVECs were incubated with or without 200 μg/ml AOPP-BSA for 12 h. The protein levels of p53 and p53 SUMOylation were detected by western blot and immunoprecipitation. (F) Quantification of p53 protein expression ( n = 4). (G) The SUMO1-p53/p53 ratio was calculated ( n = 3). (H) HUVECs were infected with K386R, WT-TP53, and Ad-GFP with or without AOPP-BSA stimulation. The expression of senescence-associated proteins p21 and p16 were detected by western blot. (I,J) Quantification of p21 and p16 protein expression ( n = 4). (K) HUVECs were treated as mentioned above, and the SA-β-gal assay was used to identify the senescent cells. Scale bar for upper panel = 20 μm, scale bar for lower panel = 50 μm. (L) The SA-β-gal positive rate was quantitated ( n = 4). (M) HUVECs were incubated with or without 200 μg/ml AOPP-BSA for 12 h, then the expression of p53 and acetylation of p53 were detected by western blot. (N) The ratio of acetylated p53 and total p53 was calculated ( n =6 ). Two-tailed unpaired Student's t -test for (N) . One-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons for (B,C,E,G,I,J,L) . Data are shown as mean ± SEM. ns, non-significant ( P > 0.05). Ad-GFP, control adenovirus containing GFP; WT-TP53, wide-type TP53 adenovirus; K386R, TP53 K386-deficient adenovirus; Ac-p53 (K386), acetylated p53 at K386.

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Infection, Western Blot, Control, Expressing, Incubation, Immunoprecipitation, Two Tailed Test

Journal: Frontiers in Cardiovascular Medicine

Article Title: p53 SUMOylation Mediates AOPP-Induced Endothelial Senescence and Apoptosis Evasion

doi: 10.3389/fcvm.2021.795747

Figure Lengend Snippet: AOPP-BSA promotes vascular senescence through vascular autophagy inhibition. ApoE −/− mice fed with high-fat diet were used to establish the model of vascular_senescence. Saline, BSA (50 mg/kg), AOPP-BSA (50 mg/kg), or AOPP-BSA (50 mg/kg) + Rapa (1 mg/kg) was injected intraperitoneally in 200 μl for consecutive 100 days or 200 days. (A) The expression of p16, p21, p62, and LC3II in aortic tissue were detected by western blot. GAPDH was used as loading control. (B–E) Quantification of p16 ( n = 6), p21 ( n = 3), p62 ( n = 3), and LC3II ( n = 3) protein expression. (F,G) Immunoprecipitation was used for p53 purification, and the levels of p53 and p53 SUMOylation in aortic tissue were detected by western blot. (H,J,L,N) Aortas isolated from mice in different groups were fixed and immunostained with RAGE, LC3, SUMO1 and p16. Images were photographed with Zeiss Imager Z2 microscope (Zeiss, Germany) and analyzed by Image J software. Representative images were showed and red arrows indicated the aortic endothelium. Scale bar for upper panel = 20 μm, scale bar for lower panel = 50 μm. (I,K,M,O) Quantification of RAGE, LC3, SUMO1 and p16 positive area in aortic tissue ( n = 4 in Control group, n = 5 in BSA group, n = 4 in AOPP-BSA group, n = 4 in AOPP-BSA+Rapa group). (P) The aortic plaque area of high-fat-fed ApoE −/− mice was revealed by Oil Red O staining and analyzed by Image-Pro Plus Software. Representative images were showed. (Q) Quantification of plaque area in aortic tissue ( n = 3 in Control group, n = 3 in BSA group, n = 3 in AOPP-BSA group, n = 4 in AOPP-BSA+Rapa group). One-way ANOVA with LSD or Dunnett's T3 post-hoc multiple comparisons was used for statistical analysis. All data are shown as mean ± SEM.

Article Snippet: The ApoE −/− mice aged 4–6 weeks were randomly divided into four groups, and injected intraperitoneally with saline (0.9% NaCl 200 μl), BSA (50 mg/kg/d), AOPP-BSA (50 mg/kg/d), or AOPP-BSA (50 mg/kg/d) + Rapamycin (MedChemExpress, 1 mg/kg/d) for 100 days and 200 days respectively.

Techniques: Inhibition, Saline, Injection, Expressing, Western Blot, Control, Immunoprecipitation, Purification, Isolation, Microscopy, Software, Staining