VEGF modulation of retinal pigment epithelium resistance

Abstract

Fluid accumulation into the subretinal space and the development of macular edema is a common condition in age-related macular degeneration, diabetic retinopathy, and following ocular surgery, or injury. Vascular endothelial growth factor (VEGF) and other cytokines have been implicated in the disruption of retinal pigment epithelium (RPE) barrier function and a reduction in the regulated removal of subretinal fluid; however, the cellular and molecular events linking these agents to the disruption of barrier function have not been established. In the current study, cultures of ARPE-19 and primary porcine retinal pigment epithelium (RPE) cells were utilized to investigate the effects of the VEGF-induced modifications to the barrier properties of the RPE. The barrier function was determined by transepithelial resistance (TER) measurements and morphology of the RPE monolayers. In both ARPE-19 and primary porcine RPE cells the administration of VEGF produced a significant drop in TER, and this response was only observed following apical administration. Maximum reduction in TER was reached 5 hours post VEGF administration. These responses were concentration-dependent with an EC 50 of 502 pg/mL in ARPE-19 cells and 251 pg/mL in primary porcine cells. In both ARPE-19 and primary RPE cells, the response to VEGF was blocked by pretreatment with the relatively selective VEGF-R2 antagonists, SU5416 or ZM323881, or the protein tyrosine kinase inhibitor, genistein. Administration of the relatively selective VEGF-R2 agonist, VEGF-E, also reduced TER in a concentration-dependent manner (EC 50 of 474 pg/mL), while VEGF-R1 agonist, placental growth factor (PlGF), did not significantly alter the TER. Immunolocalization studies demonstrated that confluent monolayers exhibited continuous cell-tocell ZO-1 protein contacts and apical localization of the VEGF-R2 receptors. These data provide evidence that the VEGF-induced breakdown of RPE barrier function is mediated by the activation of apically-oriented VEGF-R2 receptors. Thus, VEGF-mediated increases in RPE permeability are initiated by a rise in intraocular levels of VEGF.

Sublytic Membrane-Attack-Complex (MAC) Activation Alters Regulated Rather than Constitutive Vascular Endothelial Growth Factor (VEGF) Secretion in Retinal Pigment Epithelium Monolayers

“…Activity in these two pathways might mediate the VEGF-induced reduction in TER, which plateaus within 1-4 h after stimulation (18,29). The inhibitor experiments presented in Fig.…”

“…Ref. 29,32) via a saturable process (57). Regulated secretion of polypeptides present in granular vesicles occurs via exocytosis triggered by Ca 2ϩ .…”

“…Cells were grown in DMEM-F12 (Invitrogen) with 10% fetal bovine serum (FBS) and 1ϫ penicillin:streptomycin as monolayers on Transwell filters. FBS was removed completely for the final 5-7 days (2-3 media changes) prior to measurements, which we have previously shown does not alter survival or monolayer formation in these cells (29). TER of the cell monolayer on the Transwell filters was determined by measuring the resistance across the monolayer with an EVOM volt-ohmmeter (World Precision Instruments) (18).…”

“…The value for cell monolayers was determined by subtracting the TER for filters without cells and then multiplying by the surface area of the filters. Cell monolayers were considered stable when TER was repeatedly measured as ϳ40 -45 ⍀/cm 2 (29). TER measurements, which are proportional to membrane permeability, are an accepted readout for the barrier function of an RPE monolayer (27,29).…”

Smoke Exposure Causes Endoplasmic Reticulum Stress and Lipid Accumulation in Retinal Pigment Epithelium through Oxidative Stress and Complement Activation

“…TER values reach a stable plateau of 40 -45 ⍀ cm 2 within 2-3 weeks after confluence (46). FBS was removed completely for the final days prior to measurements, which does not alter survival or monolayer formation (47), such that complement-sufficient normal human serum can be used as a source of complement. TER was determined by measuring the resistance across the monolayer with an EVOM volt-ohmmeter (World Precision Instruments) as described previously, subtracting the TER for filters without cells and then multiplying by the surface area of the filters.…”