Abstract
PurposeTo evaluate the effects and underlying mechanisms of early and late subconjunctival injection of bevacizumab on the inhibition of corneal neovascularization (NV).MethodsCorneal 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.ResultsEarly 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.ConclusionsEarly 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.
Highlights
Vascular endothelial growth factor (VEGF) induces corneal NV under pathological scenarios. [2,5,11,12,13,14] Numerous studies have shown that VEGF is a critical mediator of retinal and iris NV following injury and ischemia, and in diabetic retinopathy too. [15,16] Increased VEGF mRNA levels in the epithelium were observed in a rabbit model of closed eye contact lens (CL)-induced corneal NV
Several studies have shown that anti-VEGF agents can inhibit corneal NV. [22,23,24,25,26,27,28,29] One such inhibitor is bevacizumab, a humanized murine monoclonal antibody against all VEGF isoforms. [23,30] Bevacizumab has been used to treat metastatic colorectal cancer, [31] diabetic retinopathy, [32,33,34] choroidal NV in pathologic myopia, [35] exudative age-related macular degeneration (ARMD), [36,37,38] and corneal NV in some circumstances. [39,40,41] Recently, we reported that subconjunctival injection of bevacizumab inhibits the formation of corneal NV in various rabbit corneal NV models [23,27] and showed that bevacizumab can be used to effectively treat lipid keratopathy in certain patients
In which subconjunctival injection of bevacizumab was administered weekly from day 0 until 1 month after closed eye CL wear, a significant decrease in corneal NV compared to the control group was observed from week 3 to 4 after treatment (PICS, centricity, and extent of corneal NV: all p,0.01) (Table 1)
Summary
[39,40,41] Recently, we reported that subconjunctival injection of bevacizumab inhibits the formation of corneal NV in various rabbit corneal NV models [23,27] and showed that bevacizumab can be used to effectively treat lipid keratopathy in certain patients. [29] in rabbit corneas, we found that administration of bevacizumab injection immediately after limbal injury has better inhibitory effects on corneal NV than late treatment. [27] Papathanassiou et al found that subconjunctival administration of bevacizumab effectively inhibits corneal neovascularization in an experimental rabbit model, especially if administered early. Immunohistochemistry was performed to evaluate intracorneal bevacizumab diffusion and the expression of VEGF, VEGFR1, VEGFR2, AM-3K (an anti-macrophage antibody), and vascular endothelial apoptosis. A better understanding of the effects and underlying mechanisms of early and late subconjunctival injection of bevacizumab may help establishing guidelines for bevacizumab use in corneal NV treatment
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