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Hyewon Chung, Ae Jin Choi, Eun Sook Lee, Gum-Yong Kang, Soyoung Choi, Hyunjung Lim; Exosomal Proteins in the Aqueous Humor as Novel Biomarkers in Patients with Neovascular Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3654.
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Understanding molecular and proteomic changes of retinal pigment epithelium (RPE) during the disease process of age-related macular degeneration (AMD) is important since failure or decrease of RPE adaptive mechanism to aging or stress could be the major pathogenesis of developing AMD. We discovered and validated the secretory proteins in the aqueous humor (AH) from patients with neovascular AMD. We speculate that secretory proteins from the RPE, possibly in the form of secretory vesicles like exosomes, could be identified in the AH of patients.
We adopted a comparative proteomic approach to find novel biomarkers for AMD. We performed the whole proteomic profiling of AH of patients, conditioned media (CM) from ARPE-19 cells exposed to 400 µM paraquat 24 h and exosomes derived from AH and CM by LC-MS/MS. Exosome pellets from AH and CM were obtained with ExoQuickTM Exosome Precipitation Solution. To confirm exosome and exosomal proteins, transmission electron microscopy (TEM) and Western blot analysis for exosomal marker proteins (CD63 and Hsp70) were performed. Finally, liquid chromatography multiple reaction monitoring (LC-MRM) analysis of AH of 15 treatment-naïve patients with neovascular AMD and control subjects were performed to validate the target proteins.
TEM revealed that exosomes from the AH of patients and CM from ARPE-19 cells as round shaped membrane vesicles sized 50-100 nm in diameter. CD63 and Hsp70 in exosomes from AH and CM were detected by Western blot analysis. Among 70 proteins identified both in AH and CM, six proteins including cathepsin D and 26S proteasome non-ATPase were found in exosomes of AH. Seven proteins including cytokeratin 8 and Hsp70 were found in exosomes of CM. Two proteins were detected in exosomes from both. In total, 11 exosomal proteins were increased in AH of 15 patients with various levels of decrease after intravitreal injection of ranibizumab by LC-MRM.
The present study has identified potential biomarkers and therapeutic target proteins of AMD and provided an effective approach to identify AMD-associated proteins that are secreted by RPE cells in vivo. In addition, we provide new evidence that the secretory mechanisms of these proteins might be closely related to exocytic activity as in the formation of drusens which are considered a risk factor for developing AMD.
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