Peroxisome proliferator-activated receptor-β/δ regulates angiogenic cell behaviors and oxygen-induced retinopathy

Abstract

Purpose: To develop new therapies against ocular neovascularization (NV), we tested the effect of peroxisome proliferator-activated receptor-β/δ (PPAR-β/δ) agonism and antagonism on angiogenic behaviors and in human retinal microvascular endothelial cells (HRMEC) and on preretinal NV in rat oxygen-induced retinopathy (OIR).

Methods: HRMECs were treated with the PPAR-β/δ agonist GW0742 and the antagonist GSK0660. Messenger RNA levels of a PPAR-β/δ target gene, angiopoietin-like-4 (angptl4) were assayed by qRT-PCR. HRMEC proliferation and tube formation were assayed according to standard protocols. OIR was induced in newborn rats by exposing them to alternating 24-hour episodes of 50% and 10% oxygen for 14 days. OIR rats were treated with GW0742 or GSK0660. Angptl4 protein levels were assessed by ELISA and preretinal NV was quantified by adenosine diphosphatase staining.

Results: GW0742 significantly increased angptl4 mRNA, and GSK0660 significantly decreased angptl4 mRNA. GW0742 had no effect on HRMEC proliferation, but caused a significant and dose-responsive increase in tube formation. GSK0660 significantly reduced serum-induced HRMEC proliferation and tube formation in a dose-dependent manner. Intravitreal injection of GW0742 significantly increased total retinal Angptl4 protein, but intravitreal injection of GSK0660 had no effect. Intravitreal injection of GW0742 significantly increased retinal NV, as did GW0742 administered by oral gavage. Conversely, both intravitreal injection and intraperitoneal injection of GSK0660 significantly reduced retinal NV.

Conclusions: PPAR-β/δ activation exacerbates, and its inhibition reduces, preretinal NV. PPAR-β/δ may regulate preretinal NV through a prodifferentiation/maturation mechanism that depends on Angptl4. Pharmacologic inhibition of PPAR-β/δ may provide a rational basis for therapeutic targeting of ocular NV.

Keywords: angiogenesis; nuclear transcription factor; peroxisome proliferator-activated receptor; retinopathy of prematurity; vascular endothelial growth factor.

Figure 1

The effect of GW0742 and GSK0660 on angptl4 expression in HRMEC. (A) Quantitative RT-PCR analysis of angptl4 mRNA revealed significant activation of PPAR-β/δ with GW0742 treatment. (B) GSK0660 treatment led to a significant reduction in angptl4 expression. Each bar represents the mean ± SEM.

Figure 2

The effect of GW0742 and GSK0660 on HRMEC proliferation. (A) GW0742 exhibited no effect on HRMEC proliferation. GSK0660 treatment led to a dose-dependent reduction of serum-induced proliferation. (B) GSK0660 treatment results in a dose-dependent reduction of VEGF-induced proliferation. Each bar represents the mean ± SEM.

Figure 3

The effect of GW0742 and GSK0660 on HRMEC tube formation. (A) Representative images of tube formation in HRMECs treated with SF medium with vehicle, 2% serum with vehicle, 1.0 μM GW0742 in SF media, and 1.0 μM GSK0660 in 2% serum media. (B) GW0742 treatment induced tube formation in a dose-dependent manner. HRMEC tube formation was induced by 2% serum, and this induction was significantly inhibited with increasing concentrations of GSK0660. (C) GSK0660 inhibits VEGF-induced tube formation. Each bar represents the mean ± SEM.

Figure 4

The effect of local administration of GW0742 and GSK0660 on OIR-induced NV. GW0742 and GSK0660 were intravitreally injected. GW0742 significantly increased OIR-induced NV at the lowest and highest concentrations. At all concentrations, GSK0660 significantly decreased retinal NV. Each bar represents the mean ± SEM.

Figure 5

The effect of systemic administration of GW0742 and GSK0660 on OIR-induced NV. (A) Representative images of retinal quadrants in rats treated with vehicle by oral gavage, 10 mg/kg GW0742 by oral gavage, and 1.0 mg/kg GSK0660 by intraperitoneal injection. (B) GW0742 was administered by oral gavage (1.0 and 10 mg/kg) and GSK0660 by intraperitoneal injection (0.2 and 1.0 mg/kg). GW0742 significantly increased the NV area at the highest concentration tested, whereas GSK0660 significantly decreased the NV area at both concentrations tested. Each bar represents the mean ± SEM.

Figure 6

The effect of intravitreally injected GW0742 and GSK0660 on total retinal Angptl4 production. GW0742 and GSK0660 were injected at 500 nM concentration and retinas were collected 1 day later. Following GW0742 injection, Angptl4 was significantly increased. GSK0660 had no effect on Angptl4 production in the retina. Each bar represents the mean ± SEM.