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.


| INTRODUC TI ON
Macrovascular complications develop in over a half of the diabetic individuals, leading to high mortality. 1,2 Endothelial dysfunction is the critical first step of diabetic macrovascular complications. 3,4 It is therefore essential to improve diabetic endothelial dysfunction, which could prevent or slowdown the development of diabetic macrovascular complications.
Recent findings shed light on a critical role of P53 in endothelial dysfunction. 5,6 P53 is highly expressed in the endothelium/endothelial cells (ECs) under hyperglycaemia/high glucose (HG) conditions. 5,6 Acetylation of P53 is critical for its stabilization and function. 8 We and others have observed hyperacetylation of P53 in the HG-treated ECs and in the aortas of diabetic mice. 5,9 This effect enhanced endothelial oxidative stress and inflammation, resulting in endothelial senescence and dysfunction. 5,9 Notably, deacetylation of P53 by sirtuin 1 (SIRT1) mitigated the HG-induced endothelial oxidative stress and inflammation, 5 and improved endothelial dysfunction. 5,9 On the contrary, forced activation of P53 by nutlin3a increased aortic contractility in healthy mice and generated endothelial oxidative stress and inflammation in both the normal glucose (NG)-cultured ECs and the aortas of the healthy mice, demonstrating that P53 plays a crucial pathogenic role in endothelial dysfunction. 5 Collectively, these findings provide a rationale for investigating the effect of P53 inhibition on diabetic endothelial dysfunction.
Despite the identification of P53's pathogenic effect on endothelial dysfunction, little is known for the mechanism by which P53 induces endothelial dysfunction. P53 closely correlates with SIRT1 which functions to protect against diabetic endothelial dysfunction. 5,9 SIRT1 deacetylates P53, inactivating the transcription of P53-dependent genes, 10,11 including miR-34a. 13,14 In the cytoplasm, miR-34a directly targets Sirt1 mRNA without degrading Sirt1 mRNA, inhibiting SIRT1 protein production. 13,14 Previously, we found that the protein levels of P53 and acetylated P53 (ac-P53) were significantly elevated by HG in ECs, accompanied by a drastic decrease in SIRT1 protein level. 5 The diabetic mice also had higher ac-P53 level and lower SIRT1 protein level in the aortas compared with the non-diabetic control mice. 5 Therefore, we hypothesized that miR-34a may mediate P53's pathogenic effect on diabetic endothelial dysfunction by decreasing SIRT1 protein level.
Although Sirt1 mRNA is a direct target of miR-34a, 13,14 it is still needed to investigate whether or not SIRT1 is a major target of miR-34a in diabetic endothelial dysfunction, given that miR-34a may target multiple mRNAs. 16 To this end, inhibition of SIRT1 was achieved by utilizing siRNA-induced gene silencing and the SIRT1 inhibitor EX-527, in the presence of the specific inhibitor of miR-34a (miR-34a-I).
In summary, the present study aims to explore: (a) whether or not inhibition of P53/miR-34a attenuates diabetic endothelial dysfunction; (b) whether or not miR-34a mediates P53's pathogenic effect; and (c) whether or not SIRT1 is a major target of miR-34a in diabetic endothelial dysfunction. To study the effect of P53/miR-34a inhibition on aortic endothelial dysfunction under DM, pifithrin-α (PFT-α, 1.1 mg/kg, intraperitoneally injected three times weekly 21,22 ; MedChem Express, Shanghai, China) or miR-34a-I (2 mg/kg, subcutaneously injected once weekly 23 ; Thermo Fisher, Shanghai, China) was delivered to the diabetic mice immediately after DM was confirmed, for 24 weeks. In order to investigate the role of SIRT1 in mediating miR-34a-I's action, the diabetic mice were treated with EX-527 (2 mg/kg, 24

| Analysis of aortic morphology
The freshly harvested thoracic aortas were immediately fixed into 10% buffered formalin solution and were embedded in paraffin, followed by sectioning into 5-µm-thick sections onto glass slides. Haematoxylin and eosin (H&E) staining was performed to evaluate morphological change. The thickness of tunica media was measured. Selection of areas to photograph and scoring were done by people blind to the identity of the samples.

| Immunohistochemical staining
Immunohistochemical staining was performed as previously described, 26

| Cell culture and experiments
Endothelial cells were isolated from the aortas of 8-week-old C57BL/6 male mice, as previously described. 4,5,27 To investigate the impact of HG on P53/miR-34a/SIRT1 expression, NG (1 g/L)-cultured ECs were subjected to mannitol or HG (4.5 g/L), for 48 hours.
In order to study the effect of P53 inhibition on the expression of P53/miR-34a/SIRT, inflammatory genes and oxidative stress,

| Analysis of reactive oxygen species and lipid peroxides
Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were measured in cell lysates, using assay kits from Nanjing F I G U R E 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 Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China), following the manufacturer's instructions.

