Inhibition of P53/miR-34a improves diabetic endothelial dysfunction via activation of SIRT1

Abstract

Endothelial dysfunction contributes to diabetic macrovascular complications, resulting in high mortality. Recent findings demonstrate a pathogenic role of P53 in endothelial dysfunction, encouraging the investigation of the effect of P53 inhibition on diabetic endothelial dysfunction. Thus, high glucose (HG)-treated endothelial cells (ECs) were subjected to pifithrin-α (PFT-α)-a specific inhibitor of P53, or P53-small interfering RNA (siRNA), both of which attenuated the HG-induced endothelial inflammation and oxidative stress. Moreover, inhibition of P53 by PFT-α or P53-siRNA prohibited P53 acetylation, decreased microRNA-34a (miR-34a) level, leading to a dramatic increase in sirtuin 1 (SIRT1) protein level. Interestingly, the miR-34a inhibitor (miR-34a-I) and PFT-α increased SIRT1 protein level and alleviated the HG-induced endothelial inflammation and oxidative stress to a similar extent; however, these effects of PFT-α were completely abrogated by the miR-34a mimic. In addition, SIRT1 inhibition by EX-527 or Sirt1-siRNA completely abolished miR-34a-I's protection against HG-induced endothelial inflammation and oxidative stress. Furthermore, in the aortas of streptozotocin-induced diabetic mice, both PFT-α and miR-34a-I rescued the inflammation, oxidative stress and endothelial dysfunction caused by hyperglycaemia. Hence, the present study has uncovered a P53/miR-34a/SIRT1 pathway that leads to endothelial dysfunction, suggesting that P53/miR-34a inhibition could be a viable strategy in the management of diabetic macrovascular diseases.

Keywords: P53; aorta; diabetes; endothelial dysfunction; miR-34a.

Figure 1

HG increased P53 and miR‐34a levels and inhibited Sirt1 expression in ECs. NG‐cultured ECs were treated with mannitol or HG. Protein levels of (A) P53 and (B) ac‐P53 were determined by Western blot. C, Pre‐miR‐34a and (D) miR‐34a levels were measured by qPCR Sirt1(E) mRNA and (F) protein levels were determined by qPCR and Western blot respectively. GAPDH was used as an endogenous control for P53, ac‐P53 and Sirt1 expression. U6 was used as an endogenous control for pre‐miR‐34a and miR‐34a. The data were normalized to NG and are presented as means ± SD (n = 3). *P < 0.05 vs NG. Bars: orange, NG; blue, NG + M; red, HG. Abbreviations: ac‐P53, acetylated P53; EC, endothelial cell; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HG, high glucose; M, mannitol; SIRT1, sirtuin 1

Figure 2

Inhibition of P53 attenuated the HG‐induced inflammation and oxidative stress in ECs. NG‐cultured ECs were treated with HG, in the presence of PFT‐α, P53‐siRNA or its negative control siRNA. The levels of (A) P53 mRNA, (B) P53 protein, (C) ac‐P53, (D) pre‐miR‐34a, (E) miR‐34a and (F) SIRT1 protein were determined in all the groups. G, Endothelial inflammation was assessed by determining mRNA levels of Vcam‐1, Mcp‐1, Icam‐1and Sele, as summarized in the heat map. H, Heat map for ROS and MDA. GAPDH was used as an endogenous control for P53, ac‐P53, SIRT1, Vcam‐1, Mcp‐1, Icam‐1 and Sele expression. U6 was used as an endogenous control for pre‐miR‐34a and miR‐34a. The data were normalized to NG and are presented as means ± SD (n = 3). *P < 0.05 vs NG. ^† P < 0.05 vs HG. Bars: orange, NG; blue, NG + M; red, HG; green, HG + NC‐siRNA; pink, HG + P53‐siRNA; purple, HG + PFT‐α. Abbreviations: Icam‐1, intercellular adhesion molecule‐1; Mcp‐1, monocyte chemoattractant protein 1; MDA, malondialdehyde; NC, negative control; PFT‐α, pifithrin‐α; ROS, reactive oxygen species; Sele, selectin E; siRNA, small interfering RNA; Vcam‐1, vascular cell adhesion molecule‐1. Other abbreviations are the same as in Figure 1

Figure 3

P53's effect on endothelial inflammation and oxidative stress was completely mediated by miR‐34a under the HG condition. HG‐stimulated ECs were treated with NC‐I, miR‐34a‐I, PFT‐α, or PFT‐α in the presence or either NC‐M or miR‐34a‐M. (A) MiR‐34a and (B) SIRT1 protein levels were determined. C, Heatmap for mRNA expression of inflammatory genes Vcam‐1, Mcp‐1, Icam‐1and Sele. D, Heatmap for oxidative stress indicators ROS and MDA. GAPDH was used as an endogenous control for SIRT1, Vcam‐1, Mcp‐1, Icam‐1 and Sele expression. U6 was used as an endogenous control for miR‐34a. The data were normalized to HG and are presented as means ± SD (n = 3). *P < 0.05 vs HG. ^† P < 0.05 vs HG + PFT‐α + NC‐M. Bars: orange, HG; blue, HG + PFT‐α; red, HG + NC‐I; green, HG + miR‐34a‐I; pink, HG + PFT‐α + NC‐M; purple, HG + PFT‐α + miR‐34a‐M. Abbreviations: miR‐34a‐I, miR‐34a inhibitor; miR‐34a‐M, miR‐34a mimic; NC‐I, negative control for miR‐34a‐I; NC‐M, negative control for miR‐34a‐M. Other abbreviations are the same as in Figures 1 and 2

