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1.  Binding of VEGF-A is sufficient to abrogate the disturbing effects of VEGF-B together with VEGF-A on retinal endothelial cells 
Inhibition of vascular endothelial growth factor (VEGF) is a promising strategy to treat retinal complications of diabetes. In contrast to VEGF-A binding ranibizumab, aflibercept also binds to other members of the VEGF family including VEGF-B, but potential effects of this factor on permeability and angiogenic processes are unclear. Therefore, we studied how VEGF-B variants as single agents or together with VEGF-A165 might affect proliferation, migration, or barrier function of retinal endothelial cells (REC). Also investigated was the normalization of REC properties with both VEGF-inhibitors to explore if additional targeting of VEGF-B is relevant.
Stimulation of proliferation or migration of immortalized bovine REC (iBREC) and disturbance of their barrier by exposure to VEGF-B variants (as single factors or together with VEGF-A165) was determined with or without VEGF-binding proteins being added. Permeability of iBREC was assessed by measuring their transendothelial resistance (TER) and expression of the tight junction protein claudin-1.
VEGF-B167 and VEGF-B186 enhanced proliferation of iBREC but these isoforms did not affect cell migration. Interestingly, ranibizumab completely blocked both migration and proliferation induced by VEGF-A plus VEGF-B. Both VEGF-B variants did also not affect barrier function or claudin-1 expression in a normal or high-glucose environment. Accordingly, binding VEGF-A was enough to normalize a reduced TER and reinstate claudin-1 lost during treatment with this factor in combination with VEGF-B.
Important properties and functions of REC seem not to be affected by any VEGF-B variant and targeting the key factor VEGF-A is sufficient to normalize growth factor-disturbed cells of this type.
Electronic supplementary material
The online version of this article (doi:10.1007/s00417-015-2944-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4445774  PMID: 25663437
Retinal endothelial cells; VEGF-B; Diabetic retinopathy; VEGF-inhibition
2.  Ranibizumab efficiently blocks migration but not proliferation induced by growth factor combinations including VEGF in retinal endothelial cells 
Proliferation and migration of retinal endothelial cells (REC) are associated with the development of proliferative diabetic retinopathy. REC proliferation is stimulated by isoforms of vascular endothelial growth factor-A (i.e., VEGF121 and VEGF165), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1) of which VEGF165 also enhances migration of REC. Effects induced by VEGF-A can be blocked with ranibizumab, a VEGF-binding Fab fragment used in therapy of diabetic macular edema. In this study, we investigated potential angiogenic effects of placental growth factors (PlGF-1, PlGF-2) as other members of the VEGF family and whether the primary action of VEGF165 is modulated in the presence of bFGF, IGF-1 and PlGF-1/-2. We also studied how effects of growth factor combinations can be attenuated with ranibizumab.
Effects of single growth factors or their combinations on proliferation and migration of immortalized bovine retinal endothelial cells (iBREC) were studied with or without ranibizumab or the inhibitor of VEGF receptors KRN951.
Proliferation of iBREC was significantly stimulated by 1–100 ng/ml PlGF-1 or PlGF-2, but additive effects were not observed with various combinations of the tested growth factors. Ranibizumab neutralized VEGF’s effect on proliferation but was not effective when the other growth factors were used in combination with VEGF. bFGF and IGF-1 but not PlGF-1 or PlGF-2 stimulated iBREC migration as single agents, and they further enhanced VEGF-induced migration. The effects of such growth factor combinations including VEGF on migration were efficiently blocked by targeting only VEGF with ranibizumab. Migration induced by VEGF plus bFGF and IGF-1 was also almost completely inhibited by KRN951 interfering with VEGF receptor signalling.
Migration but not proliferation of iBREC induced by combinations of bFGF, IGF-1, PlGF-1 or PlGF-2 together with VEGF is efficiently suppressed by ranibizumab. VEGF-mediated signalling through VEGFR2 seems to control REC migration dominantly in the presence of other growth factors.
PMCID: PMC3777160  PMID: 23760670
Retinal endothelial cells; Growth factors; Ranibizumab; Migration; Proliferation; Proliferative diabetic retinopathy
3.  Actions of bevacizumab and ranibizumab on microvascular retinal endothelial cells: similarities and differences 
The British Journal of Ophthalmology  2012;96(7):1023-1028.
Retinal endothelial cells are crucially involved in the genesis of diabetic retinopathy which is treated with vascular endothelial growth factor (VEGF) inhibitors. Of these, ranibizumab can completely restore VEGF-induced effects on immortalised bovine retinal endothelial cells (iBREC). In most experiments supporting diabetic retinopathy therapy with bevacizumab, only non-retinal EC or retinal pigment epithelial cells have been used. Also, bevacizumab but not ranibizumab can accumulate in retinal pigment epithelial cells.
To investigate the effects of bevacizumab on VEGF-induced changes of iBREC properties and potential uptake and accumulation of both inhibitors.
Uptake of VEGF inhibitors by iBREC with or without pretreatment with VEGF165 was visualised by immunofluorescence staining and western blot analyses. Measured transendothelial resistance (TER) of iBREC (±VEGF165) showed effects on permeability, indicated also by the western blot-determined tight junction protein claudin-1. The influence of bevacizumab on proliferation and migration of iBREC was studied in the presence and absence of VEGF165.
Bevacizumab strongly inhibited VEGF-stimulated and basal migration, but was less efficient than ranibizumab in inhibiting VEGF-induced proliferation or restoring the VEGF-induced decrease of TER and claudin-1. This ability was completely lost after storage of bevacizumab for 4 weeks at 4°C. Ranibizumab and bevacizumab were detectable in whole cell extracts after treatment for at least 1 h; bevacizumab accumulated during prolonged treatment. Ranibizumab was found in the membrane/organelle fraction, whereas bevacizumab was associated with the cytoskeleton.
Both inhibitors had similar effects on retinal endothelial cells; however, some differences were recognised. Although barrier properties were not affected by internalised bevacizumab in vitro, potential adverse effects due to accumulation after repetitive intravitreal injections remain to be investigated.
PMCID: PMC3382447  PMID: 22539748
Retinal endothelial cells; VEGF inhibition; diabetic macular oedema; diabetic retinopathy; biochemistry; diagnostic tests/investigation; macula; neovascularisation; retina

Results 1-3 (3)