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Vishal Shinde, Elyse Blank, David M Wu; VEGFR1 localization in mouse RPE.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):3040 – F0411.
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© ARVO (1962-2015); The Authors (2016-present)
VEGF plays a central role in the physiology of the eye as well as the pathology of AMD. Receptors for VEGF exist in the RPE, but their exact role in the physiology of the RPE is not well characterized. VEGFR1 is a member of the VEGF receptor family that can have contradictory effects on VEGF signaling. It can negatively regulate VEGF-mediated angiogenesis through sequestration of VEGF. At the same time, although it possesses weaker tyrosine kinase activity than VEGFR2, signaling through VEGFR1 has been found to be important in the mediation of inflammation. As inflammation of the RPE is a key component of AMD pathogenesis, we elected to more closely examine the localization of VEGFR1 in the RPE.
VEGFR1 expression was assessed in RPE cells of adult c57/bl6, cd1, and rd10 mice by immunostaining of RPE flat mounts and cryosections with two different antibodies against VEGFR1. In addition, we examined VEGFR1 expression in primary RPE cell culture from c57/bl6 mice retina. In some cases, we treated the RPE cells with VEGFR1 siRNA or shRNA to further validate VEGFR1 expression.
We found VEGFR1 staining in c57/bl6 and cd1 RPE flat mounts across 3 time points (P30, P50, P70). In contrast, it was significantly reduced in rd10 RPE flat mounts checked at 3 time points (P30, P100 and P750). VEGFR1 staining could be cytoplasmic or nuclear. This was further examined in cryosections of wild type mouse eyes, where nuclear staining co-localized with immunostaining for Otx2, a transcription factor found in RPE nuclei. A blocking peptide eliminated this staining. RPE cells freshly isolated from c57/bl6 mice in primary culture also showed staining for VEGFR1 in the nucleus and cytoplasm, and these levels could be reduced after transfection of either VEGFR1 siRNA or a VEGFR1 miR30 shRNA construct. A scrambled shRNA construct that does not target VEGFR1 RNA did not show a reduction of VEGFR1.
VEGFR1 is expressed in the RPE of wild type mice, and can be found in both the nucleus and the cytoplasm. VEGFR1 is significantly reduced in the rd10 mouse model of retinal degeneration. The interesting pattern of VEGFR1 localization suggests the value of studying the role of VEGFR1 beyond its role in VEGF sequestration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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