Erythropoietin (EPO) overexpression induces a DME-like phenotype in the mouse retina

Abstract

Purpose : Diabetic macular edema (DME) is the leading cause of blindness in diabetic patients, with unmet medical need despite the success of anti-VEGF therapy. Intraocular, but not systemic EPO levels are elevated in diabetic patients. We characterized the effect of EPO overexpression to understand its role in retinal vascular leakage (RVL) and dilation (RVD) in the mouse retina.

Methods : We injected young mice (sub-retinal, C57Bl/6J) with either an adeno-associated virus with the Epo gene (AAV2-EPO) or a null vector (AAV2-Null) in two independent studies. AAV2-EPO was tested at 2×10⁹ (study #1 was bilateral, study #2 unilateral) or 5×10⁸ vg/eye (study #2, unilateral). We co-injected (IP) indocyanine green (ICG) and fluorescein (F) to quantify RVL at baseline, 4 and 8-weeks post-injection. The properties of ICG and F enabled us to quantify RVL by using MatLab to subtract ICG-delineated vessels from images obtained in the F channel. This analysis captured F RVL (reported as arbitrary units, AU). Two masked operators assessed RVD from randomized scanning laser ophthalmoscopy (SLO) images. Optical coherence tomography (OCT) and histology were used to further characterize the injected retinas. Epo and VEGF levels were measured with commercially available kits.

Results : Average baseline RVL was 0.028 and 0.013 AU in study #1 and 2 respectively. In study 1, RVL increased to 0.078 AU and 0.14 AU at 4 and 8 weeks post-injection respectively. Dose responsive elevation of RVL was observed in Study 2 (.039 and .048 AU, low dose and 0.106 and 0.069 AU for the high dose at weeks at 4 and 8, respectively) RVL also increased (0.076 AU) in the non-injected eye of mice unilaterally dosed with 2×10⁹ vg/eye AAV2-EPO. OCT analysis detected thickened retinas compared to baseline (213.2 µm) only in the un-injected eyes of study 2 at 4-weeks post-injection (220, p<.01 and 222 µm, p<.001 for each dose). SLO and histology identified dilated retinal and choroidal vessels. EPO concentration was elevated in eyes injected with AAV2-EPO (1623.4 and 3063 ng/mL, low and high dose respectively) compared to mice injected with AAV2-Null (42.8 ng/mL). In study #2, EPO was modestly elevated in non-injected contralateral eyes of mice injected with AAV2-EPO (64 ng/mL). In contrast, VEGF did not increase on either study.

Conclusions : EPO overexpression in mice induces RVL and RVD, features that are also present in DME.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.