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Endoplasmic reticulum stress effector CCAAT/enhancer-binding protein homologous protein (CHOP) regulates chronic kidney disease-induced vascular calcification.

Miyazaki-Anzai S, Masuda M, Demos-Davies KM, Keenan AL, Saunders SJ, Masuda R, Jablonski K, Cavasin MA, Kendrick J, Chonchol M, McKinsey TA, Levi M, Miyazaki M - J Am Heart Assoc (2014)

Bottom Line: Treatment with simvastatin plus ezetimibe strongly reduced levels of serum oxysterols and attenuated CKD-dependent atherosclerosis, vascular cell death, vascular calcification, and cardiac dysfunction.This therapy also reduced aortic endoplasmic reticulum stress induced by CKD.These data reveal that the cholesterol-lowering therapy of simvastatin plus ezetimibe attenuates CKD-dependent vascular diseases through a reduction of oxysterol-mediated endoplasmic reticulum stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, CO (S.M.A., M.M., A.L.K., S.J.S., R.M., K.J., J.K., M.C., M.L., M.M.).

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Treatment with simvastatin plus ezetimibe reduces endoplasmic reticulum stress and cell death induced by CKD. A, Aortic mRNA levels and B, aortic, p‐PERK, ATF4, and CHOP protein expression in 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. C, Representative micrographs show less CHOP‐positive signal (green) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. D, Immunofluorescence detection of CHOP in the aortic sinuses of 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. E, Representative micrographs show less TUNEL‐positive signal (red, arrows) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. F, Quantitative analysis of TUNEL‐positive nuclei conducted on lesions from 5/6 nx ApoE−/− mice treated with either vehicle or simvastatin plus ezetimibe. 5/6 nx ApoE−/− mice were treated with either vehicle or 20 mg/kg simvastatin plus 10 mg/kg ezetimibe for 12 weeks. N=6, *P<0.05 vs sham‐operated mice with vehicle. #P<0.05 vs 5/6 nx ApoE−/− mice treated with vehicle. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; BiP, binding immunoglobulin protein; CKD, chronic kidney disease; CHOP, C/EBP homology protein; OPN, osteopontin; p‐PERK, phospho protein kinase‐like endoplasmic reticulum kinase; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling. ***P<0.001.
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fig04: Treatment with simvastatin plus ezetimibe reduces endoplasmic reticulum stress and cell death induced by CKD. A, Aortic mRNA levels and B, aortic, p‐PERK, ATF4, and CHOP protein expression in 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. C, Representative micrographs show less CHOP‐positive signal (green) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. D, Immunofluorescence detection of CHOP in the aortic sinuses of 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. E, Representative micrographs show less TUNEL‐positive signal (red, arrows) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. F, Quantitative analysis of TUNEL‐positive nuclei conducted on lesions from 5/6 nx ApoE−/− mice treated with either vehicle or simvastatin plus ezetimibe. 5/6 nx ApoE−/− mice were treated with either vehicle or 20 mg/kg simvastatin plus 10 mg/kg ezetimibe for 12 weeks. N=6, *P<0.05 vs sham‐operated mice with vehicle. #P<0.05 vs 5/6 nx ApoE−/− mice treated with vehicle. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; BiP, binding immunoglobulin protein; CKD, chronic kidney disease; CHOP, C/EBP homology protein; OPN, osteopontin; p‐PERK, phospho protein kinase‐like endoplasmic reticulum kinase; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling. ***P<0.001.

Mentions: Since we previously reported that the aortic PERK‐eukaryotic initiation factor 2α‐ATF4‐CHOP axis of the ER stress signal contributes to CKD‐dependent cardiovascular diseases,21 we next analyzed whether the treatment with simvastatin plus ezetimibe affects ER signaling in the aortas of ApoE−/− mice with CKD. In agreement with our previous report, CKD significantly induced the mRNA expression of ER stress makers such as ATF4, CHOP, and Binding immunoglobulin protein, osteogenic markers such as alkaline phosphatase and osteopontin, and inflammatory markers such as TNFα (Figure 4A). Treatment with simvastatin plus ezetimibe significantly reduced mRNA levels of these ER stress, osteogenic, and inflammatory markers. In addition, CKD increased levels of aortic p‐PERK, p‐ATF4, total ATF4, and CHOP proteins, which were significantly reduced by simvastatin plus ezetimibe treatment (Figures 4B and S1). Immunohistological analysis confirmed that aortic CHOP expression was significantly reduced by this treatment (Figure 4C and 4D). Since CHOP is a pro‐apoptotic effector of ER stress, we performed terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining of the aortas of ApoE−/− mice with CKD to examine whether: (1) CKD induces cell death in the aorta and (2) this cholesterol‐lowering therapy reduces CKD‐induced aortic cell death. As hypothesized, CKD significantly increased aortic cell death (Figure 4E and 4F), whereas simvastatin/ezetimibe treatment attenuated CKD‐induced cell death (Figure 4E and 4F).


Endoplasmic reticulum stress effector CCAAT/enhancer-binding protein homologous protein (CHOP) regulates chronic kidney disease-induced vascular calcification.

