Abstract
Purpose:
Bevacizumab is internalized by immortalized bovine retinal endothelial cells (iBREC) and unspecifically blocks their migration in serum-free medium. Here we investigated its intracellular localization more closely and whether internalized bevacizumab is secreted back into the culture medium. The effect of fetal calf serum (FCS) or VEGF was also studied.
Methods:
Intracellular localization of bevacizumab was assessed by Western blotting of fractionated extracts and immunofluorescence staining 2 h to 1 d after uptake. Culture medium contained 0% to 5% FCS or 100 ng/ml VEGF. Uptake of bevacizumab with or without 1 µg/ml fibronectin at 4°C or 37°C for 4 h was also analyzed. To study transport through iBREC, cells were grown on membrane inserts placed in multi-well plates. After addition of bevacizumab to the lower chamber, aliquots of the supernatant in the upper chamber were analyzed at various time points (2 h to 1 d).
Results:
Transport of bevacizumab through iBREC was enhanced in medium with 5% FCS in both chambers. At 0% FCS in the lower and 5% FCS in the upper chamber - a situation resembling uptake through the vessel wall - less bevacizumab was transported. At 0.25% FCS, VEGF-A increased the transport rate. In the presence of FCS, bevacizumab was associated with proteins from membranes, organelles and the cytoskeleton 2, 4 or 24 h after uptake. It was uniformly distributed in the cell, a considerable amount localized close to α-tubulin fibers but not with marker proteins for Golgi, mitochondria, late endosomes, endoplasmatic reticulum or Weibel-Palade bodies. Without FCS, a vast amount of bevacizumab associated with insoluble proteins or the cytoskeleton; an observation also made when iBREC were kept at 4°C for 4 h. A lot of bevacizumab close to the nucleus but less in the cytoplasm was oberserved without FCS. Fibronectin did not affect the intracellular localization of bevacizumab. Cell migration in FCS presence was also not inhibited by bevacizumab.
Conclusions:
Serum components modulate cellular transport of bevacizumab in REC. Absence of FCS and low temperature drastically block this process, leading to accumulation of bevacizumab in insoluble protein complexes in iBREC. After intra-vitreal injection, first uptake of bevacizumab by REC takes place through the vessel wall. This might be dangerous for these cells due to potentially strong accumulation of the drug.