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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.


Overexpression of miR-133a abrogates lovastatin-preserved endothelial function in Apoe–/– mice fed with a high-fat diet (HFD). The protocol and experimental designs are described in the online-only Data Supplement Methods and Figure VA. (A) The miR-133a expression in the aortic arteries was detected by fluorescence in situ hybridization. Green, miR-133a; Red, vWF. (B) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (C) ACh-induced vasorelaxation was assayed in descending aortic arteries using an organ chamber. n=10 to 15 per group. *P<0.05 versus scr-miR plus RD (regular diet). #P<0.05 versus scr-miR plus HFD. Ach indicates acetylcholine; GCH1, GTP cyclohydrolase 1; miR, microRNA; NS, no significance; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and vWF, von Willebrand factor.
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Figure 5: Overexpression of miR-133a abrogates lovastatin-preserved endothelial function in Apoe–/– mice fed with a high-fat diet (HFD). The protocol and experimental designs are described in the online-only Data Supplement Methods and Figure VA. (A) The miR-133a expression in the aortic arteries was detected by fluorescence in situ hybridization. Green, miR-133a; Red, vWF. (B) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (C) ACh-induced vasorelaxation was assayed in descending aortic arteries using an organ chamber. n=10 to 15 per group. *P<0.05 versus scr-miR plus RD (regular diet). #P<0.05 versus scr-miR plus HFD. Ach indicates acetylcholine; GCH1, GTP cyclohydrolase 1; miR, microRNA; NS, no significance; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and vWF, von Willebrand factor.

Mentions: The localization of miR-133a in the vessel sections was determined by fluorescence in situ hybridization. As indicated in Figure 5A, the basal expression of miR-133a in the aortic artery was mainly located in the vascular medium, other than the endothelium. However, miR-133a expression was clearly detected in the vascular endothelium and was inhibited by lovastatin in hyperlipidemic mice infected with lentiviruses harboring scr-miR. Infection with lentiviruses expressing the premiR-133a dramatically increased the expression of miR-133a in both the vascular endothelium and in the medium.


Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo
Overexpression of miR-133a abrogates lovastatin-preserved endothelial function in Apoe–/– mice fed with a high-fat diet (HFD). The protocol and experimental designs are described in the online-only Data Supplement Methods and Figure VA. (A) The miR-133a expression in the aortic arteries was detected by fluorescence in situ hybridization. Green, miR-133a; Red, vWF. (B) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (C) ACh-induced vasorelaxation was assayed in descending aortic arteries using an organ chamber. n=10 to 15 per group. *P<0.05 versus scr-miR plus RD (regular diet). #P<0.05 versus scr-miR plus HFD. Ach indicates acetylcholine; GCH1, GTP cyclohydrolase 1; miR, microRNA; NS, no significance; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and vWF, von Willebrand factor.
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Related In: Results  -  Collection

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Figure 5: Overexpression of miR-133a abrogates lovastatin-preserved endothelial function in Apoe–/– mice fed with a high-fat diet (HFD). The protocol and experimental designs are described in the online-only Data Supplement Methods and Figure VA. (A) The miR-133a expression in the aortic arteries was detected by fluorescence in situ hybridization. Green, miR-133a; Red, vWF. (B) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (C) ACh-induced vasorelaxation was assayed in descending aortic arteries using an organ chamber. n=10 to 15 per group. *P<0.05 versus scr-miR plus RD (regular diet). #P<0.05 versus scr-miR plus HFD. Ach indicates acetylcholine; GCH1, GTP cyclohydrolase 1; miR, microRNA; NS, no significance; premiR, preliminary miR-133a; RT-PCR, reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and vWF, von Willebrand factor.
Mentions: The localization of miR-133a in the vessel sections was determined by fluorescence in situ hybridization. As indicated in Figure 5A, the basal expression of miR-133a in the aortic artery was mainly located in the vascular medium, other than the endothelium. However, miR-133a expression was clearly detected in the vascular endothelium and was inhibited by lovastatin in hyperlipidemic mice infected with lentiviruses harboring scr-miR. Infection with lentiviruses expressing the premiR-133a dramatically increased the expression of miR-133a in both the vascular endothelium and in the medium.

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.