Proteomic approach to identification of novel angiogenic factors in subretinal fluid from advanced retinopathy of prematurity

Abstract

Abstract: : Purpose: Advanced retinopathy of prematurity (ROP) is characterized by retinal neovascularization leading to traction retinal detachment and formation of a retrolental fibrovascular membranes resulting in total retinal detachment and collection of proteinaceous subretinal fluid (SRF). Since the retina is the target of intense angiogenesis, we postulate that the SRF may be a good source for analysis of putative pro–angiogenic agents. Methods: SRF was collected from 10 patients with advanced ROP (stages 4 and 5) and compared with SRF from eyes with rhegmatogenous retinal detachement (RRD). SRF samples were albumin and globulin depleted, size fractionated and used in an in vitro angiogenesis assay to determine which fraction exhibited more robust pro–angiogenic activity. Three–dimensional collagen 1 and fibrinogen gels were used to assay for capillary formation by primary human dermal capillary endothelial cells in presence or absence of SRF fractions or exogenous VEGF. Capillary formation was graded using an established imaging protocol. The SRF fractions were protein equalized and separated by 2–D gel analysis. Candidate proteins will be subjected to further analysis by mass spectroscopy (MS). Results: We have previously shown that subretinal fluid from these eyes contains significant amounts of known growth factors including VEGF and HGF/SF using standard ELISA assay and PDGF A/B on SDS PAGE. Fractions of SRF from ROP induce capillary formation in an in vitro angiogenesis model and cause a modest (2–fold) increase in proliferative activity of endothelial cells. 2–D gel analysis shows that numerous proteins are differentially expressed in ROP as compared with RRD. Candidate proteins are now being subjected to further analysis using MS. Conclusions: Advanced ROP is heralded by an intense angiogenic process that leads to retinal detachment and blindness in the newborn. Characterization of the proteome from SRF in ROP and RRD would allow us to to identify and charaterize novel candidate proteins that may participate in the intense angiogenesis observed in advanced ROP.

Keywords: retinopathy of prematurity • proteomics • growth factors/growth factor receptors