Limits...
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

Inhibition of GCH1 or eNOS bypasses the protective effects of lovastatin on endothelial dysfunction ex vivo. (A) Isolated mice aortic rings were incubated with lovastatin (10 μmol/L), ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 6 hours. ACh-induced EDR in aortic rings was assayed using an organ chamber. n=5 per group. *P<0.05 versus control. (B through D) Aortic rings isolated from mice were preincubated with L-NAME (1 mmol/L) or DAHP (10 mmol/L) for 30 minutes and then exposed to lovastatin (10 μmol/L) for 6 hours in the presence of ox-LDL in B, IL-6 in C, or TNFα in D. Vasorelaxation was elicited by ACh in an organ bath. n=5 per group. *P<0.05 versus ox-LDL, IL-6, or TNFα alone. #P<0.05 versus lovastatin treatment. Ach indicates acetylcholine; DAHP, 3-deoxy-d-arabinoheptulosonate 7-phosphate; EDR, endothelium-dependent vasorelaxation; GCH1, GTP cyclohydrolase 1; IL-6, interleukin 6; L-NAME, NG-nitro-l-arginine methyl ester; ox-LDL, oxidized low-density lipoprotein; and TNFα, tumor necrosis factor-α.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120771&req=5

Figure 4: Inhibition of GCH1 or eNOS bypasses the protective effects of lovastatin on endothelial dysfunction ex vivo. (A) Isolated mice aortic rings were incubated with lovastatin (10 μmol/L), ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 6 hours. ACh-induced EDR in aortic rings was assayed using an organ chamber. n=5 per group. *P<0.05 versus control. (B through D) Aortic rings isolated from mice were preincubated with L-NAME (1 mmol/L) or DAHP (10 mmol/L) for 30 minutes and then exposed to lovastatin (10 μmol/L) for 6 hours in the presence of ox-LDL in B, IL-6 in C, or TNFα in D. Vasorelaxation was elicited by ACh in an organ bath. n=5 per group. *P<0.05 versus ox-LDL, IL-6, or TNFα alone. #P<0.05 versus lovastatin treatment. Ach indicates acetylcholine; DAHP, 3-deoxy-d-arabinoheptulosonate 7-phosphate; EDR, endothelium-dependent vasorelaxation; GCH1, GTP cyclohydrolase 1; IL-6, interleukin 6; L-NAME, NG-nitro-l-arginine methyl ester; ox-LDL, oxidized low-density lipoprotein; and TNFα, tumor necrosis factor-α.

Mentions: We have reported that uncoupled eNOS contributes to endothelial dysfunction, which is defined by impairment of ACh-induced EDR.2,3,26 To determine whether lovastatin prevents endothelial dysfunction via eNOS recoupling, an ex vivo model of endothelial dysfunction was generated by incubating isolated descending aortic rings from mice with ox-LDL, IL-6, and TNFα (Figure 4A). Treatment with lovastatin alone did not alter the ACh-induced EDR in aortic arteries. Preincubation of aortic rings with lovastatin bypassed the detrimental effects induced by ox-LDL, IL-6, and TNFα (Figure 4B through 4D). Importantly, the addition of either the eNOS inhibitor NG-nitro-l-arginine methyl ester or the GCH1 inhibitor 3-deoxy-d-arabinoheptulosonate 7-phosphate abolished the protective effects of lovastatin on improving ACh-induced EDR. Moreover, ox-LDL, IL-6, TNFα, lovastatin, NG-nitro-l-arginine methyl ester, and 3-deoxy-d-arabinoheptulosonate 7-phosphate did not alter single-nucleotide polymorphism–induced endothelium-independent relaxation in mice aortic rings (online-only Data Supplement Figure IVA through IVD). These data reveal that lovastatin prevents endothelial dysfunction via GCH1/eNOS signaling ex vivo.


