Yonsei Med J.  2015 Nov;56(6):1678-1685. 10.3349/ymj.2015.56.6.1678.

Resveratrol Inhibits Hypoxia-Induced Vascular Endothelial Growth Factor Expression and Pathological Neovascularization

• Lee CS
• Choi EY
• Lee SC
• Koh HJ
• Lee JH
• Chung JH

• KMID: 2345900
• DOI: http://doi.org/10.3349/ymj.2015.56.6.1678

Abstract

PURPOSE
To investigate the effects of resveratrol on the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) in human adult retinal pigment epithelial (ARPE-19) cells, and on experimental choroidal neovascularization (CNV) in mice.
MATERIALS AND METHODS
ARPE-19 cells were treated with different concentrations of resveratrol and then incubated under hypoxic conditions with subsequent evaluation of cell viability, expression of HIF-1alpha, and expression of VEGF. The effects of resveratrol on the synthesis and degradation of hypoxia-induced HIF-1alpha were evaluated using inhibitors of the PI3K/Akt/mTOR and the ubiquitin proteasome pathways. In animal studies, CNV lesions were induced in C57BL/6 mice by laser photocoagulation. After 7 days of oral administration of resveratrol or vehicle, which began one day after CNV induction, image analysis was used to measure CNV areas on choroidal flat mounts stained with isolectin IB4.
RESULTS
In ARPE-19 cells, resveratrol significantly inhibited HIF-1alpha and VEGF in a dose-dependent manner, by blocking the PI3K/Akt/mTOR signaling pathway and by promoting proteasomal HIF-1alpha degradation. In mice experiments, orally administered resveratrol significantly inhibited CNV growth in a dose-dependent manner.
CONCLUSION
Resveratrol may have therapeutic value in the management of diseases involving pathological neovascularization.

Keyword

Choroidal neovascularization; hypoxia-inducible factor-1; resveratrol; retinal pigment epithelium; vascular endothelial growth factor

MeSH Terms

Adult
Animals
Anoxia/metabolism/physiopathology
Cell Survival/drug effects
Choroidal Neovascularization/*metabolism/pathology
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/*drug effects/metabolism
Mice
Mice, Inbred C57BL
Phosphatidylinositol 3-Kinases/antagonists & inhibitors/*physiology
Proteasome Endopeptidase Complex
Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*physiology
Retinal Pigment Epithelium/*drug effects/metabolism
Signal Transduction
Stilbenes/administration & dosage/*pharmacology
TOR Serine-Threonine Kinases/antagonists & inhibitors/*physiology
Ubiquitin
Vascular Endothelial Growth Factor A/*drug effects/metabolism
Phosphatidylinositol 3-Kinases
Proteasome Endopeptidase Complex
Proto-Oncogene Proteins c-akt
Hypoxia-Inducible Factor 1, alpha Subunit
Stilbenes
TOR Serine-Threonine Kinases
Ubiquitin
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Resveratrol inhibited choroidal neovascularization (CNV) growth in animal model. Effects of orally-administered resveratrol on lesion size in laser-induced CNV mice at Bruch membrane rupture sites. Representative photographs of choroidal flat mounts 7 days after laser photocoagulation are shown: (A) control group, (B) low dose group (1 mg/kg/day), and (C) high dose group (10 mg/kg/day). (D) Image analysis shows reduced CNV size from the resveratrol-treated mice. *p<0.001 and †p<0.0001 vs. the vehicle-treated control.

• 

  Fig. 2 Resveratrol inhibited hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) via the PI3K/Akt pathway. ARPE-19 cells in complete medium were pretreated with 10 µmol/L, 50 µmol/L, and 100 µmol/L resveratrol for 1 hour, and then exposed to normoxia or hypoxia for 16 hours. Resveratrol significantly reduced VEGF protein and mRNA levels as determined by ELISA (A) and RT-PCR (B) in a dose-dependent manner. (C) Pretreatment with resveratrol in varying concentration abrogated the hypoxia-induced HIF-1α protein accumulation in a dose-dependent manner. (D) Pretreatment with resveratrol resulted in decrease of phospho-Akt (p-Akt) and phospho-mTOR (p-mTOR) levels in a dose-dependent manner. *p<0.0001 vs. the normoxia group, †p<0.001 vs. the hypoxia group. ELISA, Enzyme-Linked Immunosorbent Assay.

• 

  Fig. 3 (A) Inhibition of the mTOR pathway using rapamycin and everolimus prevented HIF-1α induction under hypoxic conditions in ARPE-19 cells as determined by immunoblotting of HIF-1α and p-p70S6 kinase. (B) Transfection of ARPE-19 cells with human mTOR siRNA resulted in HIF-1α, phosphomTOR, mTOR, and phospho-p70S6 kinase as determined by western blot. (C) Silencing of mTOR using siRNA resulted in inhibition of hypoxia-induced vascular endothelial growth factor (VEGF) in ARPE-19 cells. *p<0.0001 vs. the control group. HIF, hypoxia-inducible factor; p-mTOR, phospho-mTOR.

• 

  Fig. 4 ARPE-19 cells were exposed to hypoxia for 4 hours, followed by treatment with cycloheximide (CHX) in the absence (A) or presence (B) of 100 µmol/L of resveratrol (RSV), for different time periods, and then analyzed by western blotting for HIF-1α protein. Hypoxia-induced HIF-1α protein accumulation gradually decreased to an undetectable level at 120 minutes in the presence of resveratrol (B). (C) Inhibition of hypoxia-induced HIF-1α protein accumulation by resveratrol was significantly blocked in the presence of MG132, a 26S proteasome inhibitor. HIF, hypoxia-inducible factor.

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