Expression of angiogenic and inflammatory factors in CNV-derived RPE

Purpose: Anti-angiogenic treatment is well established in the management of exudative AMD, but not sufficient in all patients. Characterization of factors driving this chronic disease could serve to identify additional treatment options. The purpose of this study was to assess gene expression patterns and distinct changes in cells derived from surgically extracted CNV (chorioidal neovascularization) membranes. Materials and Methods: Expression of >11.000 genes was analyzed by means of a microarray in cells cultured from two late-stage CNV membranes, compared to primary human RPE and ARPE-19 cells. A pathway analysis was performed to identify gene expression patterns associated with exudative AMD. Results: The analysis revealed significant alterations in gene sets associated with inflammatory processes in CNV-derived cells, involving up-regulation of pro-inflammatory factors IL6, C3, and C5 and downregulation of anti-inflammatory CFB and CFI. Factors associated with angiogenesis, such as VEGFA or ANGPT2, were not significantly regulated in the two RPE-derived cell lines. Conclusion: In late-stage CNV membrane-derived RPE, gene expression was shifted towards a pro-inflammatory state. Angiogenesis-associated factors were regulated differently in the two CNV-derived RPE membranes. While inflammation seems to be continuously stimulated by RPE associated with late exudative AMD, this seems not the case with regard to angioregulatory mechanisms. The surgical extraction of CNV membranes was a therapeutical option for patients with advanced neovascular AMD before the availability of anti-VEGF agents for intravitreal injection. Patients underwent a standard 20 gauge vitrectomy after signing informed consent. CNV membranes were removed from the subretinal space after a retinotomy. The retinotomy was secured by a laser retinopexy and gas endotamponade. The extracted CNV membranes were cultured in DMEM / Ham’s F-12 medium and characterized for RPE features, such as expression of RPE markers cytokeratine 18, vimentin, CRALBP and RPE65, as well as absence of glial fibrillary acidic protein (GFAP) or alpha smooth muscle actin (SMA). hRPE cells were obtained from ScienCell Research Laboratories (HRPEpiC, Cat. No 6540, Lot No. 0562, Carlsbad, CA, USA) at passage one. ARPE-19 is a widely used immortalized cell line obtained from ATCC (#CRL-2302).