Simvastatin promotes endothelial dysfunction by activating the Wnt/β‑catenin pathway under oxidative stress

Abstract

Atherosclerosis is a major pathogenic factor in patients with cardiovascular diseases, and endothelial dysfunction (ED) plays a primary role in its occurrence and development. Simvastatin is a lipid‑lowering drug, which is commonly used to prevent or treat risk factors of cardiovascular diseases with a significant anti‑atherogenic effect. However, its impact on endothelial cells under conditions of oxidative stress and broader mechanisms of action remain unclear. The present study evaluated the effect of simvastatin on human umbilical vein endothelial cells (HUVECs) under oxidative stress with H2O2, and the associated mechanisms. At a high dose (1 µM), simvastatin exacerbated H2O2‑induced endothelial cell dysfunction. Moreover, inhibition of the Wnt/β‑catenin pathway by salinomycin significantly suppressed the simvastatin‑associated HUVEC dysfunction. Western blot analysis further demonstrated that simvastatin promoted the phosphorylation of low‑density lipoprotein receptor‑related protein 6 (LRP6) and activated the Wnt/β‑catenin pathway. Simvastatin also activated endoplasmic reticulum (ER) stress, which was reversed by salinomycin treatment. Based on these results, it was hypothesized that simvastatin may promote ER stress by facilitating LRP6 phosphorylation and the subsequent activation of the Wnt/β‑catenin pathway, thereby enhancing H2O2‑induced ED. Therefore, high‑dose simvastatin treatment could have potential toxic side effects, indicating the need for close clinical management, monitoring and patient selection.

Figure 1

Effect of simvastatin on HUVECs and its possible mechanisms. HUVECs were treated with simvastatin in two concentration series (0, 0.2, 0.4, and 0.8 µM; 0, 1, 2, and 4 µM) for 24 h. (A) Western blot analysis of the expression levels of Bax and Bcl-2 after exposure to simvastatin at 0-0.8 µM. (B) Assessment of endothelial dysfunction based on LDH release. (C) Cell viability tested by the MTT assay. (D) Western blot analysis of the expression levels of Bax and Bcl-2 after exposure to simvastatin at 0-4 µM. (E) Western blot analysis of the expression levels of β-catenin, phospho-β-catenin, GRP78 and ATF6. (F) The ratio of phospho-β-catenin/β-catenin. All values are presented as the mean ± SD; n=3 (in A, B, D, E and F) and n=6 (in C). ^*P<0.05 or ^**P<0.01 vs. respective 0 µM group. HUVEC, human umbilical vein endothelial cell; LDH, lactate dehydrogenase; phosphor, phosphorylated; GRP78, 78 kDa glucose-regulated protein; ATF6, cyclic AMP-dependent transcription factor ATF-6α.

Figure 2

Simvastatin promotes ED by inducing the Wnt/β-catenin pathway. (A) Western blot analysis of Bax and Bcl-2 protein levels. (B) Acridine orange/ethidium bromide staining of human umbilical vein endothelial cells; magnification, ×20. The ED level of cells was reflected by the (C) MTT assay, (D) LDH levels, (E) SOD activity and (F) NOS activity. All values are presented as the mean ± SD. ^*P<0.05, ^**P<0.01 vs. C; ^#P<0.05, ^##P<0.01 vs. Sim + Sal. n=3 (in A, B, D, E and F) and n=6 (in C). Sim, simvastatin; Sal, salinomycin; LDH, lactate dehydrogenase; ED, endothelial dysfunction; NOS, nitric oxide synthase; SOD, superoxide dismutase; C, control.

Figure 3

Simvastatin increases the adhesion ability of HUVECs by inducing the Wnt/β-catenin pathway. (A) Adhesion rate of HUVECs to THP-1 cells; magnification, ×20. (B) Western blot analysis of VCAM-1, ICAM-1 and MCP-1 protein levels (mean ± SD). ^*P<0.05, ^**P<0.01 vs. C; ^#P<0.05, ^##P<0.01 vs. Sim + Sal. n=3. HUVEC, human umbilical vein endothelial cell; VCAM-1, vascular cell adhesion protein 1; ICAM-1, intercellular adhesion molecule 1; MCP-1, monocyte chemoattractant protein 1; C, control; Sim, simvastatin; Sal, salinomycin.

Figure 4

Simvastatin activates the Wnt/β-catenin pathway by enhancing LRP6 phosphorylation. The protein levels of (A) β-catenin, phospho-β-catenin, (B) LRP6 and phospho-LRP6 were measured by western blotting (mean ± SD). (C) The ratio of phospho-β-catenin/β-catenin and (D) phospho-LRP6/LRP6. ^*P<0.05, ^**P<0.01 vs. C; ^#P<0.05, ^##P<0.01 vs. Sim + Sal. n=3. LRP6, low-density lipoprotein receptor-related protein 6; phospho, phosphorylated; Sim, simvas-tatin; Sal, salinomycin; C, control.

Figure 5

Simvastatin augments endoplasmic reticulum stress through activation of the Wnt/β-catenin pathway. The protein expression levels of GRP78, ATF6 and CHOP were measured by western blotting (mean ± SD). ^*P<0.05, ^**P<0.01 vs. respective C; ^#P<0.05, ^##P<0.01 vs. respective Sim + Sal. n=3. GRP78, 78 kDa glucose-regulated protein; ATF6, cyclic AMP-dependent transcription factor ATF-6α; Sim, simvastatin; Sal, salinomycin; C, control; CHOP, C/EBP-homologous protein.

Figure 6

Proposed mechanism of action of simvastatin in promoting ED under oxidative stress. Simvastatin facilitates LRP6 phosphorylation and subsequent activation of the Wnt/β-catenin pathway, which promotes downstream ER stress, thereby leading to H₂O₂-induced ED. LRP6, low-density lipoprotein receptor-related protein 6; ED, endothelial dysfunction; ER, endoplasmic reticulum; SM, sphingomyelin; P, phosphate.