| Analysis of SIRT1 activity
Sirtuin 1 activity was analysed in cell lysates using a fluorometric assay kit from BioVision (Milpitas, CA), following the manufacturer's instructions.

| Statistical analysis
Cell experiments were performed in triplicate. Eight mice per group were studied. Western blot images were analysed by Image Studio Lite (LI-COR Biosciences, Lincoln, NE). Immunohistochemical positive area was quantified using Image Pro Plus 6.0 software (Media Cybernetics, Rockville, MD). One-way ANOVA was performed for the comparisons among the groups. The measurements for each group were summarized as means ± SD. A test is significant if P < 0.05.

| HG increased P53 and miR-34a levels and inhibited Sirt1 expression in ECs
To determine whether or not P53/miR-34a/SIRT1 pathway is altered under HG condition, ECs were treated with NG, NG + mannitol or HG for 48 hours. The protein levels of P53 and ac-P53 were significantly increased under the HG condition ( Figure 1A,B). This  Figure 1 led to an enhanced expression of the miR-34a gene, as shown by the increased levels of pre-miR-34a and miR-34a ( Figure 1C,D).
HG inhibited Sirt1 expression at both mRNA and protein levels ( Figure 1E,F) in ECs.

| Inhibition of P53 attenuated the HG-induced inflammation and oxidative stress in ECs
The following study investigated the effect of P53 inhibition on HG-induced inflammation and oxidative stress in ECs. Inhibition of P53 was achieved by using either P53-siRNA or PFT-α. P53-siRNA, but not PFT-α, decreased P53 mRNA level that was elevated by HG ( Figure 2A); however, the protein levels of P53 and ac-P53 were both significantly decreased by P53-siRNA and PFT-α ( Figure 2B,C). Both P53-siRNA and PFT-α inhibited miR-34a expression ( Figure 2D,E) and rescued SIRT1 protein level that was decreased by HG ( Figure 2F). HG treatment induced the mRNA levels of Vcam-1, Mcp-1, Icam-1 and Sele ( Figure 2G), as well as the levels of ROS and MDA ( Figure 2H). All these effects were remarkably reduced by P53-siRNA or PFT-α ( Figure 2G,H). These results demonstrate that inhibition of P53 alters miR-34a and SIRT1 expression, mitigating HG-induced inflammation and oxidative stress in ECs.

| P53's effect on endothelial inflammation and oxidative stress was completely mediated by miR-34a under the HG condition
To investigate whether to what extent miR-34a mediates P53's effect on endothelial inflammation and oxidative stress under HG, the HG-treated ECs were co-treated with PFT-α, in the presence or absence of miR-34a-M. In addition, miR-34a-I was tested for its effect on HG-induced endothelial inflammation and oxidative stress.

| SIRT1 is a major target of miR-34a in HGinduced endothelial inflammation and oxidative stress
In order to investigate whether or not SIRT1 is required for miR-34a's role in mediating HG-induced endothelial inflammation and oxidative stress, the ECs were treated with HG and miR-34a-I, in  Figures 1 and 2 the presence of either Sirt1-siRNA or EX-527. Sirt1-siRNA, but not EX-527, significantly reduced Sirt1 mRNA and protein levels ( Figure 4A,B). However, the miR-34a-I-enhanced SIRT1 activity was remarkably inhibited by both Sirt1-siRNA and EX-527 ( Figure 4C), abolishing the inhibition of inflammation and oxidative stress produced by miR-34a-I ( Figure 4D,E). These results indicate that SIRT1 is a major target of miR-34a in HG-induced endothelial inflammation and oxidative stress.

| Inhibition of P53 or miR-34a attenuated the hyperglycaemia-induced aortic inflammation and oxidative stress
The following study explored the effect of P53 or miR-34a inhibition on hyperglycaemia-induced aortic inflammation and oxidative stress in mice. EX-527 was administered to the miR-34a-I-treated diabetic mice, with the aim of verifying whether SIRT1 mediates miR-34a-I's effect in vivo. Blood glucose level was not affected by PFT-α, miR-34a-I and EX-527 in the diabetic mice ( Figure 5A).
These effects of miR-34a-I were completely reversed by EX-527.
These findings demonstrate the beneficial effects of P53 or miR-34a inhibition on hyperglycaemia-induced aortic inflammation and oxidative stress. In addition, SIRT1 was found to predominantly mediate miR-34a-I's effects in vivo.

