VEGFR–1 and VEGFR–2 are Required for VEGF–165 Mediated Blood–Retinal–Barrier Breakdown in Rabbits

Abstract

Abstract: : Purpose: Intravitreal injection of VEGF–165 in rabbits causes retinal vasodilation and vessel tortuousity (presumably increased blood flow), breakdown of the inner blood–retinal barrier, and retinal edema. These responses are completely blocked by intravitreal triamcinolone acetonide (TAA). The current study used selective VEGFR–1 and VEGFR–2 agonists in the presence or absence of TAA to identify receptor–selective vascular effects and potential differences in VEGF receptor pathway inhibition by TAA. Methods: To compare VEGFR–selective agonists to VEGF–165 in a previously characterized model, recombinant human VEGF–165 (VEGFR–1 and VEGFR–2 agonist), VEGFR–1 selective placental growth factor (PlGF) or VEGFR–2 selective orf virus–VEGF–E, were injected intradermally (50–500 ng) five minutes after i.v. Evans blue dye (EBD) in guinea pigs. Twenty minutes later, skins were excised and assessed for EBD extravasation. In female Dutch–Belt rabbits, 500 ng VEGF–165, PlGF, VEGF–E, or a combination of PlGF and VEGF–E were injected intravitreally. Scanning ocular fluorophotometry was used to measure vitreoretinal fluorescein leakage two days after growth factor injection. Fundus photos and fluorescein angiograms were obtained either 2 or 3 days post injection for subjective scoring of vasodilation/vessel tortuousity and fluorescein leakage. In a separate study, rabbits received 1 mg TAA intravitreally or vehicle four days prior to growth factor injection.Results: In guinea pigs, 50–500 ng PlGF does not induce dermal vascular leakage. In contrast, VEGF–E dose–dependently promotes dermal extravasation (equal potency to VEGF–165). As reported previously in rabbits, 500 ng VEGF–165 causes vasodilation/vessel tortuousity and severe retinal leakage. Similar to VEGF–165, VEGFR–1 selective PlGF or VEGFR–2 selective VEGF–E alone provokes marked retinal vasodilation/vessel tortuousity. In contrast, PlGF has no effect on retinal leakage, whereas VEGF–E stimulates blood–retinal barrier breakdown by ∼50% compared to VEGF–165. The combination of PlGF and VEGF–E (500 ng each) produces robust vasodilation/tortuousity and vascular leakage that is similar to or greater than that elicited by VEGF–165. Intravitreal administration of 1 mg TAA completely blocks all responses elicited by VEGF–165, VEGF–E, or PlGF. Conclusions: In contrast to VEGFR–2 mediated dermal extravasation, both VEGFR–1 and VEGFR–2 are required for maximal breakdown of the blood–retinal barrier.

Keywords: retina • growth factors/growth factor receptors • corticosteroids