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Ascorbic acid prevents VEGF-induced increases in endothelial barrier permeability

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Abstract

Vascular endothelial growth factor (VEGF) increases endothelial barrier permeability, an effect that may contribute to macular edema in diabetic retinopathy. Since vitamin C, or ascorbic acid, can tighten the endothelial permeability barrier, we examined whether it could prevent the increase in permeability due to VEGF in human umbilical vein endothelial cells (HUVECs). As previously observed, VEGF increased HUVEC permeability to radiolabeled inulin within 60 min in a concentration-dependent manner. Loading the cells with increasing concentrations of ascorbate progressively prevented the leakage caused by 100 ng/ml VEGF, with a significant inhibition at 13 µM and complete inhibition at 50 µM. Loading cells with 100 µM ascorbate also decreased the basal generation of reactive oxygen species and prevented the increase caused by both 100 ng/ml VEGF. VEGF treatment decreased intracellular ascorbate by 25 %, thus linking ascorbate oxidation to its prevention of VEGF-induced barrier leakage. The latter was blocked by treating the cells with 60 µM L-NAME (but not D-NAME) as well as by 30 µM sepiapterin, a precursor of tetrahydrobiopterin that is required for proper function of endothelial nitric oxide synthase (eNOS). These findings suggest that VEGF-induced barrier leakage uncouples eNOS. Ascorbate inhibition of the VEGF effect could thus be due either to scavenging superoxide or to peroxynitrite generated by the uncoupled eNOS, or more likely to its ability to recycle tetrahydrobiopterin, thus avoiding enzyme uncoupling in the first place. Ascorbate prevention of VEGF-induced increases in endothelial permeability opens the possibility that its repletion could benefit diabetic macular edema.

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Abbreviations

D-NAME:

N ω-nitro-d-arginine methyl ester hydrochloride

eNOS:

Endothelial nitric oxide synthase

Hepes:

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid

KRH:

Krebs–Ringer Hepes

L-NAME:

N ω-nitro-l-arginine methyl ester hydrochloride

NO:

Nitric oxide

ROS/RNS:

Reactive oxygen and nitrogen species

VEGF:

Vascular endothelial growth factor

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Acknowledgments

This work was supported by the National Institutes of Health grant DK050435 and by the Cell Culture Core of the Vanderbilt Diabetes Research and Training Center (DK020593).

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Authors and Affiliations

  1. Department of Medicine, Vanderbilt University School of Medicine, 7465 Medical Research Building IV, Nashville, TN, 37232-0475, USA

    Esad Ulker, William H. Parker, Amita Raj, Zhi-chao Qu & James M. May

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  1. Esad Ulker
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  2. William H. Parker
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  3. Amita Raj
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Correspondence to James M. May.

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Ulker, E., Parker, W.H., Raj, A. et al. Ascorbic acid prevents VEGF-induced increases in endothelial barrier permeability. Mol Cell Biochem 412, 73–79 (2016). https://doi.org/10.1007/s11010-015-2609-6

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