Mechanisms controlling the effects of bevacizumab (avastin) on the inhibition of early but not late formed corneal neovascularization.
OBJECTIVE
To evaluate the effects and underlying mechanisms of early and late subconjunctival injection of bevacizumab on the inhibition of corneal neovascularization (NV).


METHODS
Corneal NV was induced by closed eye contact lens wear followed by a silk suture tarsorrhaphy in rabbits. Weekly subconjunctival injections of bevacizumab (5.0 mg) for 1 month were started immediately (early treatment group) or 1 month after induction of corneal NV with continuous induction (late treatment group). The severity of corneal NV was evaluated. Immunostaining was used to evaluate the intracorneal diffusion of bevacizumab, and the existence of pericytes and smooth muscle cells around the NV. The expression of AM-3K, an anti-macrophage antibody, vascular endothelial growth factor (VEGF) with its receptors (VEGFR1 and VEGFR2), and vascular endothelial apoptosis were also evaluated. Western blot analysis was performed to quantify the expression level of VEGF, VEGFR1 and VEGFR2 on corneal epithelium and stroma in different groups.


RESULTS
Early treatment with bevacizumab inhibited corneal NV more significantly than late treatment. Intracorneal diffusion of bevacizumab was not different among different groups. Immunostaining showed pericytes and smooth muscle cells around newly formed vessels as early as 2 weeks after induction. Immunostaining and Western blot analysis showed that VEGF, VEGFR1, and VEGFR2 on corneal stroma increased significantly in no treatment groups and late treatment groups, but not in early treatment group. Bevacizumab significantly inhibited macrophage infiltration in the early but not late treatment group. Sporadic vascular endothelial apoptosis was found at 4 weeks in the late but not early treatment group.


CONCLUSIONS
Early but not late injection of bevacizumab inhibited corneal NV. Late injection of bevacizumab did not alter macrophage infiltration, and can't inhibit the expression of VEGF, VEGFR1, and VEGFR2 on corneal vessels. The inhibition of corneal NV in early treatment group does not occur via vascular endothelial apoptosis.
Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan; Center of Corneal Tissue Engineering and Stem Cell Biology, National Taiwan University Hospital, Taipei, Taiwan.
Wei-Li
WL
Chen
Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan; Department of Ophthalmology, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan.
Yan-Ming
YM
Chen
Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan.
Hsiao-Sang
HS
Chu
Department of Veterinary Medicine, College of Bio-Resource and Agriculture, National Taiwan University, Taipei, Taiwan.
Chung-Tien
CT
Lin
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Lu-Ping
LP
Chow
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chih-Ta
CT
Chen
Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan; Center of Corneal Tissue Engineering and Stem Cell Biology, National Taiwan University Hospital, Taipei, Taiwan.
Fung-Rong
FR
Hu
D000199:Actins / Q000378:metabolism; D020533:Angiogenesis Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000008:administration & dosage / Q000494:pharmacology*; D000951:Antigens, Neoplasm / Q000378:metabolism; D000068258:Bevacizumab; D003315:Cornea / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D016510:Corneal Neovascularization / Q000188:drug therapy / Q000209:etiology* / Q000473:pathology; D007150:Immunohistochemistry; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D011817:Rabbits; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D000099216:Chondroitin Sulfate Proteoglycan 4
D000199:Actins; D020533:Angiogenesis Inhibitors; D061067:Antibodies, Monoclonal, Humanized; D000951:Antigens, Neoplasm; D000068258:Bevacizumab; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D000099216:Chondroitin Sulfate Proteoglycan 4