In vitro induction of protein complexes between bevacizumab, VEGF-A¹⁶⁵ and heparin: explanation for deposits observed on endothelial veins in monkey eyes.
OBJECTIVE
By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin.


METHODS
Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies.


RESULTS
Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells.


CONCLUSIONS
Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.
Section of Experimental Vitreoretinal Surgery, Centre for Ophthalmology, Schleichstr. 12/1, Tuebingen 72076, Germany. Sylvie.Julien@med.uni-tuebingen.de
Sylvie
S
Julien
Antje
A
Biesemeier
Ulrich
U
Schraermeyer
D020533:Angiogenesis Inhibitors / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism* / Q000494:pharmacology; D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology; D000936:Antigen-Antibody Complex / Q000378:metabolism* / Q000648:ultrastructure; D000068258:Bevacizumab; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D007150:Immunohistochemistry; D058449:Intravitreal Injections; D008252:Macaca fascicularis; D008854:Microscopy, Electron; D000069579:Ranibizumab; D012169:Retinal Vein / Q000378:metabolism* / Q000648:ultrastructure; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism* / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D061067:Antibodies, Monoclonal, Humanized; D000925:Anticoagulants; D000936:Antigen-Antibody Complex; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab; D006493:Heparin; D000069579:Ranibizumab