Effect of subconjuctival and intraocular bevacizumab injection on angiogenic gene expression levels in a mouse model of corneal neovascularization.
This study sought to characterize the expression of angiogenesis-related genes in a mouse model of corneal neovascularization, either untreated or after treatment with a single injection of bevacizumab by three different routes. In addition, the effectiveness of the treatment was compared to a rabbit model.



A chemical burn was induced in the mid-cornea of the right eye in 119 mice; 56 of them were untreated and 63 were bevacizumab-treated. Neovascularization was evaluated 2, 4, 8, 10, and 14 days later using digital photos, angiography and India ink perfusion. The relative area of new blood vessels was analyzed using slit-lamp examination in vivo and on histological and flat-mount sections. The levels of gene expression involved in the angiogenic process vascular endothelial growth factor [VEGF], insulin-like growth factor-1 [IGF-1], pigment epithelium derived factor [PEDF], and macrophage-inflammatory protein-2 [MIP-2]) were measured by a real-time polymerase chain reaction. Six rabbits underwent the same injury and treatment, and the response was compared to the mouse model.



Neovascularization was first observed two days after injury. The affected section increased from 11.24% (+/-7.0) of the corneal area to 47.42% (+/-25.4) on day 8 and 50.62% (+/-24.7) on day 10. In the mice treated with bevacizumab, the relative area of neovascularization was significantly lower at the peak time points (p<0.005): 24.90% (+/-21.8) on day 8 and 28.29% (+/-20.9) on day 10. Spontaneous regression was observed on day 14 in both groups, to 26.98% (+/-19.9) in the untreated mice and 10.97% (+/-10.8) in the bevacizumab-treated mice (p<0.005). Rabbits also showed peak corneal neovascularization on days 8-10, with significant regression of the vessels following intracameral bevacizumab injection. In the mice, intraocular (intravitreal, intracameral) injection was more effective than subconjuctival injection. VEGF gene expression was upregulated in both the untreated and treated mice, but was slightly less in the treated mice. PEDF gene expression decreased in both the treated and untreated mice. In the untreated group, gene expression peaked (above baseline) at 14 days, and in the untreated mice, it had already peaked by day 8. IGF-1 was upregulated early in the model; at 8 days, there was only a slight change in the untreated group compared to a significant increase in the treated group. MIP-2 was upregulated in both groups in the early stage and returned to baseline on day 14.



Bevacizumab treatment partially inhibits the progressive corneal neovascularization induced by chemical damage in a mouse model. Treatment is more effective when administered via the intraocular than the subconjunctival route. The clinical findings are compatible with the angiographic and histologic data and are supported by molecular analysis showing a partial change in expression of proangiogenic genes. The molecular mechanisms involved in corneal neovascularization and inflammation warrant further exploration. These findings may have important therapeutic implications in the clinical setting.
Dratviman-Storobinsky
Olga
O
The Krieger Eye Research Laboratory, Felsenstein Medical Research Center, Petah Tiqwa, Israel.
Avraham-Lubin
Bat-Chen R
BCR
Hasanreisoglu
Murat
M
Goldenberg-Cohen
Nitza
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000494:pharmacology* / Q000627:therapeutic use*; D061067:Antibodies, Monoclonal, Humanized; D000068258:Bevacizumab; D015415:Biomarkers / Q000378:metabolism; D002244:Carbon; D003228:Conjunctiva / Q000187:drug effects* / Q000473:pathology*; D016510:Corneal Neovascularization / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005451:Fluorescein Angiography; D005786:Gene Expression Regulation / Q000187:drug effects*; D056965:Injections, Intraocular; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010477:Perfusion; D011817:Rabbits; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D015415:Biomarkers; C028433:chinese ink; D000068258:Bevacizumab; D002244:Carbon