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Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo

View Article: PubMed Central - PubMed

ABSTRACT

Background:: GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation.

Methods:: Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization.

Results:: We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin.

Conclusions:: Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

No MeSH data available.


Related in: MedlinePlus

Lovastatin recouples eNOS in endothelial cells in a miR-133a–dependent manner.(Athrough C) HUVECs infected with lentivirus containing scr-miR or premiR-133a for 48 hours followed by ox-LDL (100 μg/mL) or lovastatin treatment (10 μmol/L, 24 hours). Cell lysates were used to detect BH4 content by HPLC in A, NO production by measuring diaminofluorescein fluorescence intensity in B, and ROS production by measuring DHE fluorescence intensity in C. n=3 per group. *P<0.05 versus scr-miR alone. #P<0.05 versus scr-miR plus ox-LDL. NS indicates no significance. (Dthrough F) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα for 24 hours. Cells were used to determine BH4 content in D, NO production in E, and ROS production in F. n=3 per group. *P<0.05 versus control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. BH4 indicates tetrahydrobiopterin; DAF, diaminofluorescein; DHE, dihydroethidium; HPLC, high-performance liquid chromatography; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; NO, nitric oxide; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; ROS, reactive oxygen species; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.
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Figure 3: Lovastatin recouples eNOS in endothelial cells in a miR-133a–dependent manner.(Athrough C) HUVECs infected with lentivirus containing scr-miR or premiR-133a for 48 hours followed by ox-LDL (100 μg/mL) or lovastatin treatment (10 μmol/L, 24 hours). Cell lysates were used to detect BH4 content by HPLC in A, NO production by measuring diaminofluorescein fluorescence intensity in B, and ROS production by measuring DHE fluorescence intensity in C. n=3 per group. *P<0.05 versus scr-miR alone. #P<0.05 versus scr-miR plus ox-LDL. NS indicates no significance. (Dthrough F) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα for 24 hours. Cells were used to determine BH4 content in D, NO production in E, and ROS production in F. n=3 per group. *P<0.05 versus control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. BH4 indicates tetrahydrobiopterin; DAF, diaminofluorescein; DHE, dihydroethidium; HPLC, high-performance liquid chromatography; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; NO, nitric oxide; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; ROS, reactive oxygen species; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.

Mentions: To study the role of miR-133a in statin-induced eNOS recoupling, we generated cells with enforced miR-133a expression by infecting cells with a lentivirus expressing premiR-133a. Although lovastatin treatment effectively normalized the BH4 levels (Figure 3A) and recoupled eNOS (Figure 3B and 3C) in ox-LDL–treated HUVECs infected with a lentivirus expressing scr-miR, it failed to increase the BH4 levels and recouple eNOS in HUVECs on exogenous miR-133a expression.


Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo
Lovastatin recouples eNOS in endothelial cells in a miR-133a–dependent manner.(Athrough C) HUVECs infected with lentivirus containing scr-miR or premiR-133a for 48 hours followed by ox-LDL (100 μg/mL) or lovastatin treatment (10 μmol/L, 24 hours). Cell lysates were used to detect BH4 content by HPLC in A, NO production by measuring diaminofluorescein fluorescence intensity in B, and ROS production by measuring DHE fluorescence intensity in C. n=3 per group. *P<0.05 versus scr-miR alone. #P<0.05 versus scr-miR plus ox-LDL. NS indicates no significance. (Dthrough F) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα for 24 hours. Cells were used to determine BH4 content in D, NO production in E, and ROS production in F. n=3 per group. *P<0.05 versus control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. BH4 indicates tetrahydrobiopterin; DAF, diaminofluorescein; DHE, dihydroethidium; HPLC, high-performance liquid chromatography; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; NO, nitric oxide; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; ROS, reactive oxygen species; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.
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Figure 3: Lovastatin recouples eNOS in endothelial cells in a miR-133a–dependent manner.(Athrough C) HUVECs infected with lentivirus containing scr-miR or premiR-133a for 48 hours followed by ox-LDL (100 μg/mL) or lovastatin treatment (10 μmol/L, 24 hours). Cell lysates were used to detect BH4 content by HPLC in A, NO production by measuring diaminofluorescein fluorescence intensity in B, and ROS production by measuring DHE fluorescence intensity in C. n=3 per group. *P<0.05 versus scr-miR alone. #P<0.05 versus scr-miR plus ox-LDL. NS indicates no significance. (Dthrough F) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα for 24 hours. Cells were used to determine BH4 content in D, NO production in E, and ROS production in F. n=3 per group. *P<0.05 versus control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. BH4 indicates tetrahydrobiopterin; DAF, diaminofluorescein; DHE, dihydroethidium; HPLC, high-performance liquid chromatography; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; NO, nitric oxide; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; ROS, reactive oxygen species; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.
Mentions: To study the role of miR-133a in statin-induced eNOS recoupling, we generated cells with enforced miR-133a expression by infecting cells with a lentivirus expressing premiR-133a. Although lovastatin treatment effectively normalized the BH4 levels (Figure 3A) and recoupled eNOS (Figure 3B and 3C) in ox-LDL–treated HUVECs infected with a lentivirus expressing scr-miR, it failed to increase the BH4 levels and recouple eNOS in HUVECs on exogenous miR-133a expression.

View Article: PubMed Central - PubMed

ABSTRACT

Background:: GTP cyclohydrolase 1 (GCH1) deficiency is critical for endothelial nitric oxide synthase uncoupling in endothelial dysfunction. MicroRNAs (miRs) are a class of regulatory RNAs that negatively regulate gene expression. We investigated whether statins prevent endothelial dysfunction via miR-dependent GCH1 upregulation.

Methods:: Endothelial function was assessed by measuring acetylcholine-induced vasorelaxation in the organ chamber. MiR-133a expression was assessed by quantitative reverse transcription polymerase chain reaction and fluorescence in situ hybridization.

Results:: We first demonstrated that GCH1 mRNA is a target of miR-133a. In endothelial cells, miR-133a was robustly induced by cytokines/oxidants and inhibited by lovastatin. Furthermore, lovastatin upregulated GCH1 and tetrahydrobiopterin, and recoupled endothelial nitric oxide synthase in stressed endothelial cells. These actions of lovastatin were abolished by enforced miR-133a expression and were mirrored by a miR-133a antagomir. In mice, hyperlipidemia- or hyperglycemia-induced ectopic miR-133a expression in the vascular endothelium, reduced GCH1 protein and tetrahydrobiopterin levels, and impaired endothelial function, which were reversed by lovastatin or miR-133a antagomir. These beneficial effects of lovastatin in mice were abrogated by in vivo miR-133a overexpression or GCH1 knockdown. In rats, multiple cardiovascular risk factors including hyperglycemia, dyslipidemia, and hyperhomocysteinemia resulted in increased miR-133a vascular expression, reduced GCH1 expression, uncoupled endothelial nitric oxide synthase function, and induced endothelial dysfunction, which were prevented by lovastatin.

Conclusions:: Statin inhibits aberrant miR-133a expression in the vascular endothelium to prevent endothelial dysfunction by targeting GCH1. Therefore, miR-133a represents an important therapeutic target for preventing cardiovascular diseases.

No MeSH data available.


Related in: MedlinePlus