Critical role of vascular peroxidase 1 in regulating endothelial nitric oxide synthase

Figures

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  Fig. 1. Effects of AngⅡ on VPO1 and eNOS in HUVECs. (A–D): AngⅡ treatment up-regulated protein and mRNA of expression of VPO1 in a time and dose dependent manner (**P<0.01) vs control group. (E) AngⅡ (100 nM) treatment inhibited cGMP production. (*P<0.05) vs control group. (F–G) incubation of HUVECs with Ang (100 nM) for 24 h decreased eNOS expression and activity. (**P<0.01) vs control group. (n=3 per group).

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  Fig. 2. VPO1 regulates eNOS expression through PRMT1/ADMA pathway. HUVECs were treated with AngⅡ for 24 h after transfected with siVPO1. (A) VPO1 gene knockdown reversed AngⅡ induced decrease of cGMP generation. (B–C) the eNOS protein and mRNA expression. (D–E) the PRMT1 protein and mRNA expression. (F) AMDA was measured by HPLC(n=3 per group) (**P<0.01) vs control group. (##P<0.01) vs AngⅡ group, (n=3 per group).

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  Fig. 3. The effect of VPO1 on eNOS activity in AngⅡ treated HUVECs. (A) eNOS activity was measured by Flow CytoMetry. VPO1-siRNA attenuated the decreased eNOS activity induced by AngⅡ. (B) The eNOS dimer and monomer were measured by low-temperature SDS-PAGE. (C) The level of p-eNOS. (D) The level of intracellular H2O2. (E–F) the 3-chlorotyrosine expression assessed by western blot and immunohistochemistry. (**P<0.01) vs control group. (##P<0.01) vs AngⅡgroup. (n=3 per group).

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  Fig. 4. HOCl directly decreases eNOS expression and activity in HUVECs. HUVECs were incubated with HOCl (100 µmol/L) for 2 h, and then cultured with DMEM for 24 h. (A) cGMP production measured by ELISA. (B) eNOS activity. (C)PRMT1, total eNOS, eNOS dimer and monomer were analyzed by western blot. (D) The eNOS and PRMT1 mRNA expression. (E) ADMA production. n=3 per group (** P<0.01, * P<0.05) vs control group. (n=3 per group).

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  Fig. 5. The proposed pathway of VPO1 regulation of eNOS. AngⅡ up-regulates H2O2 production and VPO1 expression. VPO1-derived HOCl decreases eNOS expression through PRMT1/ADMA pathway and down-regulates eNOS activity via inducing eNOS uncoupling and eNOS ser1177 dephosphorylation.