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John Sweigard, Ryoji Yanai, Kip Connor, Philipp Gaissert, Aris Thanos; The Alternative Complement System Mediates Neovascularization in Retinopathy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5575.
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© ARVO (1962-2015); The Authors (2016-present)
A defining feature in retinopathy of prematurity (ROP) is the formation of neovessels (NV) in the superficial vasculature of the ganglion cell layer. Often the NV spontaneously regress while the established vasculature is kept intact, returning the retina to a relatively normal state. The mechanisms for the selective removal of NV are not fully understood and would be of great therapeutic value.
C57BL/6 mice and alternative pathway deficient mice (fb-/-) were used in the oxygen induced retinopathy (OIR) model to mimic the effects of ROP. Retinas were collected from postnatal (P) day 14 to P25 and used for immunohistochemistry (IHC), RNA, and protein evaluation of complement activity and NV development and regression. Retinal flatmounts were used to quantitate the number of NV. Laser capture micro-dissection (LCM) was preformed to isolate NV and mature vessels to compare complement regulator expression. Cobra venom factor was used to deplete systemic complement in order to study if complement activation is controlled by the local retinal microenvironment.
Flatmounts from the OIR model show that fb-/- mice develop more NV along with delayed regression compared to control mice. Real time PCR on disease retinas isolated from P14 - P17 of the OIR model show a gradual increase in factor b expression, compared to normoxic controls, with a maximum difference at P17 (P=0.001). IHC for factor b shows expression on NV, which co-localizes with TUNEL positive cells. After depleting systemic complement using cobra venom factor no difference was observed in disease severity. LCM of NV versus established vessels analyzed by RTPCR shows that CD55 levels are decreased on NV relative to established vessels (P=0.005).
Our results indicate that the alternative arm of complement is involved in the clearance of NV in the mouse model of ROP. This is a local phenomenon where the retinal microenvironment regulates complement activity. Expression levels of CD55, a regulator of the alternative arm, are decreased on NV but maintained on mature vessels. This allows the targeting NV by complement for elimination while leaving the established vasculature intact. Our findings help in the understanding of how the retina maintains such organized vessel architecture and will potentially lead to new disease markers or novel ways for selective removal of NV.
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