Effects of high glucose concentration on the barrier function and the expression of tight junction proteins in human retinal pigment epithelial cells
Introduction
The retinal pigment epithelium (RPE) is a highly specialized epithelium that serves as a multifunctional and indispensable component of the vertebrate eye (Strauss, 2005). RPE forms the outer blood retinal barrier (BRB), thus controlling the flow of solutes and fluid from the choroidal vasculature into the outer retina (Erickson et al., 2007, Strauss, 2005). The inner BRB is constituted by the blood vessels of the retina and directly controls the flux into the inner retina (Erickson et al., 2007, Strauss, 2005). The strict control of fluid and solutes that cross the BRB is achieved through well-developed tight junctions. Over 40 proteins have been found to be associated with tight junctions (Gonzalez-Mariscal et al., 2003). Zonula occludens-1 (ZO-1), claudins and occludin are the most studied of these proteins, especially regarding how they are related to the BRB.
Diabetic macular edema is one of the primary causes of poor visual acuity in patients with diabetic retinopathy (Congdon et al., 2003, Lightman and Towler, 2003). The breakdown of the BRB due to the disruption of the tight junctions is the main factor accounting for diabetic macular edema (Joussen et al., 2007). While extensive work has been carried out to identify the factors involved in the disruption of the tight junctions of the inner BRB, the mechanisms implicated in the outer BRB regulation have been poorly explored.
Treatment of RPE cells with either serum, interferon-γ, tumor necrosis factor-α, hepatocyte growth factor (HGF), interleukin (IL)-1β or placental growth factor-1 (PLGF-1) decreased transepithelial electrical resistance (TER), increased permeability and altered the expression or content of tight junction molecules (Abe et al., 2003, Chang et al., 1997, Jin et al., 2002, Miyamoto et al., 2007, Zech et al., 1998). However, to the best of our knowledge the direct effect of high glucose concentrations has never been reported.
The aim of the study was to explore the effect of 5.5 mM d-glucose and 25 mM d-glucose on the permeability and the expression of tight junction proteins (occludin, ZO-1 and claudin-1) in a human RPE line (ARPE-19).
Section snippets
Human RPE cell cultures
ARPE-19 was obtained from American Type Culture Collection (Manassas, VA, USA). The cells used in these experiments were between passages 16 and 19. Initially, the culture was started at a concentration of 80,000 RPE cells/well (800,000 cells/mL) in a medium with 10% FBS and 5.5 mM d-glucose (0.2 mL on the apical side and 0.6 mL on the basolateral side). Seven days after the culture was started, half of the transwells were maintained with d-glucose 5.5 mM and the other half were switched to d
Measurement of TER
The cells grown at 25 mM of d-glucose showed higher TER values than the ones maintained at 5.5 mM of d-glucose (Fig. 1A). These differences were already evident at 7 days of switched on 25 mM (p < 0.001) and continued to be significantly different for at least two weeks (p ≤ 0.002 at days 14, 16, 18, 21, 23, 25, 28, 30).
Permeability assay
Permeability was significantly lower in cultures under 25 mM of d-glucose in comparison with 5.5 mM of d-glucose. These results were similar when using 70 kDa dextran (p < 0.05
Discussion
The effect of the RPE on the properties of the neighboring cells is well documented but the effects of neighboring environments on RPE are less well studied (King and Suzuma, 2000, Peng et al., 2003). Intercellular junction integrity of RPE can be impaired by several proinflammatory cytokines, HGF and PLGF-1 (Abe et al., 2003, Jin et al., 2002, Miyamoto et al., 2007, Zech et al., 1998). However, the specific effects of high glucose concentrations on the function and molecular constituents of
Competing interests
None.
Acknowledgments
This study was supported by grants from Novo Nordisk Pharma S.A, Fundación para la Diabetes, the Generalitat de Catalunya (2005SGR0030), and Ministerio de Ciencia y Tecnología (SAF2006-05284). CIBER for Diabetes and Associated Metabolic Diseases is an initiative of the Instituto de Salud Carlos III.
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