Complement factor B knockdown by short hairpin RNA inhibits laser-induced choroidal neovascularization in rats

Abstract

AIM: To evaluate whether recombinant complement factor B (CFB) short hairpin RNA (shRNA) reduces laser-induced choroidal neovascularization (CNV) in rats. METHODS: Laser-induced rat CNV model was established, and then the animals underwent fundus fluorescence angiography (FFA) and hematoxylin and eosin (HE) staining. On day 3 and 7 after photocoagulation, the expression of CFB and membrane attack complex (MAC) was detected by immunhischemistry. A recombinant CFB-shRNA plasmid was constructed. CFB and scrambled shRNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment, respectively. On day 7, the incidence of CNV was determined by FFA, and the expression of CFB and vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)/choroidal tissues was detected by immunhischemistry, Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) in CFB and scrambled shRNA groups. The possible adverse effects of CFB-shRNA injection were assessed by transmission electron microscopy and electroretinography. RESULTS: FFA and HE results indicated that a laser-induced rat CNV model was successfully established on day 7 after photocoagulation. The expression of CFB and MAC was extremely weak in normal retina and choroid, and increased on day 3 after photocoagulation. However, it started to reduce on day 7. CFB shRNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues. FFA showed CFB knockdown significantly inhibited incidence of CNV in rats. Moreover, CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues. CFB shRNA caused no obvious side effects in eyes. CONCLUSION: CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF, which is a potential therapy target. The alternative pathway of complement activation plays an important role in CNV formation.

Keywords

• choroidal neovascularization
• complement factor b
• short hairpin rna
• membrane attack complex
• vascular endothelial growth factor