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 A 165 (VEGF 165 ) 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. VEGF 121 , placental growth factor (P l GF-1 and P l GF-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 P l GF-1, P l GF-2 or VEGF 121 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 P l GF. Whereas 1 ng/ml VEGF-E were sufficient to impair the iBREC barrier, a higher concentration of 100 ng/ml VEGF 121 was needed to reduce TER and expression of claudin-1 over 2 days. By P l GF-1 or P l GF-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 VEGF 165 . The weak barrier derangement caused by VEGF 121 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 P l GF impairs the barrier function of iBREC and that the longer isoform VEGF 165 is more potent than VEGF 121 . 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 VEGF 165 , but not P l GF-1/-2 reduced TER or claudin-1 expression.