Atorvastatin downregulates HSP22 expression in an atherosclerotic model in vitro and in vivo

Abstract

One of the pathological functions of heat shock protein 22 (HSP22) is the association with inflammatory diseases and atherosclerosis. However, the effects of a high‑fat diet (HFD) or oxidized low‑density lipoprotein (ox‑LDL) combined with atorvastatin (ATV) on HSP22 expression are entirely unknown. The present study investigated the effects of ATV on HSP22 expression in HFD‑induced atherosclerotic apolipoprotein E‑deficient (ApoE‑/‑) mice and in ox‑LDL‑induced human umbilical vein endothelial cells (HUVECs). Furthermore, the influence of HSP22‑knockdown on the HFD- or ox‑LDL‑induced atherosclerotic model was also examined. It was found that HFD or ox‑LDL treatment significantly increased HSP22 expression in the serum and aorta, accompanied by decreased phosphorylated (p)‑endothelial nitric oxide synthase (p-eNOS) activity and activated p38 mitogen‑activated protein kinase (MAPK). However, these effects were suppressed by treatment with ATV. Furthermore, HSP22-knockdown showed reduced ox‑LDL‑induced lesions, evidenced by increased p‑eNOS activity and inactivated p38 MAPK, while suppression of cell proliferation inhibition and cell cycle arrest were also observed. Taken together, the results of this study suggest that HFD or ox‑LDL increased the expression of HSP22 and p‑p38 MAPK, and decreased the p‑eNOS activity in vitro and in vivo, and ATV could reduce the effects by downregulating HSP22 expression.

Figure 1

Atherosclerotic lesion formation in aortic sections from ApoE^−/− mice. (A) There were significantly increased TC, TG and LDL levels, and decreased HDL levels in the mice in the HFD group. (B) Atherosclerotic lesions in the aortic root, as measured by hematoxylin and eosin staining. Data are presented as the mean ± standard deviation (n=6). ^*P<0.05 and ^**P<0.01 compared with the ND group; ^#P<0.05 and ^##P<0.01 compared with the HFD group. Scale bars, 40 µm. ND, normal diet; HFD, high-fat diet; ApoE^−/−, apolipoprotein E-deficient; TC, total cholesterol; TG, triglycerides; LDL, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; ATV, atorvastatin.

Figure 2

Effect of HFD on HSP22, eNOS and p38 MAPK in atherosclerotic lesions. The expression of HSP22 in ApoE^−/− mice with an HFD and/or ATV treatment was measured by (A) immunohistochemistry, (B) enzyme-linked immunosorbent assay and (C and D) western blot assay, respectively. (C and E) The expression level of p-eNOS was examined by western blot assay in ApoE^−/− mice with an HFD and/or ATV treatment. The expression level of p-p38 MAPK was examined by (C and E) western blot assay and (F) immunohistochemistry in ApoE^−/− mice with an HFD and/or ATV treatment. Data are presented as the mean ± standard deviation (n=6). ^**P<0.01 compared with the ND group; ^##P<0.01 compared with the HFD group. Scale bars, 100 µm. ND, normal diet; HFD, high-fat diet; HSP22, heat shock protein 22; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated protein kinase; ApoE^−/−, apolipoprotein E-deficient; ATV, atorvastatin; p-phosphorylated.

Figure 3

Effect of ox-LDL and ATV on cell proliferation of HUVECs. Cell proliferation was measured by MTT assay in HUVECs with different concentrations of (A) ox-LDL or (B) ATV treatment for 24, 48 and 72 h. ^*P<0.05 and ^**P<0.01 compared with the control group. ox-LDL, oxidized low-density lipoprotein; ATV, ATV, atorvastatin; HUVECs, human umbilical vein endothelial cells; OD, optical density.

Figure 4

Effect of ATV on HSP22, eNOS and p38 MAPK in HUVECs. The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by (A) reverse transcription-quantitative polymerase chain reaction and (B) western blot assay, respectively. (C and D) The expression levels of p-eNOS and p-p38 MAPK were examined by western blot assay in HUVECs with an HFD and/or ATV treatment. ^**P<0.01 compared with the control group; ^#P<0.05 and ^##P<0.01 compared with the ox-LDL group; ^△△P<0.01 compared with the ATV group. ox-LDL, oxidized low-density lipoprotein; ATV, atorvastatin; HUVECs, human umbilical vein endothelial cells; HSP22, heat shock protein 22; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated kinase; HFD, high-fat diet; p-, phosphorylated; t-, total.

Figure 5

HSP22-knockdown inhibits ox-LDL-induced p-eNOS decrease and p-p38 increase in HUVECs. (A) The transfection efficiency of HSP22 shRNAs in HUVECs was measured by western blot assay. (B) The expression of HSP22 in HUVECs with an HFD and/or ATV treatment was measured by western blot assay. (C and D) The expression of p-eNOS and p-p38 in HUVECs with an HFD and/or ATV treatment in the absence or presence of HSP22 shRNA was measured by western blot assay. ^*P<0.05 and ^**P<0.01 compared with the corresponding NC group. ox-LDL, oxidized low-density lipoprotein; ATV, atorvastatin; HUVECs, human umbilical vein endothelial cells; HSP22, heat shock protein 22; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated kinase; HFD, high-fat diet; p-, phosphorylated; t-, total; NC, negative control; shRNA, short hairpin RNA.

Figure 6

HSP22-knockdown reduces ox-LDL-induced cell proliferation inhibition and cell cycle arrest in HUVECs. Cell proliferation was measured by MTT assay in HUVECs with an HFD and/or ATV treatment in the absence or presence of HSP22 shRNA for (A) 24 h, (B) 48 h and (C) 72 h. (D and E) The cell cycle was measured by flow cytometry assay in HUVECs with an HFD and/or ATV treatment in the absence or presence of HSP22 shRNA. ^*P<0.05 and ^**P<0.01 compared with the corresponding NC group. ox-LDL, oxidized low-density lipoprotein; ATV, atorvastatin; HUVECs, human umbilical vein endothelial cells; HSP22, heat shock protein 22; eNOS, endothelial nitric oxide synthase; MAPK, mitogen-activated kinase; HFD, high-fat diet; p-, phosphorylated; t-, total; NC, negative control; shRNA, short hairpin RNA; OD, optical density.