Figure 4

SIRT1 is a major target of miR‐34a in HG‐induced endothelial inflammation and oxidative stress. The effect of SIRT1 inhibition was investigated in the presence of miR‐34a‐I in HG‐treated ECs. A, Sirt1 mRNA, (B) protein and (C) activity were determined. D, Heatmap for mRNA expression of inflammatory genes Vcam‐1, Mcp‐1, Icam‐1and Sele. E, Heatmap for oxidative stress indicators ROS and MDA. GAPDH was used as an endogenous control for Sirt1, Vcam‐1, Mcp‐1, Icam‐1 and Sele expression. The data were normalized to HG and are presented as means ± SD (n = 3). *P < 0.05 vs HG; ^† P < 0.05 vs HG + miR‐34a‐I + NC‐siRNA; ^‡ P < 0.05 vs HG + miR‐34a‐I + DMSO. Bars: orange, HG; blue, HG + NC‐I; red, HG + miR‐34a‐I; green, HG + miR‐34a‐I + NC‐siRNA; pink, HG + miR‐34a‐I + Sirt1‐siRNA; purple, HG + miR‐34a‐I + DMSO; grey, HG + miR‐34a‐I + EX‐527. Abbreviations are the same as in Figures 1, 2, 3

Figure 5

Inhibition of P53/miR‐34a attenuated the hyperglycaemia‐induced aortic inflammation and oxidative stress. C57BL/6 male mice were induced to DM by streptozotocin. The effects of P53/miR‐34a inhibition on the diabetic mice were investigated by treatments with PFT‐α and miR‐34a‐I. The miR‐34a‐I‐treated diabetic mice were co‐treated with EX‐527 to study the role of SIRT1 in mediating miR‐34a's effect. A, Blood glucose levels were recorded 0, 4, 8, 12, 16, 20 and 24 wk post DM onset. B, The level of miR‐34a was determined in the aortas of the mice. U6 was used as an endogenous control for miR‐34a. IHC staining (bar = 100 µmol/L) was performed to detect the protein expression of (C) ac‐P53, (D) SIRT1, (E) VCAM‐1 and (F) 4‐HNE. For (B‐F), the data were normalized to Ctrl and are presented as means ± SD (n = 8). *P < 0.05 vs Ctrl; ^† P < 0.05 vs DM; ^‡ P < 0.05 vs DM + miR‐34a‐I + DMSO. Plots and bars: orange, Ctrl; blue, DM; red, DM + PFT‐α; green, DM + NC‐I; pink, DM + miR‐34a‐I; purple, DM + miR‐34a‐I + DMSO; grey, DM + miR‐34a‐I + EX‐527. Abbreviations: 4‐HNE, 4‐hydroxynonenal; Ctrl, control; DM, diabetes mellitus. Other abbreviations are the same as in Figures 1, 2, 3

Figure 6

Inhibition of P53 or miR‐34a improved the hyperglycaemia‐induced aortic endothelial dysfunction via activation of SIRT1. A, H&E staining was performed to evaluate aortic morphology, with tunica media thickness measured (bar = 100 µmol/L). B, Aortic contraction in response to PE and (C) relaxation in response to ACh were determined to assess endothelial dysfunction. The doses of PE and ACh were 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, 10⁻⁵ and 10⁻⁴ mol/L. For tunica media thickness, the data were normalized to Ctrl and are presented as means ± SD (n = 8). *P < 0.05 vs Ctrl; ^† P < 0.05 vs DM; ^‡ P < 0.05 vs DM + miR‐34a‐I + DMSO. Plots: orange, Ctrl; blue, DM; red, DM + PFT‐α; green, DM + NC‐I; pink, DM + miR‐34a‐I; purple, DM + miR‐34a‐I + DMSO; grey, DM + miR‐34a‐I + EX‐527. D, Schematic diagram: P53 is hyperacetylated under diabetic condition. This elevates oxidative stress and inflammation, leading to endothelial dysfunction which is the critical first step for vascular complications (red pathway). Acetylation is essential for P53's stabilization and function. In the nucleus, ac‐P53 activates the transcription of the miR‐34a gene, the effect of which can be prohibited by PFT‐α (purple pathway). Ac‐P53‐directed miR‐34a production inhibits SIRT1 protein translation through binding the 3′ untranslated region of the Sirt1 mRNA (green pathway). This effect can be modulated by miR‐34a‐I, miR‐34a‐M and Sirt1‐siRNA. SIRT1 deacetylates P53, facilitating degradation of P53 (blue pathway). This effect can be reversed by EX527. Therefore, a P53/miR‐34a/SIRT1 circuit may contribute to diabetic endothelial dysfunction. Abbreviations: ACh, acetylcholine; H&E, haematoxylin and eosin; PE, phenylephrine. Other abbreviations are the same as in Figures 1, 2, 3 and 5