Effects of Parstatin on Ocular Angiogenesis, Inflammation, and BRB Breakdown

Abstract

Purpose: : Parstatin, a 41-mer peptide which is cleaved upon activation of Proteinase-Activated Receptor 1, suppresses angiogenesis in several in vitro and in vivo models. This study investigates whether parstatin can inhibit ocular angiogenesis, inflammation, and BRB breakdown associated with VEGF. The mechanisms of inhibition by parstatin will be investigated using a variety of analogues.

Methods: : Parstatin, its analogues, and scrambled parstatin were delivered alone or with VEGF into the vitreous cavity with a microinjection apparatus and glass micropipets. C57BL6 mice were exposed to 75% O₂ at P7 for 5 days and then placed in room air for 5 days, inducing ischemic retinopathy. Resulting retinal neovascularization (NV) was stained with CD31 or GSA-lectin. Laser-induced rupture of Bruch’s membrane generated choroidal NV (CNV). Choroidal flat mounts were prepared following perfusion of FITC-dextran. Parstatin effects on corneal NV in rats were assessed 7 days after burn-induced corneal NV with a semiautomatic software developed in MATLAB 7.5 and histologically. CNV and retinal NV were quantified with Image-Pro Plus and Photoshop. Leukostasis and BRB breakdown were assessed following perfusion with FITC-ConA and with a [³H]mannitol tracer assay, respectively.

Results: : Parstatin potently inhibited CNV with an IC₅₀ of approximately 1µg and maximum inhibition of 73% at 10µg. A dose of 30µg parstatin did not provide additional benefit. A 10µg dose of the hydrophilic analog, parstatin1-26, also significantly inhibited CNV. Parstatin significantly inhibited retinal NV with a 0.5µg dose close to the IC₅₀ and a 3µg dose inhibiting retinal NV by 64%. Ten and 30µg doses appear to be toxic to the neonatal retina. Similarly, 10µg parstatin1-26 effectively inhibited CNV and retinal NV. Subconjunctival parstatin inhibited corneal NV with 200µg being most effective (54% inhibition). Scrambled parstatin had no effect on choroidal, retinal, or corneal NV. Parstatin inhibited VEGF-induced retinal leukostasis and had no pro-inflammatory activity. Preliminary results showed parstatin did not suppress VEGF-induced BRB breakdown.

Conclusions: : Parstatin and/or its hydrophobic analog, parstatin1-26, is a potent anti-angiogeneic agent of ocular angiogenesis and may have clinical potential in the treatment of angiogenesis and inflammation in ocular disorders, such as diabetic retinopathy and AMD.

Keywords: neovascularization • inflammation • retina