Miyazaki-Anzai S, Masuda M, Demos-Davies KM, Keenan AL, Saunders SJ, Masuda R, Jablonski K, Cavasin MA, Kendrick J, Chonchol M, McKinsey TA, Levi M, Miyazaki M - J Am Heart Assoc (2014)

Treatment with simvastatin plus ezetimibe reduces endoplasmic reticulum stress and cell death induced by CKD. A, Aortic mRNA levels and B, aortic, p‐PERK, ATF4, and CHOP protein expression in 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. C, Representative micrographs show less CHOP‐positive signal (green) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. D, Immunofluorescence detection of CHOP in the aortic sinuses of 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. E, Representative micrographs show less TUNEL‐positive signal (red, arrows) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. F, Quantitative analysis of TUNEL‐positive nuclei conducted on lesions from 5/6 nx ApoE−/− mice treated with either vehicle or simvastatin plus ezetimibe. 5/6 nx ApoE−/− mice were treated with either vehicle or 20 mg/kg simvastatin plus 10 mg/kg ezetimibe for 12 weeks. N=6, *P<0.05 vs sham‐operated mice with vehicle. #P<0.05 vs 5/6 nx ApoE−/− mice treated with vehicle. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; BiP, binding immunoglobulin protein; CKD, chronic kidney disease; CHOP, C/EBP homology protein; OPN, osteopontin; p‐PERK, phospho protein kinase‐like endoplasmic reticulum kinase; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling. ***P<0.001.
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fig04: Treatment with simvastatin plus ezetimibe reduces endoplasmic reticulum stress and cell death induced by CKD. A, Aortic mRNA levels and B, aortic, p‐PERK, ATF4, and CHOP protein expression in 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. C, Representative micrographs show less CHOP‐positive signal (green) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. D, Immunofluorescence detection of CHOP in the aortic sinuses of 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. E, Representative micrographs show less TUNEL‐positive signal (red, arrows) in nuclei (blue) of aortic sinus lesions from 5/6 nx ApoE−/− mice treated with simvastatin plus ezetimibe. F, Quantitative analysis of TUNEL‐positive nuclei conducted on lesions from 5/6 nx ApoE−/− mice treated with either vehicle or simvastatin plus ezetimibe. 5/6 nx ApoE−/− mice were treated with either vehicle or 20 mg/kg simvastatin plus 10 mg/kg ezetimibe for 12 weeks. N=6, *P<0.05 vs sham‐operated mice with vehicle. #P<0.05 vs 5/6 nx ApoE−/− mice treated with vehicle. ALP indicates alkaline phosphatase; ATF4, activating transcription factor 4; BiP, binding immunoglobulin protein; CKD, chronic kidney disease; CHOP, C/EBP homology protein; OPN, osteopontin; p‐PERK, phospho protein kinase‐like endoplasmic reticulum kinase; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling. ***P<0.001.
Mentions: Since we previously reported that the aortic PERK‐eukaryotic initiation factor 2α‐ATF4‐CHOP axis of the ER stress signal contributes to CKD‐dependent cardiovascular diseases,21 we next analyzed whether the treatment with simvastatin plus ezetimibe affects ER signaling in the aortas of ApoE−/− mice with CKD. In agreement with our previous report, CKD significantly induced the mRNA expression of ER stress makers such as ATF4, CHOP, and Binding immunoglobulin protein, osteogenic markers such as alkaline phosphatase and osteopontin, and inflammatory markers such as TNFα (Figure 4A). Treatment with simvastatin plus ezetimibe significantly reduced mRNA levels of these ER stress, osteogenic, and inflammatory markers. In addition, CKD increased levels of aortic p‐PERK, p‐ATF4, total ATF4, and CHOP proteins, which were significantly reduced by simvastatin plus ezetimibe treatment (Figures 4B and S1). Immunohistological analysis confirmed that aortic CHOP expression was significantly reduced by this treatment (Figure 4C and 4D). Since CHOP is a pro‐apoptotic effector of ER stress, we performed terminal deoxynucleotidyl transferase dUTP nick‐end labeling (TUNEL) staining of the aortas of ApoE−/− mice with CKD to examine whether: (1) CKD induces cell death in the aorta and (2) this cholesterol‐lowering therapy reduces CKD‐induced aortic cell death. As hypothesized, CKD significantly increased aortic cell death (Figure 4E and 4F), whereas simvastatin/ezetimibe treatment attenuated CKD‐induced cell death (Figure 4E and 4F).

Bottom Line: Treatment with simvastatin plus ezetimibe strongly reduced levels of serum oxysterols and attenuated CKD-dependent atherosclerosis, vascular cell death, vascular calcification, and cardiac dysfunction.This therapy also reduced aortic endoplasmic reticulum stress induced by CKD.These data reveal that the cholesterol-lowering therapy of simvastatin plus ezetimibe attenuates CKD-dependent vascular diseases through a reduction of oxysterol-mediated endoplasmic reticulum stress.

View Article: PubMed Central - PubMed

Affiliation: Division of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, CO (S.M.A., M.M., A.L.K., S.J.S., R.M., K.J., J.K., M.C., M.L., M.M.).

Show MeSH
Related in: MedlinePlus