Modulation of VEGF-induced retinal vascular permeability by peroxisome proliferator-activated receptor-β/δ

Abstract

Purpose: Vascular endothelial growth factor (VEGF)-induced retinal vascular permeability contributes to diabetic macular edema (DME), a serious vision-threatening condition. Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) antagonist/reverse agonist, GSK0660, inhibits VEGF-induced human retinal microvascular endothelial cell (HRMEC) proliferation, tubulogenesis, and oxygen-induced retinal vasculopathy in newborn rats. These VEGF-induced HRMEC behaviors and VEGF-induced disruption of endothelial cell junctional complexes may well share molecular signaling events. Thus, we sought to examine the role of PPARβ/δ in VEGF-induced retinal hyperpermeability.

Methods: Transendothelial electrical resistance (TEER) measurements were performed on HRMEC monolayers to assess permeability. Claudin-1/Claudin-5 localization in HRMEC monolayers was determined by immunocytochemistry. Extracellular signal-regulated protein kinases 1 and 2 (Erk 1/2) phosphorylation, VEGF receptor 1 (VEGFR1) and R2 were assayed by Western blot analysis. Expression of VEGFR1 and R2 was measured by quantitative RT-PCR. Last, retinal vascular permeability was assayed in vivo by Evans blue extravasation.

Results: Human retinal microvascular endothelial cell monolayers treated with VEGF for 24 hours showed decreased TEER values that were completely reversed by the highest concentration of GSK0660 (10 μM) and PPARβ/δ-directed siRNA (20 μM). In HRMEC treated with VEGF, GSK0660 stabilized tight-junctions as evidenced by Claudin-1 staining, reduced phosphorylation of Erk1/2, and reduced VEGFR1/2 expression. Peroxisome proliferator-activated receptor β/δ siRNA had a similar effect on VEGFR expression and Claudin-1, supporting the specificity of GSK0660 in our experiments. Last, GSK0660 significantly inhibited VEGF-induced retinal vascular permeability and reduced retinal VEGFR1and R2 levels in C57BL/6 mice.

Conclusions: These data suggest a protective effect for PPARβ/δ antagonism against VEGF-induced vascular permeability, possibly through reduced VEGFR expression. Therefore, antagonism/reverse agonism of PPARβ/δ siRNA may represent a novel therapeutic methodology against retinal hyperpermeability and is worthy of future investigation.

Keywords: vascular permeability, PPARβ; δ, VEGF.

Figure 1

Both GSK0660 and PPARβ/δ-directed siRNA inhibit VEGF-induced permeability of HRMEC monolayers. (A) Human retinal microvascular endothelial cells were treated with vehicle (0.1% BSA), 25, 50, or 75 ng/mL VEGF for 24 hours, resulting in dose-dependent decreases in electrical resistance of HRMEC monolayers at 8, 12, and 24 hours; ‡P < 0.0001. (B) The GSK0660 completely blocked VEGF-induced decreases in electrical resistance of HRMECs (vehicle versus VEGF [75 ng/mL]; ‡P < 0.0001). There was no significant difference between monolayers treated with VEGF (75 ng/mL) + 10 μM GSK0660 and control monolayers not treated with VEGF. (C) Peroxisome proliferator-activated receptor β/δ–directed siRNA blocked VEGF-induced decreases in the electrical resistance in HRMEC monolayers. Vascular endothelial growth factor decreased the electrical resistance in HRMEC monolayers transfected (vehicle with negative control siRNA versus VEGF with negative control siRNA [75 ng/mL]; ‡P < 0.0001). There were no significant decreases in electrical resistance between monolayers transfected with PPARβ/δ-directed siRNA + VEGF (75 ng/mL) and controls not treated with VEGF.

Figure 2

Plasma membrane localization of Claudin-1 is lost after VEGF treatment (24 hours), and GSK0660 (24 hours) restores this localization. Human retinal microvascular endothelial cells were cultured in (A) 10% FBS medium, (B) 75 ng/mL VEGF, or (C) 75 ng/mL VEGF plus 10 μM GSK0660.

Figure 3

Plasma membrane localization of Claudin-1, but not Claudin-5, is lost after VEGF treatment (24 hours), and PPARβ/δ-directed siRNA restores membrane localization. Human retinal microvascular endothelial cells were cultured in (A) 10% FBS medium plus 20 μM negative control oligomer, (B) 20 μM negative control oligomer plus 75 ng/mL VEGF, or (C) 20 μM PPARβ/δ-directed siRNA plus 75 ng/mL VEGF. Top: Claudin-1, bottom: Claudin-5.

Figure 4

Both GSK0660 and PPARβ/δ-directed siRNA inhibit activation of VEGF signaling. Vascular endothelial growth factor (75 ng/mL) significantly upregulated phosphorylation of Erk 1/2 (p-Erk 1/2) when compared with control conditions. Treatment with (A) 10 μM GSK0660 or (C) 20 μM PPARβ/δ-directed siRNA significantly reduced VEGF-induced p-Erk 1/2. *P < 0.05; †P < 0.01. Quantification of three independent experiments is shown in (B, D).

Figure 5

GSK0660 or PPARβ/δ-directed siRNA inhibit constitutive expression of VEGFR1 and VEGFR2 in HRMECs. Human retinal microvascular endothelial cells were cultured in 10% FBS plus (A) 10 μM GSK0660 for 6 hours or (B) 20 μM negative control siRNA oligomers or 20 μM PPARβ/δ-directed siRNA for 24 hours. Expression of VEGFR1 and VEGFR2 was measured using qRT-PCR. *P < 0.05; †P < 0.01.

Figure 6

GSK0660 inhibits VEGF-induced retinal vascular permeability. Intraocular injection of 50 ng/mL VEGF significantly increased Evans blue extravasation when compared with vehicle injection. Treatment with 10 μM GSK0660 reduced VEGF-induced retinal extravasation. Permeability was analyzed via Evans blue extravasation after 24 hours of 50 ng/mL VEGF and/or 10 μM GSK0660 injections. Data are normalized to the vehicle group. **P < 0.001.

Figure 7

GSK0660 reduces retinal VEGFR1 and R2 levels. Mice were injected with 50 ng/mL VEGF and/or 10 μM GSK0660. Retinal levels of VEGFR1 (A) and VEGFR2 (C) were analyzed 24 hours later by Western blot analysis. Bar graphs represent quantification of at least two independent experiments (B, D). *P < 0.05.