Inhibition of Aberrant MicroRNA-133a Expression in Endothelial Cells by Statin Prevents Endothelial Dysfunction by Targeting GTP Cyclohydrolase 1 in Vivo
Inhibition of GCH1 or eNOS bypasses the protective effects of lovastatin on endothelial dysfunction ex vivo. (A) Isolated mice aortic rings were incubated with lovastatin (10 μmol/L), ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 6 hours. ACh-induced EDR in aortic rings was assayed using an organ chamber. n=5 per group. *P<0.05 versus control. (B through D) Aortic rings isolated from mice were preincubated with L-NAME (1 mmol/L) or DAHP (10 mmol/L) for 30 minutes and then exposed to lovastatin (10 μmol/L) for 6 hours in the presence of ox-LDL in B, IL-6 in C, or TNFα in D. Vasorelaxation was elicited by ACh in an organ bath. n=5 per group. *P<0.05 versus ox-LDL, IL-6, or TNFα alone. #P<0.05 versus lovastatin treatment. Ach indicates acetylcholine; DAHP, 3-deoxy-d-arabinoheptulosonate 7-phosphate; EDR, endothelium-dependent vasorelaxation; GCH1, GTP cyclohydrolase 1; IL-6, interleukin 6; L-NAME, NG-nitro-l-arginine methyl ester; ox-LDL, oxidized low-density lipoprotein; and TNFα, tumor necrosis factor-α.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Inhibition of GCH1 or eNOS bypasses the protective effects of lovastatin on endothelial dysfunction ex vivo. (A) Isolated mice aortic rings were incubated with lovastatin (10 μmol/L), ox-LDL (100 μg/mL), IL-6 (10 ng/mL), or TNFα (50 ng/mL) for 6 hours. ACh-induced EDR in aortic rings was assayed using an organ chamber. n=5 per group. *P<0.05 versus control. (B through D) Aortic rings isolated from mice were preincubated with L-NAME (1 mmol/L) or DAHP (10 mmol/L) for 30 minutes and then exposed to lovastatin (10 μmol/L) for 6 hours in the presence of ox-LDL in B, IL-6 in C, or TNFα in D. Vasorelaxation was elicited by ACh in an organ bath. n=5 per group. *P<0.05 versus ox-LDL, IL-6, or TNFα alone. #P<0.05 versus lovastatin treatment. Ach indicates acetylcholine; DAHP, 3-deoxy-d-arabinoheptulosonate 7-phosphate; EDR, endothelium-dependent vasorelaxation; GCH1, GTP cyclohydrolase 1; IL-6, interleukin 6; L-NAME, NG-nitro-l-arginine methyl ester; ox-LDL, oxidized low-density lipoprotein; and TNFα, tumor necrosis factor-α.
Mentions: We have reported that uncoupled eNOS contributes to endothelial dysfunction, which is defined by impairment of ACh-induced EDR.2,3,26 To determine whether lovastatin prevents endothelial dysfunction via eNOS recoupling, an ex vivo model of endothelial dysfunction was generated by incubating isolated descending aortic rings from mice with ox-LDL, IL-6, and TNFα (Figure 4A). Treatment with lovastatin alone did not alter the ACh-induced EDR in aortic arteries. Preincubation of aortic rings with lovastatin bypassed the detrimental effects induced by ox-LDL, IL-6, and TNFα (Figure 4B through 4D). Importantly, the addition of either the eNOS inhibitor NG-nitro-l-arginine methyl ester or the GCH1 inhibitor 3-deoxy-d-arabinoheptulosonate 7-phosphate abolished the protective effects of lovastatin on improving ACh-induced EDR. Moreover, ox-LDL, IL-6, TNFα, lovastatin, NG-nitro-l-arginine methyl ester, and 3-deoxy-d-arabinoheptulosonate 7-phosphate did not alter single-nucleotide polymorphism–induced endothelium-independent relaxation in mice aortic rings (online-only Data Supplement Figure IVA through IVD). These data reveal that lovastatin prevents endothelial dysfunction via GCH1/eNOS signaling ex 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