| D ISCUSS I ON
The present study has found a P53/miR-34a/SIRT1 pathway that contributes to diabetic endothelial dysfunction ( Figure 6D). This  38 Here we report the pathogenic effect of P53 on diabetic aortic endothelial dysfunction, with miR-34a to be the mediator. Like P53, miR-34a was reported to promote multiple complications of DM, including nephropathy, endothelial dysfunction and neuropathy. 39,40 As P53/ miR-34a activation leads to DM and its complications, inhibition of P53/miR-34a may produce beneficial effects on multiple organs/tissues in diabetic individuals. This 'one stone for multiple birds' strategy has a unique advantage in future clinical management of DM and its complications.
The present study has found an unhindered effect passing from P53 to the endothelium through miR-34a and SIRT1, by supplementing miR-34a in the presence of PFT-α ( Figure 3) and inhibiting SIRT1 in the presence of miR-34-I ( Figures. 4-6). We previously studied the effect of SIRT1 activation by SRT2104 -a novel SIRT1 activatoron diabetic endothelial dysfunction. 5 Activation of SIRT1 lowered endothelial ac-P53 level that was increased under the HG/diabetic conditions. 5 Therefore, P53, miR-34a and SIRT1 may form a positive feedback loop in diabetic endothelial dysfunction ( Figure 6D).
Under HG/diabetic conditions, P53 and miR-34a are predominant factors within this circuit, whereas SIRT1 is suppressed by miR-34a.
This inactivation of SIRT1 may lead to uncontrolled activation of P53 and overexpression of miR-34a, accelerating the progression of diabetic endothelial dysfunction ( Figure 6D). P53, miR-34a and SIRT1 are key factors in diabetic endothelial dysfunction, as modulation of each of these factors produced a remarkable effect (Figures 2G,H; 3C,D; 4D,E; 5C-F; 6B,C; 5 ). This suggests P53, miR-34a and SIRT1 as important candidates for clinical intervention of diabetic endothelial dysfunction.
Previous studies have demonstrated that miR-34a negatively regulates SIRT1 expression. 14,15,43 Mechanistically, miR-34a targets Sirt1 mRNA without degrading Sirt1 mRNA, inhibiting SIRT1 protein production. 15,43 In the present study, in order to investigate how miR-34a regulates Sirt1 expression, ECs were treated with the miR-34a inhibitor (miR-34a-I) under HG condition. We observed that SIRT1 protein ( Figure 4B), but not Sirt1 mRNA ( Figure 4A), was significantly decreased by miR-34a-I ( Figure 4A,B, red columns). This result is in line with the previous findings, 43 confirming that miR-34a inhibits Sirt1 gene expression at the translational level.
In the present study, HG induced a mild decrease in Sirt1 mRNA in ECs ( Figure 1E). However, HG resulted in a steep decrease in SIRT protein in ECs ( Figure 1F). This differential regulation of the Sirt1 gene expression under the HG condition indicates the involvement of translational regulation, where miR-34a exerts its action ( Figures   1D-F; 3A,B; 5B,D; 13,14 ). Nonetheless, the decrease in Sirt1 mRNA level under the HG condition, although mild ( Figure 1E), suggests that HG induces a transcriptional inhibition of the Sirt1 gene. We observed lower SIRT1 protein level in the HG + miR-34a-I + Sirt1-siRNA group ( Figure 4B) and lower SIRT1 activities in the HG + miR-34a-I + Sirt1-siRNA and HG + miR-34a-I + EX527 groups ( Figure 4C), as compared with the HG group. However, these did not significantly increase inflammation and oxidative stress ( Figure 4D,E).
One explanation could be that the HG condition already resulted in a very low basal level of SIRT1 protein ( Figures 1F,2F). In other words, very few SIRT1 proteins were left under the HG condition. Hence, the decrease in SIRT1 protein level or activity, although statistically significant, might not be able to result in a remarkable exacerbation of inflammation and oxidative stress.
In the present study, VCAM-1 and 4-HNE were highly expressed not only in the intima, but also in the adventitia, under the diabetic condition ( Figure 5E,F). The adventitia is known to play a role in vascular inflammation and oxidative stress. 44,45 The overexpression of VCAM-1 in the adventitia could be caused by adventitial ECs which were generated during neovascularization. 46 In addition, adventitial fibroblasts have been shown to produce substantial amounts of ROS in response to vascular injury. 44 This observation should encourage future study of the role of adventitia in diabetic endothelial dysfunction and macrovascular complications.
MiR-34a targets multiple mRNAs. 16 MiR-34a was reported to target Notch mRNA and exacerbate diabetic endothelial dysfunction 41 in rats. In the present study, SIRT1 was found to mediate miR-34a's pathogenic effect on diabetic endothelial dysfunction, because inhibition of SIRT1 abrogated miR-34a-I's indicating a potential SIRT1/NOTCH pathway in protecting against diabetic endothelial dysfunction. However, more evidence has shown the negative regulatory effect of SIRT1 on NOTCH through deacetylation in ECs. 48,49 Moreover, numerous studies have demonstrated the pathogenic role of NOTCH in diabetic endothelial dysfunction. 50,51 Therefore, it is needed to investigate whether or not NOTCH mediates miR-34a's action on diabetic endothelial dysfunction by gene silencing/overexpression in future studies.
Collectively, the present study has found an unhindered P53/ miR-34a/SIRT1 pathway that plays a critical role in diabetic endothelial dysfunction, providing novel strategies for future management of diabetic macrovascular diseases.

ACK N OWLED G EM ENTS
This work was supported in part by the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflict of interests.