VEGF-Aa and angiopoietin-2 differently affect the barrier formed by retinal endothelial cells.

Exposure to VEGF-Aa over several days leads to a persistent dysfunction of the very tight barrier formed by immortalized endothelial cells of the bovine retina (iBREC). Elevated permeability of the barrier is indicated by low cell index values determined by electric cell-substrate impedance measurements, by lower amounts of claudin-1, and by disruption of the homogenous and continuous staining of vascular endothelial cadherin at the plasma membrane. Because of findings that suggest modulation of VEGF-A's detrimental effects on the inner blood-retina barrier by the angiogenic growth factor angiopoietin-2, we investigated in more detail in vitro whether this growth factor indeed changes the stability of the barrier formed by retinal endothelial cells or modulates effects of VEGF-A.

Different responses of the MIO‑M1 Mueller cell line to angiotensin II under hyperglycemic or hypoxic conditions.

Members of the renin-angiotensin aldosterone system (RAAS) are expressed by various retinal tissues including Mueller glial cells. As the RAAS is hypothesized to play an important role in the pathogenesis of diseases that threaten vision, such as diabetic macular edema or retinal vein occlusion, the possible changes induced by exposure of the human cell line MIO-M1, an established model of Mueller cells, to angiotensin II or aldosterone for 6 h under hypoxic and/or hyperglycemic conditions were investigated. The mRNA expression levels of the members of the RAAS were assessed by reverse transcription-quantitative PCR, and the secretion of cytokines was assessed by ELISA.

Beovu, but not Lucentis impairs the function of the barrier formed by retinal endothelial cells in vitro.

Because rare, but severe adverse effects, i.e. retinal vasculitis or retinal vein occlusion, have been observed after repetitive intravitreal injections of VEGF-A-binding single-chain variable fragment brolucizumab (Beovu), we investigated its possible impact on the barrier formed by immortalized bovine retinal endothelial cells (iBREC) in comparison to that of the VEGF-A-binding Fab fragment ranibizumab (Lucentis).

Impairment of the Retinal Endothelial Cell Barrier Induced by Long-Term Treatment with VEGF-A No Longer Depends on the Growth Factor's Presence.

As responses of immortalized endothelial cells of the bovine retina (iBREC) to VEGF-A depend on exposure time to the growth factor, we investigated changes evident after long-term treatment for nine days. The cell index of iBREC cultivated on gold electrodes-determined as a measure of permeability-was persistently reduced by exposure to the growth factor. Late after addition of VEGF-A protein levels of claudin-1 and CD49e were significantly lower, those of CD29 significantly higher, and the plasmalemma vesicle associated protein was no longer detected.

Blocking of VEGF-A is not sufficient to completely revert its long-term effects on the barrier formed by retinal endothelial cells.

The VEGF-A-induced functional impairment of the barrier formed by retinal endothelial cells (REC) can be prevented and even - at least temporarily - reverted by trapping the growth factor in a complex with a VEGF-binding protein or by inhibiting the activity of the VEGF receptor 2 (VEGFR2). In an approach to emulate the clinically relevant situation of constant exposure to effectors, we investigated (1) whether prolonged exposure to VEGF-A for up to six days results in a different type of disturbance of the barrier formed by immortalized bovine REC (iBREC) and (2) whether alterations of the barrier induced by VEGF-A can indeed be sustainably reverted by subsequent treatment with the VEGF-A-binding proteins ranibizumab or brolucizumab. As a measure of barrier integrity, the cell index (CI) of iBREC cultivated on gold electrodes was monitored continuously.

Type of culture medium determines properties of cultivated retinal endothelial cells: induction of substantial phenotypic conversion by standard DMEM.

