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Ines P. Rodrigues, Cristiana F. Pires, Miguel C. Seabra, Jose S. Ramalho; Uncovering the VEGF Secretory Pathway in Retinal Pigment Epithelium: the Rab Proteins’ Role. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3024. doi: https://doi.org/.
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In the healthy eye, VEGF secretion occurs from the basolateral side of retinal pigment epithelium cells (RPE), which are in close contact with the choriocapillaries. However, VEGF secretion is impaired with aging and in retinal degenerative disorders. The Rab family of GTPases is known to control several steps of intracellular vesicular transport, but their involvement in the regulated secretion of RPE cells is not yet understood. We aim at identifying the molecular regulators underlying VEGF secretion in RPE cells.
To overexpress Rab proteins, vectors encoding their GFP-tagged version were prepared. Silencing miRNA sequences against Rab genes were designed and cloned into adenoviral and lentiviral vectors. Mouse RPE primary cultures were used as an in vitro model, and the integrity of the cultures was evaluated by immunofluorescence using specific cell markers, and by transepithelial resistance. VEGF levels in cell culture media were measured by ELISA.
Expression of tight junction proteins was assessed by confocal microscopy analysis. Transepithelial resistance of mouse RPE cells monolayers was 30-60 Ω*cm2. Overexpression of secretory pathway Rab proteins in RPE cells, such as Rab3a and Rab14 led to a two-fold increase of VEGF secretion. Screening of Rab silencing vectors led to several hits leading to deregulated VEGF secretion.
Modulation of expression of some Rab GTPases affect VEGF secretion, suggesting the involvement of these Rab-dependent secretory pathways in sustaining a healthy retina.
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