Intraocular VEGF Deprivation Induces Inflammatory and Fibrogenic Response in Retina

Abstract

Backgrounds: Anti-VEGF reagents have become the first-line treatment to patients with intraocular neovascularization, although accumulative clinical data point to strong association between VEGF deprivation and adverse effects in ocular system, the underlying mechanisms remain to be explored. Methods: We employed recombinant adeno-associated virus encoding soluble Fms-related tyrosine kinase-1 (rAAV.sFLT-1) for sustained VEGF neutralization. The functional effects of sFLT-1 were confirmed both in vitro and in vivo. The retinal tissues and functions were analyzed by fundus photography, optical coherence tomography (OCT), TUNEL assay, immunofluorescence staining, fluorescence in situ hybridization (FISH) and electroretinography. Moreover, high throughput RNA-seq analysis was performed to interrogate gene expression profiles of the retina. Findings: In vitro, sFLT-1 was co-precipitated with VEGF165. rAAV-mediated expression of sFLT-1 was maintained for at least three months post-intravitreal injection in mice. rAAV.sFLT-1 treatment effectively suppressed laser-induced choroidal neovascularization, an indication of VEGF deprivation. Furthermore, we showed that intraocular deprivation of VEGF induced retinal degeneration and gliosis. Functional deficit in retinal response to visual stimulation was also recorded by electroretinogram in rAAV.sFLT-1 treated eyes. Moreover, RNA-seq analysis of the VEGF-deprived retina revealed 5356 differentially expressed genes, which suggests potential mechanisms for VEGF deprivation induced retinal pathogenesis. Interpretation: Our findings demonstrate that sustained intraocular VEGF deprivation induced substantial molecular changes, which paves way to elucidation of pathogenic mechanisms for adverse effects associated with VEGF deprivation in clinical practice and identification of potential therapeutic targets. Funding: National Natural Science Foundation of China (No. 817008280; 81470640), National Key R&D Program of China (No. 2017YFA0105300), Program for Eastern Young Scholar at Shanghai Institutions of Higher Learning (No. QD2016003), Shanghai Rising-Star Program (No. 17QA1402800), Science and Technology Commission of Shanghai Municipality (No. 16411952900, 16dz2251500, 17140902800). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: Animal procedures were approved by the Shanghai Jiao Tong University Institutional Review Board. All animal experiments conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.