Increased Shedding of Endothelial Microparticles Following Anti-VEGF Therapy of Human Proliferative Diabetic Retinopathy

Abstract

Purpose: : Development of retinal neovascularization in proliferative diabetic retinopathy (PDR) is correlated to vitreous VEGF levels. Intravitreal administration of Bevacizumab, a humanized recombinant antibody that binds all isoforms of VEGF, causes at least short-term involution of retinal neovascularization. We hypothesized that endothelial microparticles (MP), which are submicron membrane vesicles released following endothelial cell activation or apoptosis, accumulate in vitreous fluid from patients with PDR following anti-VEGF therapy.

Methods: : Undiluted vitreous fluid samples were collected at the start of standard surgery for the treatment of retinal disease in diabetic (D, n=14, 61+/-3yrs, 7.5+/-0.2% HbA1c) and non-diabetic (ND, with macular hole, epiretinal membrane or retinal detachment; n=15, 65+/-4yrs) patients. Five patients with PDR received intravitreal injection of Bevacizumab (50µL; 25µg/µL) one week before surgery. Levels and cellular origins of MP in vitreous fluid were analysed by flow cytometry, using markers for platelet (CD41), endothelial (CD144), microglial (Bandeiraea Simplicifolia Lectin; ILB4) and photoreceptor (Arachis hypogaea Lectin; PNA) cells.

Results: : Vitreous levels of endothelial and platelet MPs were markedly increased in PDR when compared to ND patients (139+/-53 vs 21+/-5 CD41+MP/µl (p=0.02); 238+/-61 vs. 62+/-12 CD144+MP/µl (p=0.004); respectively). Levels of MPs of microglial or photoreceptor origin did not differ significantly in D and ND vitreous samples (89+/-51 vs. 17+/-6 PNA+MP/µl (p=0.234); 47+/-25 vs. 20+/-13 ILB4+MP/µl (p=0.32); respectively). Intravitreal injection of anti-VEGF antibody led to an eight fold increase in endothelial MPs shedding (1980+/-602 CD144+MP/µl) and a complete disappearance of platelet-derived CD41+MPs in PDR vitreous samples. Anti-VEGF treatment also reduced microglial ILB4+MPs (3+/-3 MP/µl; p=0.16) levels.

Conclusions: : Microparticle identification in vitreous samples indicates that local anti-VEGF therapy induces massive vascular endothelial cell apoptosis. In addition, augmented platelet MPs levels in diabetic vitreous samples suggests that PDR is associated with an increased endothelial permeability, which is restored to basal level by anti-VEGF therapy.

Keywords: diabetic retinopathy • vascular endothelial growth factor • drug toxicity/drug effects