title: VEGF but not PlGF disturbs the barrier of retinal endothelial cells.
abstract: Elevated permeability of retinal endothelial cells (REC), as observed in diabetic retinopathy (DR), is induced by extended exposure to ≥25 ng/ml vascular endothelial growth factor A165 (VEGF165) for up to 3 d and this effect is more pronounced when equimolar amounts of basic fibroblast growth factor (bFGF) and insulin-like growth factor (IGF-1) are present. Down-regulation of the tight-junction protein claudin-1 and its loss from the plasma membrane is associated with induced higher permeability, whereas other tight-junction proteins (e.g. claudin-3, claudin-5, ZO-1) show only subtle changes in our experimental setting. Using immortalized bovine REC (iBREC) as a well-established model, we investigated effects of other members of the VEGF family, i.e. VEGF121, placental growth factor (PlGF-1 and PlGF-2) and viral VEGF-E which activate different sets of VEGF receptors, on barrier function after extended treatment: iBREC were incubated with 1-100 ng/ml of the growth factors for up to 2 days before barrier function was assessed by measuring transendothelial resistance (TER). Presence of TJ-proteins was determined by western blot analyses and immunofluorescence staining. Similar experiments were performed to evaluate whether the primary actions of PlGF-1, PlGF-2 or VEGF121 are modulated by bFGF or IGF-1 when all growth factors (each at 25 ng/ml, but 10 ng/ml IGF-1) act simultaneously at equimolar concentrations. We also studied the potential normalization of the barrier disturbed with combinations of growth factors by addition of the VEGF-specific Fab fragment ranibizumab or the recombinant protein aflibercept which binds VEGF and PlGF. Whereas 1 ng/ml VEGF-E were sufficient to impair the iBREC barrier, a higher concentration of 100 ng/ml VEGF121 was needed to reduce TER and expression of claudin-1 over 2 days. By PlGF-1 or PlGF-2, the barrier was not affected even at the highest concentration tested (100 ng/ml) and these factors also did not modulate the effect of VEGF165. The weak barrier derangement caused by VEGF121 was slightly enhanced by bFGF and IGF-1. After induction of the barrier breakdown with various combinations of all growth factors included in the study, normal TER and claudin-1 expression was re-established by ranibizumab. Both VEGF inhibitors ranibizumab and aflibercept similarly reinstated lost claudin-1, even when applied at a small fraction of the clinically relevant concentrations. These results show that VEGF-A, but not PlGF impairs the barrier function of iBREC and that the longer isoform VEGF165 is more potent than VEGF121. To induce barrier dysfunction in iBREC, activation of VEGF receptor 2 - probably in concert with neuropilin-1 - seems to be sufficient because VEGF-E and VEGF165, but not PlGF-1/-2 reduced TER or claudin-1 expression.
authors.affiliation: Department of Ophthalmology, University of Ulm, Prittwitzstrasse 43, 89075 Ulm, Germany. Electronic address: heidrun.deissler@uniklinik-ulm.de.
authors.forename: Heidrun L
authors.initials: HL
authors.lastname: Deissler
authors.forename: Helmut
authors.initials: H
authors.lastname: Deissler
authors.forename: Gerhard K
authors.initials: GK
authors.lastname: Lang
authors.forename: Gabriele E
authors.initials: GE
authors.lastname: Lang
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology; D001692:Biological Transport; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D002463:Cell Membrane Permeability; D062445:Claudin-1 / Q000378:metabolism; D017097:Electric Impedance; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D039942:Neuropilin-1 / Q000378:metabolism; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000494:pharmacology*; D000069579:Ranibizumab; D040262:Receptors, Vascular Endothelial Growth Factor / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012171:Retinal Vessels / Q000166:cytology; D019108:Tight Junctions / Q000187:drug effects*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D062445:Claudin-1; D011257:Pregnancy Proteins; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; D000072483:Placenta Growth Factor; D039942:Neuropilin-1; C533178:aflibercept; D007334:Insulin-Like Growth Factor I; D040262:Receptors, Vascular Endothelial Growth Factor; D000069579:Ranibizumab
keywords: (i)BREC; (immortalized) bovine retinal endothelial cells; DME; DR; EC; ECGM; ECGS/H; FCS; IGF-1; KRN951; N-(2-Chloro-4-((6,7-dimethoxy-4-quinolyl)oxy)phenyl)-N′-(5-methyl-3-isoxazolyl)urea; NRP-1; PlGF; REC; SRM; TER; TJ; VECad; VEGF; VEGF receptor 1; VEGF receptor 2; VEGF-E; VEGFR1; VEGFR2; ZO-1; aflibercept; bFGF; basic fibroblast growth factor; diabetic macular edema; diabetic retinopathy; endothelial cell growth medium; endothelial cells; endothelial cells growth supplement/H; fetal calf serum; insulin-like growth factor; neuropilin-1; placental growth factor; ranibizumab; retinal endothelial cells; serum-reduced medium; tight junction; transendothelial resistance; vascular endothelial cadherin; vascular endothelial growth factor A; vascular endothelial growth factor E; zona occludens-1