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Michael R Kozlowski; Non-vascular effects of VEGF in a model of age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3455.
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The purpose of this work is to assess whether vascular endothelial growth factor (VEGF) has effects in addition to promoting blood vessel growth that could contribute to the development of age-related macular degeneration (AMD). Previous studies have found that VEGF can weaken the connections between retinal pigment epithelial (RPE) cells, in vitro. The potential relevance of this phenomenon to the development of AMD is partially addressed in this study.
ARPE-19 cells were grown in 6-well plates on membrane-containing inserts to model the native RPE cell layer. Cells from subcultures at a lower population doubling level (PDL; 23 to 35) and at a higher PDL (56 to 96) were compared. After being grown on the inserts for two weeks, the trans-epithelial electrical resistance (TEER) of the cell layers was measured. Once baseline measurements were obtained, the cells were treated with either VEGF (20ng) or its vehicle (PBS with 0.1% BSA), and TEER was again measured. One week later, the cells, still growing on the inserts, were stained for senescence-associated β-galactosidase (SABG) activity as an indicator of cell senescence.
The baseline TEER values did not differ significantly between lower and higher PDL cells (35 ohms-cm2 and 34 ohms-cm2, respectively; t-Test, p = .74). In contrast, the effect of VEGF was much greater on the higher PDL cells than on the lower PDL cells (22% decrease in TEER vs. 6% decrease; t-Test, p > .005). SABG staining was present in both the high and low PDL ARPE-19 cells, but the pattern of the staining was more darkly mottled in the higher PDL cells.
In addition to promoting vascular growth in the retina, VEGF can also decrease the TEER of RPE cells. Since TEER is an indicator of the quality of the tight junctions between the ARPE-19 cells, this effect could interfere with the functioning of the RPE layer in situ and might be permissive to the entry of choroidal neovascularization into the retina. Here we confirm the disruptive effect of VEGF on RPE cell layer integrity and show that it is greater in higher PDL cells than in lower PDL cells. The pattern of SABG staining of the higher PDL cells suggests that they are approaching senescence. A greater disruptive effect of VEGF on senescent RPE cells, together with the proposed occurrence of RPE senescence in AMD, suggests that his additional action of VEGF could also contribute to the pathology of AMD.
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