The humanised VEGF-binding antibody bevacizumab is widely used off-label to treat DME.5
Although REC are considered the most important target cells of VEGF in the development and progression of this disease, in vitro experiments performed to understand the mechanisms of the therapeutic effects of bevacizumab have been focused almost exclusively on non-retinal EC or RPE cells.13
In this investigation we used iBREC, a retinal cell line with the distinct advantage over primary ECs that contaminating cells of other types cannot be present in the cultures.20
Studies with iBREC ideally complement investigations based on rodent models of diabetes and may even be more relevant to clinical applications of drugs under investigation, because of the higher similarity of bovine proteins to their human counterparts. In our previous studies based on the iBREC model, we showed that ranibizumab, in addition to efficient inhibition of VEGF-stimulated proliferation and migration, can completely restore the VEGF-induced impairment of the endothelial barrier; this provided a rationale for using this antibody fragment in DME therapy.4
Stimulation of iBREC was carried out in these experiments, with concentrations of VEGF similar to those measured in the vitreous fluid of DR patients; concentrations of ranibizumab and bevacizumab reflected values found to be achievable by standard therapies with these drugs.1–5
We confirmed that bevacizumab inhibits VEGF-induced proliferation but is less efficient than ranibizumab; this was also observed in experiments with human REC and could be a consequence of its much lower affinity to VEGF.12
Likewise, the ability of bevacizumab to re-establish an intact iBREC barrier that had been deranged by VEGF, was lower than that of ranibizumab. Despite being observed in vitro, this difference might be seen as a challenge to the assumption that both drugs are equivalent in the treatment of DR.5
Also of potential relevance for its clinical application is the surprising finding that bevacizumab repackaged in syringes and stored at 4°C for more than 2 weeks lost its ability to restore VEGF-induced loss of claudin-1, an indicator of an intact barrier. In one previous study, loss of bevacizumab activity was reported to be caused by freezing and thawing, after which it failed to prevent VEGF-stimulated increase of permeability of choroidal EC.21
It has been suggested that partial inactivation of bevacizumab in solution might be due to formation of antibody aggregates with potentially masked binding sites, a process more frequently observed when the solutions were stored in syringes.22
However, in solutions of bevacizumab used in this study, such aggregates were undetectable; we assume that partial loss of activity was more likely caused by yet unidentified reactions leading to modification of the protein. In contrast to the reversion of a state established during exposure to VEGF for days, reflecting the pathophysiological conditions typical for DME, prevention of the effects of VEGF on proliferation, migration and expression of claudin-1 by bevacizumab was not dependent on the time and conditions of its storage prior to the experiments. Preventing VEGF-induced processes may simply require lower amounts of an inhibitor, and it is therefore less sensitive to partial inactivation during storage than reversion of established states. In accordance with this assumption, preventing VEGF-induced loss of claudin-1 in iBREC was achieved with much lower concentrations of an inhibitor of VEGF receptor 2 than those for restoring VEGF-decreased claudin-1.14
This suggests that elimination of VEGF from the extracellular space may not be sufficient to reverse its long-term effects on ECs and that additional mechanisms contribute to the therapeutic effects of drugs like ranibizumab and bevacizumab. Potential second targets may be secreted factors like VEGF or cell surface molecules on ECs, but internalised drugs may also affect intracellular processes.
After treatment for 1 day, about 40 000 molecules of ranibizumab were calculated to be present inside iBREC. Association of ranibizumab with the organelle/membrane fraction is in accordance with the speculation that this substance is internalised by endocytosis to be degraded. Then the constant intracellular amounts of ranibizumab observed during prolonged exposure can be considered a steady state in which uptake and degradation of the protein are balanced. In contrast, a higher amount (~100 000 molecules per cell) of bevacizumab measured after one day in iBREC further increased when the cells were kept in medium containing the antibody. Most of the accumulated bevacizumab was found associated with the cytoskeleton, which might be the reason for the surprisingly observed inhibition of basal migration of iBREC. Because serum-free medium was used in this assay, this inhibitory effect could not be caused by targeting residual amounts of VEGF. The part of internalised bevacizumab found in the organelle/membrane fraction could be degraded like ranibizumab, but this process seems to be not fast enough to avoid an increase of intracellular amounts. Treatment of iBREC with VEGF165 did not alter intracellular quantities or their localisation, indicating that the endocytotic uptake of ranibizumab or bevacizumab by iBREC is not dependent on binding to the antigen. This is in accordance with a different localisation of intracellular VEGF which may be taken up through other pathways. Whether binding of the constant region of the antibody bevacizumab to the Fc receptor plays a role in its internalisation, remains to be investigated.
Neither ranibizumab nor bevacizumab affected the barrier of iBREC in the absence or presence of VEGF, even when the cells were exposed to the drugs for several days. This result contrasts with those of experiments with RPE cells in which ranibizumab transiently and bevacizumab lastingly increased the permeability, showing that in the two most important cell types involved in DR and DME, barriers of cell layers and dynamics of TJs are most likely regulated in different ways.17
In an overall assessment of therapeutically relevant processes in the eye, such differences between alternative drugs and their cell type-specific effects have to be taken into consideration.
In this study we showed that the actions of ranibizumab and bevacizumab on REC were similar but differed in some respects: bevacizumab inhibited or restored VEGF-induced effects with a slightly lower efficiency than ranibizumab and accumulated in these cells. Although the barrier properties of REC were not affected by internalised bevacizumab in our in vitro experiments, adverse effects in vivo cannot be ruled out because more antibody molecules might accumulate in these cells after repetitive intravitreal injections. Transient accumulation of bevacizumab in the RPE has been observed after its intravitreal injection into the primate eye.24
This might have some functional consequences as suggested by the finding that phagocytotic uptake of photoreceptor outer segments by porcine RPE cells is inhibited after long-term exposure to bevacizumab, an effect not observed with ranibizumab.16
Considering the typical ages of the DME-patients and duration of treatment, these results strongly suggest further investigations to exclude any harmful long-term consequences for retinal cells due to accumulation of bevacizumab after repetitive injections.