Contradictory behavior of microvascular retinal endothelial cells (REC) - a reliable in vitro model to study retinal diseases - have recently been reported which might result from cultivating the cells in standard DMEM not optimized for this cell type. Therefore, we studied DMEM's effects on phenotype and behavior of immortalized bovine REC. Cells were cultivated in endothelial cell growth medium (ECGM) until a confluent monolayer was reached and then further kept for 1-4 days in ECGM, DMEM, or mixes thereof all supplemented with 5% fetal bovine serum, endothelial cell growth supplement, 90 μg/ml heparin, and 100 nM hydrocortisone.

Transport and fate of aflibercept in VEGF-A-challenged retinal endothelial cells.

Retinal vessels are at least in part involved in clearing of Fc terminus-containing proteins from the vitreous. In vitro, the Fc fusion protein aflibercept is transported through a monolayer of unchallenged immortalized bovine retinal endothelial cells (iBREC), mediated by the neonatal Fc receptor (FcRn), but part of the Fc fusion protein is also degraded. Aflibercept's target VEGF-A not only enhances the permeability of REC by destabilization of tight junctions (TJs) thereby allowing for paracellular flow, it may also lower the intracellular stability of the Fc fusion protein by changing its binding properties to the FcRn.

Sitagliptin and the Blood-Retina Barrier: Effects on Retinal Endothelial Cells Manifested Only after Prolonged Exposure.

Inhibitors of dipeptidyl peptidase-4 (DPP-4) are widely used to treat diabetes mellitus, but data concerning their effects on the barrier stability of retinal endothelial cells (REC) in vivo and in vitro are inconsistent. Therefore, we studied whether the barrier properties of immortalized endothelial cells of the bovine retina (iBREC) were affected by the inhibitors of DPP-4 sitagliptin (10-1000 nM) and diprotin A (1-25 M). Their effects were also investigated in the presence of VEGF-A because diabetic patients often develop macular edema caused by VEGF-A-induced permeability of REC.

VEGF receptor 2 inhibitor nintedanib completely reverts VEGF-A-induced disturbances of barriers formed by retinal endothelial cells or long-term cultivated ARPE-19 cells.

Various severe ocular diseases are associated with an elevated intravitreal expression of VEGF-A which increases the permeability of retinal endothelial cells (REC) or retinal pigment epithelial (RPE) cells in vivo and in vitro. Inhibition of VEGF receptor 2 (VEGFR2) is sufficient to completely prevent VEGF-A-induced dysfunctions of barriers formed by long-term cultivated, immortal human ARPE-19 cells or immortalized bovine retinal endothelial cells (iBREC). Extended exposure to VEGF-A could result in additional activation of other growth factor receptors, potentially promoting synergistic effects of corresponding factors on various cellular processes including angiogenesis.

Fate of the Fc fusion protein aflibercept in retinal endothelial cells: competition of recycling and degradation.

Purpose: Intravitreal injection of the VEGF-binding protein aflibercept is widely used to treat various ocular diseases. In vitro, immortalized bovine retinal endothelial cells (iBREC) take up and transport aflibercept through the cell layer in a serum-dependent manner, likely mediated through the neonatal Fc receptor (FcRn), but degradation of the Fc domain-containing protein might be a competing intracellular process. Therefore, aflibercept's associations with proteins either involved in FcRn-mediated transport or in the lysosomal pathway were studied.

Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165.

Background/aims: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A165), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A165, we investigated if its signaling pathways triggered in REC are redundant.

Methods: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A165.

Neonatal Fc receptor FcRn is involved in intracellular transport of the Fc fusion protein aflibercept and its transition through retinal endothelial cells.

Retinal endothelial cells (REC) likely contribute to the clearance of intravitreally injected IgG. Because this is of high relevance to the pharmacokinetic assessment of the widely used therapeutic Fc fusion protein aflibercept, we studied its transport through immortalized bovine REC (iBREC) in detail. For shuttling of IgG or Fc fusion proteins like aflibercept, endothelial cells use the highly conserved neonatal Fc receptor (FcRn) also expressed in iBREC where it is down regulated by serum depletion.

Pre-Analytical Parameters Affecting Vascular Endothelial Growth Factor Measurement in Plasma: Identifying Confounders.

