The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4

Abstract

It is well established that 4-hydroxynonenal (HNE) plays a major role in oxidative stress-induced signaling and the toxicity of oxidants. Surprisingly our recent studies also demonstrate that low levels of HNE generated during oxidative stress promote cell survival mechanisms and proliferation. Since the expression and secretion of VEGF is known to be affected by Oxidative stress, during present studies, we have examined dose dependent effect of HNE on VEGF expression and secretion in a model of retinal pigment epithelial (RPE) cells in culture. Results of these studies showed that while inclusion of 0.1 μM HNE in the medium caused increased secretion of VEGF, its secretion and expression was significantly suppressed in the presence of >5 μM HNE in the media. These concentration dependent hormetic effects of HNE on VEGF secretion could be blocked by the over expression of GSTA4-4 indicating that these effects were specifically attributed to HNE and regulated by GSTA4-4. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVEC in matrigel when grown in media from RPE cells treated with 1 μM HNE. The corresponding media from GSTA4-4 over expressing RPE cells had no effect on migration and tube formation of HUVEC in matrigel. These results are consistent with earlier studies showing that at low concentrations, HNE promotes proliferative mechanisms and suggest that HNE induces VEGF secretion from RPE cells that acts in a paracrine fashion to induce angiogenic signaling mechanism in the endothelial cells. These findings may suggest a role of HNE and GSTA4-4 in oxidative stress induced proliferative retinopathies.

Figure 1. Effect of HNE toxicity in RPE cells

(A) RPE cells were treated with 0–70 μM of HNE for 12 h and assayed for cytotoxicity by MTT and LDH assay. (B) RPE cells were transiently transfected with empty p-Target vector (VT) and p-Target vector containing the ORF of hGSTA4 sequence (hGSTA4-Tr). VT and hGSTA4-Tr cells were treated with 0–70 μM of HNE for 12 h and assayed for cytotoxicity by MTT assay. The plots show the percent cell survival (mean ± SD, n = 4) at different concentrations of HNE. (C) Over-expression of hGSTA4-4 in transfected cells was confirmed by western blot analysis.

Figure 2. Effect of HNE on VEGF secretion and VEGFR-2 expression in RPE cells

(A) Effect of HNE on VEGF secretion: RPE cells were transiently transfected with empty p-Target vector (VT) and p-Target vector containing the ORF of hGSTA4 sequence (hGSTA4-Tr). VT and hGSTA4-Tr cells were treated with 0–20 μM of HNE for 12 h and assayed for VEGF secretion by ELISA as mentioned in Method Section. The data represent the mean ± SD (n =3). (B) Effect of 4-HNE on VEGFR-2 expression: RPE cells were treated with 0–20 μM HNE. Cell extracts prepared as described in methods section containing 70 μg protein were subjected to Western blot analysis. Western blot analysis was performed with total cell lysates using indicated antibodies. β-actin was used to control for loading differences.

Figure 3. Effect of RPE conditioned medium on HUVEC cells in wound healing assay

HUVEC cells at confluence were injured by a scratch with a 10 μL pipette tip. Wounded cells were allowed to heal for 12 h in presence of conditioned medium from control VT and hGSTA4-Tr cells and these treated with 0–1 μM of HNE for 12 h. The photograph is representative of three experiments showing similar results. The data in bar graph represents the mean ± SD (n=3).

Figure 4. Effect of RPE conditioned medium on HUVEC cells in tube formation assay

HUVEC cells were allowed to grow on matrigel for 4 h in presence of conditioned medium from VT and hGSTA4-Tr cells treated with 0–1 μM of HNE for 12 h. The photograph is representative of three experiments showing similar results. The data in bar graph represents the mean ± SD (n=3).