Inhibitory Effects of Simvastatin on Oxidized Low-Density Lipoprotein-Induced Endoplasmic Reticulum Stress and Apoptosis in Vascular Endothelial Cells

Background: Oxidized low-density lipoprotein (ox-LDL)-induced oxidative stress and endothelial apoptosis are essential for atherosclerosis. Our previous study has shown that ox-LDL-induced apoptosis is mediated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic translation initiation factor 2α-subunit (eIF2α)/CCAAT/enhancer-binding protein homologous protein (CHOP) endoplasmic reticulum (ER) stress pathway in endothelial cells. Statins are cholesterol-lowering drugs that exert pleiotropic effects including suppression of oxidative stress. This study aimed to explore the roles of simvastatin on ox-LDL-induced ER stress and apoptosis in endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with simvastatin (0.1, 0.5, or 2.5 μmol/L) or DEVD-CHO (selective inhibitor of caspase-3, 100 μmol/L) for 1 h before the addition of ox-LDL (100 μg/ml) and then incubated for 24 h, and untreated cells were used as a control group. Apoptosis, expression of PERK, phosphorylation of eIF2α, CHOP mRNA level, and caspase-3 activity were measured. Comparisons among multiple groups were performed with one-way analysis of variance (ANOVA) followed by post hoc pairwise comparisons using Tukey's tests. A value of P < 0.05 was considered statistically significant. Results: Exposure of HUVECs to ox-LDL resulted in a significant increase in apoptosis (31.9% vs. 4.9%, P < 0.05). Simvastatin (0.1, 0.5, and 2.5 μmol/L) led to a suppression of ox-LDL-induced apoptosis (28.0%, 24.7%, and 13.8%, F = 15.039, all P < 0.05, compared with control group). Ox-LDL significantly increased the expression of PERK (499.5%, P < 0.05) and phosphorylation of eIF2α (451.6%, P < 0.05), if both of which in the control groups were considered as 100%. Simvastatin treatment (0.1, 0.5, and 2.5 μmol/L) blunted ox-LDL-induced expression of PERK (407.8%, 339.1%, and 187.5%, F = 10.121, all P < 0.05, compared with control group) and phosphorylation of eIF2α (407.8%, 339.1%, 187.5%, F = 11.430, all P < 0.05, compared with control group). In contrast, DEVD-CHO treatment had no significant effect on ox-LDL-induced expression of PERK (486.4%) and phosphorylation of eIF2α (418.8%). Exposure of HUVECs to ox-LDL also markedly induced caspase-3 activity together with increased CHOP mRNA level; these effects were inhibited by simvastatin treatment. Conclusions: This study suggested that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.

destroy the vascular wall integrity. [1] Apoptotic endothelial cells are prothrombotic and pro-proliferative and lead to the ensuing atherogenic processes. [3] Elevation of low-density lipoprotein (LDL) cholesterol levels is a major risk factor for the pathogenesis of atherosclerosis. [4] The production of reactive oxygen species in vascular endothelial cells causes oxidation of LDL and results in increased levels of oxidized LDL (ox-LDL), a key mediator of the initiation and progression of atherosclerosis. [5][6][7][8][9] Ox-LDL induces oxidative stress and cell injury in endothelial cells, which in turn facilitates the progression of atherosclerosis. [1] The endoplasmic reticulum (ER) is the primary intracellular site for folding and assembly of membrane-associated and secreted proteins. Physiological and pathological perturbations may interfere with protein folding processes in the ER and lead to accumulation of unfolded or misfolded proteins, a cellular condition termed ER stress. [8,9] ER stress triggers the unfolded protein response (UPR), a transcriptional induction pathway aimed at restoring normal ER functioning. [10] If UPR is insufficient to recover ER homeostasis, cells undergo apoptosis. [11][12][13] The UPR is mediated by three ER stress receptors: protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating transcription factor-6 (ATF6) that representing three branches of the UPR. [14] PERK is an ER transmembrane protein kinase that inhibits protein translation through inactivation of eukaryotic translation initiation factor 2α-subunit (eIF2α), which results in upregulation of CCAAT/enhancer-binding protein homologous protein (CHOP), a critical factor for triggering apoptosis in response to ER stress. [8,15,16] Our previous study has shown that ox-LDL induces apoptosis in vascular endothelial cells largely through the PERK/eIF2α/CHOP ER stress pathway. [8] Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-determining enzyme in the multistep mevalonate cascade for cholesterol synthesis. [17] Statins are widely used in the prevention of cardiovascular disease by lowering cholesterol. [17] Beyond their cholesterol reduction effect, statins have been shown to have pleiotropic effects. [15][16][17] Accumulating evidences indicated that the inhibition of the mevalonate pathway by statins induced ER stress and apoptosis in cancerous and noncancerous cells. [17][18][19][20] In this study, we explored the beneficial effect of simvastatin on ox-LDL-induced ER stress and apoptosis in human vascular endothelial cells.

Ethical approval
The study was reviewed and approved by the Medical Research Ethics Committee of the China-Japan Friendship Hospital.

