Abstract
Purpose :
Soluble epoxide hydrolase (sEH), encoded by Ephx2, metabolizes epoxy fatty acids into diols. We identified sEH as a target of a novel antiangiogenic small molecule, SH-11037, and showed that intravitreal chemical inhibition of sEH suppresses choroidal neovascularization (CNV). Other studies reported that systemic knockout or inhibition of sEH also reduces CNV. However, ocular tissue specific knockdown of sEH and gene therapy approaches to target sEH to treat CNV have not been investigated to date. Here, we examined antiangiogenic effects of sEH knockdown using an adeno-associated virus (AAV) serotype 8 vector expressing shRNA against sEH, delivered intravitreally in the mouse laser-induced CNV model.
Methods :
AAV8 vectors (0.5 µl of 1x1011 gc/µl) with CMV promoter-driven Ephx2 shRNA, scrambled control shRNA (each with mCherry reporter) or 0.5 µl of vehicle (PBS) were injected intravitreally in 7 week old C57BL/6J male mice. After 7 days, mice were treated with laser to induce CNV. Transduction level (mCherry fluorescence) and neovascular volume were assessed by weekly fundoscopy and optical coherence tomography. On day 14 post laser, retina and choroid tissue of each treatment group were harvested for choroidal flatmounts, cryosectioning, protein, and RNA analyses. Expression of sEH and relevant targets in treated retina and choroid tissue was assessed by qPCR and immunoblot. CNV lesion volume was calculated using OCT measurements and by confocal Z-stack imaging of choroidal flatmounts stained with isolectin GS-IB4.
Results :
Fluorescence fundoscopy showed efficient viral transduction and microscopic assessment revealed transduced cells localized in the photoreceptors and RPE. sEH expression was reduced in retina and choroid at protein and mRNA levels. Compared to vehicle and AAV8-scrambled shRNA controls, AAV8-Ephx2 shRNA delivery significantly reduced CNV. In addition, gene expression analysis showed reduced Vegfc and inflammatory markers Tnfα, Il1β, and Vcam1 but increased cytoprotective Hmox1 in the AAV8-Ephx2 shRNA treatment group compared to scrambled control.
Conclusions :
Tissue specific knockdown of sEH in the eye inhibits CNV. Thus, depletion of sEH phenocopies the antiangiogenic effects seen with small molecule inhibitors, further demonstrating sEH as a promising therapeutic target in the treatment of CNV associated with wet AMD.
This is a 2020 ARVO Annual Meeting abstract.