Abstract
Purpose :
The plasma kallikrein kinin system has been implicated in diabetic macular edema (DME), however its downstream mediators are not fully understood. To identify factors that may contribute to BK’s effects in DME, we compared the proteome from rat retina with BK-induced edema with the proteome from DME patients. This analysis revealed an association of increased C3 with retinal thickness, which was investigated in C3 deficient (C3-/-) mice subjected to BK-induced retinal edema.
Methods :
LC-MS/MS-based proteomics was performed on retina from Sprague Dawley rats with BK (2μM)-induced retinal edema compared to PBS-injected controls. Protein changes were correlated with retinal thickening measured by optical coherence tomography (OCT) and compared with human vitreous (DME, n=10 and macular hole (MH), n=5) proteomes. Western blotting of C3 in vitreous was performed. C3-/- and wildtype (WT) mice received intravitreal (IVT) injections with BK, C3, or PBS. Retinal thickness was quantified by OCT at baseline and 24 hrs.
Results :
LC-MS/MS-based proteomics revealed that C3 was increased 4.7 fold in DME compared with MH vitreous (441 vs 94 spectral/peptide counts, p<0.001) and this increase was confirmed by western blotting (8.2 fold). C3 levels were elevated 7.8 fold (23.8 ± 7.2 vs 3.3 ± 2.8 spectral/peptide counts, p=0.027) in BK-stimulated rat retina 24hrs post IVT injection compared to control retina and C3 peptide counts correlated (R2= 0.90) with retinal thickening measured by OCT. BK-induced retinal edema measured 24hrs post IVT was reduced in C3-/- mice by 67.3% in males (thickeness change:23.5±2.7μm in WT vs 7.7±3.6μm in C3-/-, p<0.01) and 78.1% in females (21.5±2.5μm WT vs 4.7±1.6μm C3-/-, p<0.0015). VEGF (10ng/eye) induced retinal thickening 24hrs post IVT was reduced in C3-/- mice (22.5±7.4μm in WT vs 9.0 ±3.8μm in C3-/-). IVT injection of C3 (1μg/eye) increased retinal thickness by 10.6± 3.0μm compared to 6.2 ±1.1μm with vehicle alone (p=0.132).
Conclusions :
C3 levels are elevated in DME patients vitreous and increased C3 levels correlated with retinal thickness in rat retina with BK-induced edema. Mice with C3 deficiency are protected against both BK- and VEGF-induced retinal edema, however intravitreal injection of C3 alone did not significantly affect retinal thickness. These results identify C3 as a potential therapeutic target for DME.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.