Background: Vascular endothelial growth factor-A (VEGF-A) is intensively investigated in various medical fields. However, comparing VEGF-A measurements is difficult because sample acquisition and pre-analytic procedures differ between studies. We therefore investigated which variables act as confounders of VEGF-A measurements.

The Protein Kinase C Inhibitor: Ruboxistaurin.

The isozyme protein kinase C (PKC) β is involved in several processes that are deregulated in different retinal cell types by hyperglycemia. This family of serine/threonine-specific protein kinases comprises several different members, which differ in their structure, cofactor requirement and substrate specificity. Therefore, PKCβ was considered a valuable target for therapeutic intervention.

Internalization of bevacizumab by retinal endothelial cells and its intracellular fate: Evidence for an involvement of the neonatal Fc receptor.

Bevacizumab is one of the VEGF-binding proteins that are established in clinical practice to treat various ocular diseases. In view of therapeutic long-term application, potential accumulation of the antibody in retinal cells gave reason for safety concerns. Internalization of considerable amounts of bevacizumab by retinal endothelial (REC) and pigment epithelial cells has been observed which may affect their important functions.

Binding of VEGF-A is sufficient to abrogate the disturbing effects of VEGF-B together with VEGF-A on retinal endothelial cells.

Purpose: 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).

Capacity of aflibercept to counteract VEGF-stimulated abnormal behavior of retinal microvascular endothelial cells.

Members of the vascular endothelial growth factor (VEGF) family differently regulate processes in retinal endothelial cells (REC) which are crucially involved in the pathogenesis of diabetic retinopathy: Both, VEGF-A and placenta growth factor (PlGF), stimulate proliferation of primary and immortalized bovine REC ((i)BREC) but only VEGF-A165 stimulates their migration. Diabetic macular edema is most likely a consequence of an elevated permeability of REC which can be induced by VEGF-A, but not by PlGF. Binding of VEGF-A by the antibody fragment ranibizumab is sufficient to completely restore or prevent VEGF-A-induced disturbance of the iBREC barrier or migration of these cells without affecting the basal processes.

VEGF but not PlGF disturbs the barrier of retinal endothelial cells.

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.

Ranibizumab efficiently blocks migration but not proliferation induced by growth factor combinations including VEGF in retinal endothelial cells.

Background: 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.

Actions of bevacizumab and ranibizumab on microvascular retinal endothelial cells: similarities and differences.

Background: 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.

Medium-sized deletion in the BRCA1 gene: Limitations of Sanger sequencing and MLPA analyses.

We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification.

Inhibition of vascular endothelial growth factor (VEGF) is sufficient to completely restore barrier malfunction induced by growth factors in microvascular retinal endothelial cells.

Background: Deregulated expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or insulin-like growth factor-1 (IGF-1) is associated with the pathogenesis of diabetic retinopathy. The VEGF(165)-induced increase in permeability of retinal endothelial cells (REC), probably resulting in diabetic macular oedema (DME), could be completely restored by the VEGF-binding Fab fragment ranibizumab in vitro. We investigated whether bFGF and IGF-1 as single factors or in combination with VEGF(165) influence permeability and tight junctions in immortalised bovine REC (iBREC) and if these effects could be restored by inhibition of VEGF.

Inhibition of protein kinase C is not sufficient to prevent or reverse effects of VEGF165 on claudin-1 and permeability in microvascular retinal endothelial cells.

Purpose: Pathogenesis of diabetic macular edema is driven by deregulated expression of VEGF. A study of long-term exposure of immortalized bovine retinal endothelial cells (iBRECs) to VEGF(165) clearly confirmed the role of the tight junction protein claudin-1, which almost completely disappeared within 24 hours, an effect that was completely reversed by addition of the VEGF-binding Fab fragment ranibizumab. This study was conducted to investigate whether the VEGF(165)-induced loss of claudin-1 is regulated by protein kinase C (PKC) and indeed affects the barrier function of iBRECs.