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S. E. Yanni, J. C. Latham, R. M. Caprioli, J. S. Penn; Monitoring the Abundance (in vitro) and Spatial Localization (ex vivo) of Angiogenic Factors in the Rat Model of Retinopathy of Prematurity by Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2952.
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© ARVO (1962-2015); The Authors (2016-present)
Of the growth factors involved in retinal neovascularization (NV), VEGF is recognized as the principal mediator. Alternative splicing results in several VEGF splice variants. Our knowledge of the relative expression of the different VEGF splice variants under normal and pathological conditions is limited. The recent emergence of imaging mass spectrometry (IMS) allows one to simultaneously map the spatial distribution and relative abundance of numerous biomolecules directly from, and in, tissue. In this study, we used this technology to assay splice variant-specific VEGF production by retinal astrocytes and Müller cells (in vitro) and their spatial localization ex vivo in the rat model of oxygen-induced retinopathy (OIR).
Rat retinal astrocytes and Müller cells were subjected to normoxia or hypoxia for 24 hrs. Cell media and lysates were analyzed for the presence of VEGF splice variants (VEGF188, 164, and 120) using MS. After subjecting rats to OIR, eyes were enucleated and retinas dissected. Fixed retinas were flat-mounted, embedded, and sectioned. IMS images were acquired, mapping the distribution of VEGF splice variants within the retina.
Cell lysate from hypoxia-exposed astrocytes demonstrated a 400% increase in VEGF188 production, while medium from hypoxia-exposed Müller cells exhibited a 10-fold increase in VEGF164. Ex vivo flat-mount images acquired by IMS also provide evidence of distinct localization of these two VEGF splice variants in retinal tissues.
For the first time, we have monitored the retinal production of specific VEGF splice variants at the protein level in vitro and ex vivo. In vitro, hypoxia-exposed astrocytes and Müller cells preferentially expressed different VEGF splice variants. The locations of these cell types within the retina, and the IMS evidence of distinct VEGF splice variant localization ex vivo, suggests complex signaling pathways associating stimuli with both physiological and pathological cellular responses.
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