Rosuvastatin protects against endothelial cell apoptosis in vitro and alleviates atherosclerosis in ApoE-/- mice by suppressing endoplasmic reticulum stress

Abstract

The development of abnormal lipid-induced atherosclerosis is initiated with endothelial cell apoptosis. Vascular endothelial cells possess highly developed endoplasmic reticulum (ER), which is involved in lipid metabolism, indicating that ER stress may contribute chiefly to the induction of endothelial cell apoptosis. Based on its ability to reduce cholesterol levels, rosuvastatin may play an endothelial and vascular protective role by regulating ER stress. In the present study, the involvement of the inhibition of the ER stress-induced endothelial injury was investigated in combination with the lipid lowering effects of rosuvastatin. This compound can be used to inhibit cholesterol synthesis in atherosclerosis. Rosuvastatin decreased the apoptotic rates of human umbilical vascular endothelial cells (HUVECs) that had been stimulated with ox-low density lipoprotein (LDL) in vitro and repressed the mRNA levels of CHOP, sXBP1 and caspase-12, and decreased caspase-12 activity, as well as the content of glucose-regulated protein 78 (GRP78), phosphorylated (p)-protein kinase RNA-like ER kinase (PERK), p-inositol-requiring protein 1α (IRE1α) and p-eIF2α proteins. In addition, ApoE^-/- mice were fed with atherogenic chow for 8 weeks for atherosclerosis induction and rosuvastatin was provided by intragastric administration for an additional 4 weeks. Subsequently, the atherosclerotic plaque formation in the aorta was evaluated by Oil Red O and hematoxylin and eosin staining, and the serum LDL, high-density lipoprotein, total cholesterol (TC) and triacylglycerol (TG) levels were measured. In addition, the induction of apoptosis of endothelial cells and the expression levels of GRP78, p-PERK, p-IRE1α and p-eIF2α were assessed in the aorta. Rosuvastatin repressed atherosclerotic plaque formation and endothelial apoptosis in the aorta and decreased LDL and TG levels in the serum, as determined by in vivo results. Furthermore, it downregulated the expression levels of protein chaperone GRP78, p-PERK, p-IRE1α and p-eIF2α in the aortic intima. The data indicated that rosuvastatin could protect HUVECs from ER stress-induced apoptosis triggered by oxidized LDL. It could also inhibit atherosclerosis formation in ApoE^-/- mice aorta by regulating the PERK/eIF2α/C/EBPα-homologous protein and IRE1α/sXBP1 signaling pathways. Taken collectively, the present study demonstrated the preventive and therapeutic effects of rosuvastatin in protecting from the development of endothelial cell dysfunction diseases.

Keywords: atherosclerosis; endoplasmic reticulum stress; endothelial cell apoptosis; rosuvastatin.

Figure 1

Effect of rosuvastatin on ox-LDL-induced HUVECs apoptosis. (A) Detection of apoptosis rate by flow cytometry. (B) The values of apoptosis rate analysis were expressed as the mean ± standard deviation. (C) Effects of rosuvastatin on the mRNA levels of CHOP, sXBP1, caspase-12 in HUVECs induced by ox-LDL. The fold of mRNA levels compared with control or ox-LDL group expressed as the mean ± standard deviation. (D) Effects of rosuvastatin on the activity of caspase-12 in HUVECs induced by ox-LDL. The fold of caspase-12 activity compared with control or ox-LDL group expressed as the mean ± standard deviation. ^**P<0.01 vs. control group; ^#P<0.05 vs. ox-LDL group; ^##P<0.01 vs. ox-LDL group. ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells.

Figure 2

Effect of rosuvastatin on the phosphorylation of PERK and eIF2α in HUVECs induced by ox-LDL. (A) The expression of p-PERK and p-eIF2α in HUVECs. (B and C) The densitometric analysis of p-PERK and p-eIF2α, which expressed as the ratio of p-PERK/p-eIF2α to total PERK/eIF2α. GAPDH was the loading control. The densitometric analysis values expressed as the mean ± standard deviation. ^**P<0.01 vs. control group; ^##P<0.01 vs. ox-LDL group. PERK, protein kinase RNA-like ER kinase; eIF2α, inositol-requiring protein 1α; HUVECs, human umbilical vascular endothelial cells; ox-LDL, oxidized low density lipoprotein; p-, phosphorylated.

Figure 3

Effect of rosuvastatin on the expression of GRP78 and p-IRE1α in HUVECs induced by ox-LDL. (A) The expression of GRP78 and p-IRE1α in HUVECs. (B and C) The densitometric analysis of GRP78 and p-IRE1α, the phosphorylation of IRE1α expressed as the ratio of p-IRE1α to total IRE1α. GAPDH was the loading control and the subjacent band is the target band of GRP78. The densitometric analysis values expressed as the mean ± standard deviation. ^**P<0.01 vs. control group; ^#P<0.05 vs. ox-LDL; ^##P<0.01 vs. ox-LDL group. GRP78, glucose-regulated protein 78; p-, phosphorylated; IRE1α, p-inositol-requiring protein 1α; HUVECs, human umbilical vascular endothelial cells.

Figure 4

Effect of rosuvastatin on atherogenesis in aorta of ApoE^-/- mice induced by high-fat diet. (A) Oil red O staining of total aorta. Lesion area (%) = (plaque area/total area of aorta) x 100%. The values expressed as the mean ± standard deviation. (B) Representative HE staining of aortic cross sections, magnification, x 400, scale bar = 50 µm. ^**P<0.01 vs. Control group; ^##P<0.01 vs. Model group. HE, hematoxylin and eosin.

Figure 5

Effect of rosuvastatin on aortic endothelial cell apoptosis. Representative immunofluorescence staining of aortic cross sections. Endomucin⁽⁺⁾/TUNEL⁽⁺⁾ colocalization ratio were counted and expressed as the mean ± standard deviation. Magnification, x 200, scale bar = 100 µm.^**P<0.01 vs. Control group; ^##P<0.01 vs. Model group.

Figure 6

Effect of rosuvastatin on ER stress in aortic intima of atherosclerotic mice. Representative immunohistochemical staining and content quantification of GRP78, p-PERK, p-IRE1α and p-elF2α in aortic intima and plaque, magnification, x 400, scale bar = 50 µm. The density means expressed as the mean ± standard deviation. ^**P<0.01 vs. Control group; ^#P<0.01 vs. Model group. ER GRP78, glucose-regulated protein 78; p-, phosphorylated; PERK, protein kinase RNA-like ER kinase; IRE1α, p-inositol-requiring protein 1α; eIF2α, inositol-requiring protein 1α.

Figure 7

Schematic overview shows that rosuvastatin reverse the HUVECs apoptosis triggered by ox-LDL and endothelial injury induced by high-fat diets specifically by downregulating PERK/p-eIF2α/CHOP and IRE1α/sXBP1 signaling pathways.