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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

Cytokines/oxidants induce aberrant miR-133a expression in endothelial cells to inhibit GCH1 gene expression, which is abolished by statin. (A) MiR-133a levels were measured in cultured primary cells by RT-qPCR. EC indicates endothelial cells; VSMC, vascular smooth muscle cells; CMC, cardiomyocytes; and SC, skeletal cells from hamstrings. n=5 per group. *P<0.05 versus EC. (B) HUVECs were pretreated with lovastatin (10 μmol/L) for 30 minutes followed by coincubation with ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 24 hours. Cells were subjected to RT-qPCR for detecting miR-133a levels. n=3 per group. *P<0.05 versus Vehicle alone. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. (Cthrough E) HUVECs were infected with lentivirus harboring the scr-miR or premiR-133a for 48 hours followed by lovastatin treatment (10 μmol/L) for 24 hours. Cells were then used for detecting miR-133a levels by RT-qPCR in C, GCH1 mRNA levels by RT-PCR in D, and GCH1 protein levels by Western blotting in E. n=3 per group. *P<0.05 versus Scr-miR alone. NS indicates no significance. (F) GCH1 protein levels were detected by Western blotting analysis in cells from B. (G) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα. Cells were subjected to Western blotting analysis for GCH1 protein levels. n=3 per group. *P<0.05 versus Control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. GCH1 indicates GTP cyclohydrolase 1; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; RT-qPCR, quantitative reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.
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Figure 2: Cytokines/oxidants induce aberrant miR-133a expression in endothelial cells to inhibit GCH1 gene expression, which is abolished by statin. (A) MiR-133a levels were measured in cultured primary cells by RT-qPCR. EC indicates endothelial cells; VSMC, vascular smooth muscle cells; CMC, cardiomyocytes; and SC, skeletal cells from hamstrings. n=5 per group. *P<0.05 versus EC. (B) HUVECs were pretreated with lovastatin (10 μmol/L) for 30 minutes followed by coincubation with ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 24 hours. Cells were subjected to RT-qPCR for detecting miR-133a levels. n=3 per group. *P<0.05 versus Vehicle alone. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. (Cthrough E) HUVECs were infected with lentivirus harboring the scr-miR or premiR-133a for 48 hours followed by lovastatin treatment (10 μmol/L) for 24 hours. Cells were then used for detecting miR-133a levels by RT-qPCR in C, GCH1 mRNA levels by RT-PCR in D, and GCH1 protein levels by Western blotting in E. n=3 per group. *P<0.05 versus Scr-miR alone. NS indicates no significance. (F) GCH1 protein levels were detected by Western blotting analysis in cells from B. (G) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα. Cells were subjected to Western blotting analysis for GCH1 protein levels. n=3 per group. *P<0.05 versus Control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. GCH1 indicates GTP cyclohydrolase 1; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; RT-qPCR, quantitative reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.

Mentions: To determine the role of miR-133a/b in regulating GCH1 gene expression in endothelial cells, we examined the expression of miR-133a/b in different cells. As indicated in Figure 2A, the basal level of miR-133a in endothelial cells was particularly low, ≈1/500 to 1/100 of that in skeletal cells, cardiomyocytes, and vascular smooth muscle cells, wherein miR-133a/b was highly expressed as reported.21–23 miR-133b was undetectable in endothelial cells. These observations suggest that miR-133a/b is not required for maintaining endothelial function under physiological conditions.


Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo
Cytokines/oxidants induce aberrant miR-133a expression in endothelial cells to inhibit GCH1 gene expression, which is abolished by statin. (A) MiR-133a levels were measured in cultured primary cells by RT-qPCR. EC indicates endothelial cells; VSMC, vascular smooth muscle cells; CMC, cardiomyocytes; and SC, skeletal cells from hamstrings. n=5 per group. *P<0.05 versus EC. (B) HUVECs were pretreated with lovastatin (10 μmol/L) for 30 minutes followed by coincubation with ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 24 hours. Cells were subjected to RT-qPCR for detecting miR-133a levels. n=3 per group. *P<0.05 versus Vehicle alone. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. (Cthrough E) HUVECs were infected with lentivirus harboring the scr-miR or premiR-133a for 48 hours followed by lovastatin treatment (10 μmol/L) for 24 hours. Cells were then used for detecting miR-133a levels by RT-qPCR in C, GCH1 mRNA levels by RT-PCR in D, and GCH1 protein levels by Western blotting in E. n=3 per group. *P<0.05 versus Scr-miR alone. NS indicates no significance. (F) GCH1 protein levels were detected by Western blotting analysis in cells from B. (G) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα. Cells were subjected to Western blotting analysis for GCH1 protein levels. n=3 per group. *P<0.05 versus Control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. GCH1 indicates GTP cyclohydrolase 1; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; RT-qPCR, quantitative reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.
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Figure 2: Cytokines/oxidants induce aberrant miR-133a expression in endothelial cells to inhibit GCH1 gene expression, which is abolished by statin. (A) MiR-133a levels were measured in cultured primary cells by RT-qPCR. EC indicates endothelial cells; VSMC, vascular smooth muscle cells; CMC, cardiomyocytes; and SC, skeletal cells from hamstrings. n=5 per group. *P<0.05 versus EC. (B) HUVECs were pretreated with lovastatin (10 μmol/L) for 30 minutes followed by coincubation with ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 24 hours. Cells were subjected to RT-qPCR for detecting miR-133a levels. n=3 per group. *P<0.05 versus Vehicle alone. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. (Cthrough E) HUVECs were infected with lentivirus harboring the scr-miR or premiR-133a for 48 hours followed by lovastatin treatment (10 μmol/L) for 24 hours. Cells were then used for detecting miR-133a levels by RT-qPCR in C, GCH1 mRNA levels by RT-PCR in D, and GCH1 protein levels by Western blotting in E. n=3 per group. *P<0.05 versus Scr-miR alone. NS indicates no significance. (F) GCH1 protein levels were detected by Western blotting analysis in cells from B. (G) HUVECs were transfected with the miR-133a antagomir for 24 hours followed by coincubation with ox-LDL, IL-6, or TNFα. Cells were subjected to Western blotting analysis for GCH1 protein levels. n=3 per group. *P<0.05 versus Control. #P<0.05 versus ox-LDL, IL-6, or TNFα alone. GCH1 indicates GTP cyclohydrolase 1; HUVEC, human umbilical vein endothelial cell; IL-6, interleukin 6; miR, microRNA; ox-LDL, oxidized low-density lipoprotein; premiR-133a, preliminary miR-133a; RT-qPCR, quantitative reverse transcription polymerase chain reaction; scr-miR, scrambled miR; and TNFα, tumor necrosis factor-α.
Mentions: To determine the role of miR-133a/b in regulating GCH1 gene expression in endothelial cells, we examined the expression of miR-133a/b in different cells. As indicated in Figure 2A, the basal level of miR-133a in endothelial cells was particularly low, ≈1/500 to 1/100 of that in skeletal cells, cardiomyocytes, and vascular smooth muscle cells, wherein miR-133a/b was highly expressed as reported.21–23 miR-133b was undetectable in endothelial cells. These observations suggest that miR-133a/b is not required for maintaining endothelial function under physiological conditions.

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