Statistical analysis
All continuous variable values were expressed as mean ± standard deviation (SD). Statistical analyses were performed with SPSS for Windows version 19.0 (IBM Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) was administered to compare the means of multiple groups. A two-tailed P < 0.05 was considered statistically significant.

Simvastatin reduced oxidized low-density lipoproteininduced CHOP mRNA level and caspase-3 activity in human umbilical vein endothelial cells
Therefore, we examined the effect of ox-LDL and simvastatin on the CHOP mRNA level in HUVECs. As shown in Figure 4, exposure of HUVECs to ox-LDL (100 μg/ml) resulted in approximately 7-fold changes in the mRNA level of CHOP as compared with that in the control (F = 10.825, P < 0.05). Simvastatin treatment attenuated ox-LDL-induced

dIscussIon
Our present study suggested that statins could inhibit ox-LDL-induced PERK/eIF2α/CHOP ER stress signaling in vascular endothelial cells; this effect led to inhibition of ox-LDL-induced vascular endothelial apoptosis and caspase-3 activation. [8,[26][27][28] Both oxidative stress and endothelial cell dysfunction contribute to the development of atherosclerosis. [29] Ox-LDL is the key factor to induce oxidative stress and endothelial apoptosis in EC, which is essential for the initiation and progression of atherosclerosis. [6,7] Our previous study has shown that ox-LDL induced apoptosis in vascular endothelial cells largely through the PERK/eIF2α/CHOP ER stress pathway. [8] Statins are effective cholesterol-lowering drugs that exert pleiotropic effects including inhibitory effects on inflammation, oxidative stress, and tissue repair. [30] In the present study, we provided the evidence that statins could inhibit ox-LDL-induced ER stress and apoptosis in vascular endothelial cells. Our previous study showed that ox-LDL treatment at 100 μg/ml for 24 h effectively induced ER stress and apoptosis in HUVECs, similar to the results of other studies. [8,31,32] The ER responds to ER stress by activating UPR. If UPR is insufficient to recover ER homeostasis, cells undergo apoptosis. [8,9] The PERK branch of the UPR is strongly protective at modest levels of signaling but can contribute signals to cell death pathways if under irreversible ER stress, for its downstream effector CHOP has a proapoptotic activity and is critical for triggering apoptosis in response to ER stress. [8,9,26] Among various ER responses, eIF2α phosphorylation primarily protects cells from stress by  attenuating global translation and specifically upregulating chaperone proteins; however, it can lead to apoptosis if under prolonged and severe ER stress. [33] In our study, ox-LDL treatment resulted in activation of PERK/eIF2α/CHOP signaling as well as significant apoptosis in HUVECs, suggesting that ox-LDL was an effective inducer of irreversible ER stress in endothelial cells. [8] The severe ER stress induced by ox-LDL was reflected in induced expression of PERK, increased phosphorylation of eIF2α, elevated CHOP mRNA level, and upregulated caspase-3 activity, which culminated in triggering cell apoptosis.
Simvastatin treatment inhibited each step in this process, starting from inhibiting ox-LDL-induced PERK expression. This provided a mechanistic explanation for the protective effect of simvastatin on ox-LDL-induced ER stress and endothelial apoptosis observed in this study. In addition, our results showed that simvastatin at 0.5 and 2.5 μmol/L significantly inhibited ox-LDL-induced PERK/eIF2α/CHOP signaling and apoptosis in HUVECs, suggesting that statin was a potent inhibitor of ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.
Statins inhibit HMG-CoA reductase, which converts HMG-CoA to mevalonate. [34,35] In cancerous cells and noncancerous cells, statins have been reported to inhibition of the mevalonate pathway and induction of ER stress. [17][18][19][20]36] Inconsistent with these studies, our study has demonstrated that simvastatin treatment significantly inhibited ER stress in the ox-LDL-treated EC. The seemingly discrepancy might be due to the presence of chronic ox-LDL treatment and the endothelial cell stress. In addition, it was possible that the chronic ox-LDL treatment might have altered the signaling downstream of simvastatin. Further studies are warranted to uncover the underlying mechanisms of actions.
The UPR is mediated by three ER stress receptors: PERK, IRE1, and ATF6, representing three branches of the UPR. [14] In this study, we only explored the effect of simvastatin on ox-LDL-induced PERK/eIF2α/CHOP signaling. The ATF6 and the IRE1 branches of UPR were not examined. In future studies, we will explore whether simvastatin and ox-LDL regulate the ATF6 and/or the IRE1 ER stress pathways. In addition, it will be important to verify the effects of other clinically applied statins besides simvastatin on ox-LDL-induced ER stress and apoptosis in vascular endothelial cells.
In conclusion, this study has shown that simvastatin could inhibit ox-LDL-induced ER stress and apoptosis in HUVECs, which might add new insights into the pharmacological effects of statins.

Financial support and sponsorship
This study was supported by a grant from the State Key Clinical Specialty Construction Project Funding, China.

Conflicts of interest
There are no conflicts of interest.