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Jeffrey Sundstrom, Mariano Russo, Yuanjun Zhao; Molecular pathways altered in retinal disease are detectable in vitreous.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5488.
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© ARVO (1962-2015); The Authors (2016-present)
The pathogenesis of many retinal diseases remains poorly understood and animal models of retinal disease often fail to accurately reflect the underlying disease sate. While it is not possible to obtain retinal tissue, prior studies of the vitreous and effective intravitreal treatment suggest that the interrogation of the vitreous may reflect changes in retinal tissue at the molecular level. As such, we hypothesize that in-depth proteomic analysis of vitreous fluid will allow for the identification of molecular pathways altered in retinal disease.
Deep proteomic analysis of clinical vitreous samples was developed using LC MS/MS. Several methods and instruments were compared to optimize vitreous proteome depth.
Depletion of abundant proteins (Top14 Depletion Column, Agilent) and increased sample prefractionation are critical to obtain sufficient proteome depth. For each experiment, 800-1000 proteins were identified from each 200ul sample with an false discover rate of <0.1%. Ingenuity Pathway Analysis revealed the presence of extracellular and membrane components of numerous retinal signaling pathways. This approach was validate by application to samples of proliferative diabetic retinopathy (PDR) vitreous. As anticipated, well established pathways critical to the development of PDR were identified through molecular pathway analysis. These pathways include VEGF signaling, oxidative stress, the complement system and the kininogen pathway. Each of these pathways were noted to be elevated in PDR compared to control. In addition, pathways potentially related to the neurodegenerative component of PDR were identified. These pathways include the semaphorin pathway and ROBO signaling pathways that are involved in dendritic remodeling and axonal growth. Both of these pathways were down regulated in PDR compared to control vitreous.
Proteomic analysis of vitreous fluid is an effective tool to directly identify molecular pathways altered in retinal disease. Our data suggest that the vitreous contains proteins derived from both ocular tissue and plasma that are associated with the development of retinal disease. Identification of pathways altered in disease directly from patients will promote translational research and lead the way toward novel therapeutic approaches to retinal disease.
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