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Mol Vis. 2017 Aug 10;23:579-587. eCollection 2017.

Heparanase mediates vascular endothelial growth factor gene transcription in high-glucose human retinal microvascular endothelial cells.

Author information

1
The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.

Abstract

PURPOSE:

To observe the nuclear expression and interaction of heparanase and RNA polymerase II (RNA Pol II), an enzyme that catalyzes the transcription of DNA in eukaryotic cells) in human retinal microvascular endothelial cells (HRECs) under high glucose condition and to investigate the association of heparanase with the transcription activity of the vascular endothelial growth factor (VEGF) gene promoter.

METHODS:

Cultured HRECs were maintained for 3 days in media with high or normal glucose. The expressions of heparanase and RNA Pol II in each group were analyzed with immunofluorescence. Co-immunoprecipitation was applied to detect the interaction of heparanase and Pol II proteins. Cells in both groups were used for chromatin immunoprecipitation (ChIP) with anti-heparanase and anti-RNA Pol II antibodies to identify high-confidence heparanase-binding regions across the entire VEGF gene promoter. Moreover, real-time PCR was used to demonstrate the interaction between heparanase and the VEGF gene promoter region.

RESULTS:

The immunofluorescence studies showed that the nuclear expression of heparanase was intense in high-glucose HRECs but faint in the normal group; RNA Pol II in the nucleus was also intense in high glucose HRECs, and the distribution of heparanase was consistent with that of RNA Pol II. The co-immunoprecipitation data showed that heparanase combined with RNA Pol II in HRECs cells treated with high glucose, and the molecular size of HPA interacted with RNA Pol II was 50 kDa, while no combination of two proteins was evident in normal HRECs cells. Real-time PCR-based ChIP results showed that the high-confidence HPA-binding region was -1155 to -1018 (containing hypoxia response element) in the VEGF gene promoter, and the cells treated with high glucose showed increases in heparanase and RNA Pol II in the VEGF gene promoter region compared with the normal glucose treated cells (t = -3.244, p = 0.032; t = -6.096, p = 0.004, respectively).

CONCLUSIONS:

Nuclear heparanase combines directly with the VEGF gene promoter and is involved in the regulation of VEGF gene transcription in high-glucose HRECs.

PMID:
28848320
PMCID:
PMC5561135
[Indexed for MEDLINE]
Free PMC Article

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