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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

In vivo antagonism of miR-133a improves endothelial dysfunction in STZ-induced diabetic mice. The protocol and experimental designs are described in online-only Data Supplement Methods and Figure VIA. (A) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (B) BH4 content in aortic tissues was determined by HPLC. (C) ACh-induced vasorelaxation in descending aortic artery was assayed using an organ chamber. Serum NO levels (D), serum SOD activities (E), and serum MDA levels (F) were measured in mice. n=10 to 15 per group. *P<0.05 versus control mice alone. #P<0.05 versus diabetic mice. Ach indicates acetylcholine; BH4, tetrahydrobiopterin; GCH1, GTP cyclohydrolase 1; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; miR, microRNA; NO, nitric oxide; NS, no significance; RT-PCR, reverse transcription polymerase chain reaction; SOD, superoxide dismutase; and STZ, streptozotocin.
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Figure 7: In vivo antagonism of miR-133a improves endothelial dysfunction in STZ-induced diabetic mice. The protocol and experimental designs are described in online-only Data Supplement Methods and Figure VIA. (A) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (B) BH4 content in aortic tissues was determined by HPLC. (C) ACh-induced vasorelaxation in descending aortic artery was assayed using an organ chamber. Serum NO levels (D), serum SOD activities (E), and serum MDA levels (F) were measured in mice. n=10 to 15 per group. *P<0.05 versus control mice alone. #P<0.05 versus diabetic mice. Ach indicates acetylcholine; BH4, tetrahydrobiopterin; GCH1, GTP cyclohydrolase 1; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; miR, microRNA; NO, nitric oxide; NS, no significance; RT-PCR, reverse transcription polymerase chain reaction; SOD, superoxide dismutase; and STZ, streptozotocin.

Mentions: If statin prevents endothelial dysfunction via inhibition of miR-133a, miR-133a antagonism might produce effects similar to those produced by statin in vivo. To test this notion, streptozotocin-induced diabetic C57B16 mice received in vivo delivery of miR-133a antagomir through Alzet osmotic minipumps for 4 weeks to establish a loss-of-function model for miR-133a (online-only Data Supplement Figure VIA and Table II). In comparison with those in control mice, hyperglycemia reduced the levels of GCH1 mRNA and protein (Figure 7A), and the BH4 content (Figure 7B) in aortic arteries, which were normalized by the miR-133a antagomir. Functional analysis indicated that hyperglycemia impaired the ACh-induced EDR, decreased serum NO levels and superoxide dismutase activities, and increased serum malondialdehyde levels in mice infused with the control antagomir, as well (Figure 7C through 7F). Importantly, the miR-133a antagomir rescued these abnormal phenotypes in diabetic mice. These data suggest that antagonism of miR-133a upregulates GCH1 expression and rescues endothelial function in diabetes mellitus.


Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo
In vivo antagonism of miR-133a improves endothelial dysfunction in STZ-induced diabetic mice. The protocol and experimental designs are described in online-only Data Supplement Methods and Figure VIA. (A) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (B) BH4 content in aortic tissues was determined by HPLC. (C) ACh-induced vasorelaxation in descending aortic artery was assayed using an organ chamber. Serum NO levels (D), serum SOD activities (E), and serum MDA levels (F) were measured in mice. n=10 to 15 per group. *P<0.05 versus control mice alone. #P<0.05 versus diabetic mice. Ach indicates acetylcholine; BH4, tetrahydrobiopterin; GCH1, GTP cyclohydrolase 1; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; miR, microRNA; NO, nitric oxide; NS, no significance; RT-PCR, reverse transcription polymerase chain reaction; SOD, superoxide dismutase; and STZ, streptozotocin.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5120771&req=5

Figure 7: In vivo antagonism of miR-133a improves endothelial dysfunction in STZ-induced diabetic mice. The protocol and experimental designs are described in online-only Data Supplement Methods and Figure VIA. (A) Western blotting analysis of GCH1 protein and RT-PCR analysis of GCH1 mRNA in aortic tissues. (B) BH4 content in aortic tissues was determined by HPLC. (C) ACh-induced vasorelaxation in descending aortic artery was assayed using an organ chamber. Serum NO levels (D), serum SOD activities (E), and serum MDA levels (F) were measured in mice. n=10 to 15 per group. *P<0.05 versus control mice alone. #P<0.05 versus diabetic mice. Ach indicates acetylcholine; BH4, tetrahydrobiopterin; GCH1, GTP cyclohydrolase 1; HPLC, high-performance liquid chromatography; MDA, malondialdehyde; miR, microRNA; NO, nitric oxide; NS, no significance; RT-PCR, reverse transcription polymerase chain reaction; SOD, superoxide dismutase; and STZ, streptozotocin.
Mentions: If statin prevents endothelial dysfunction via inhibition of miR-133a, miR-133a antagonism might produce effects similar to those produced by statin in vivo. To test this notion, streptozotocin-induced diabetic C57B16 mice received in vivo delivery of miR-133a antagomir through Alzet osmotic minipumps for 4 weeks to establish a loss-of-function model for miR-133a (online-only Data Supplement Figure VIA and Table II). In comparison with those in control mice, hyperglycemia reduced the levels of GCH1 mRNA and protein (Figure 7A), and the BH4 content (Figure 7B) in aortic arteries, which were normalized by the miR-133a antagomir. Functional analysis indicated that hyperglycemia impaired the ACh-induced EDR, decreased serum NO levels and superoxide dismutase activities, and increased serum malondialdehyde levels in mice infused with the control antagomir, as well (Figure 7C through 7F). Importantly, the miR-133a antagomir rescued these abnormal phenotypes in diabetic mice. These data suggest that antagonism of miR-133a upregulates GCH1 expression and rescues endothelial function in diabetes mellitus.

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