Transcript The Inhibitory Effect of Bevacizumab eyedrops on NGF expression

Eun Chul Kim, M.D., Man Soo Kim, M.D.
Department of Ophthalmology & Visual Science,
College of Medicine, Catholic University of Korea, Seoul, Korea
The authors have no financial interest in the subject matter of this poster.
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potent growth factor that stimulates the proliferation of blood vessels
Injured states, VEGF expression upregulated and corneal
neovascularization
important player in nerve growth
In vitro :VEGF and its receptor are expressed by neurons and stimulate
neurogenic, neuroprotective, and neurotrophic activities
VEGF
 proliferation of cortical neurons,
 protection of central and peripheral neurons from hypoxia-induced
death
 promotion of axonal outgrowth in peripheral neurons
Systemic inhibition of VEGF with bevacizumab may cause apoptosis in
retinal neurons (Invest Ophthalmol Vis Sci. 2007;48:1773–1781.)
Nerve growth factor promotes corneal healing: structural,
biochemical, and molecular analyses of rat and human
corneas.
Invest Ophthalmol Vis Sci. 2000 Apr;41(5):1063-9.
 The Use of Nerve Growth Factor in Surgical Wound
Healing of the Cornea Ophthalmic Res 2006;38:177–181
 Use of nerve growth factor to treat congenital
neurotrophic corneal ulceration.
Cornea. 2006 Apr;25(3):352-5
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Reciprocal angiogenic and neurotrophic effects on blood vessels and
neurons (Curr Pharm Des 2006;12:2609–2622.)
Capillary sprouting was promoted by NGF via the release of VEGF. (Proc
Natl Acad Sci U S A 2001;98:4160–4165.)
Overexpression of NGF in brown adipose tissue of NGF-transgenic mice :
elevated both mRNA and protein levels of VEGF and VEGFRs. (Histochem
Cell Biol. 2006;125:637-649.)
Activated VEGF stimulated angiogenesis : blocked by NGF neutralizing
antibodies. (Histochem Cell Biol. 2006;125:637-649.)
NGF stimulated the expression of VEGF isoforms in epithelial ovarian
cancer and the effect was dose-dependent and inhibited by an NFG
antibody and by a TrkA inhibitor. (Brain Res 2002;953:12–16.)
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To investigate whether anti–VEGF antibodies such as
bevacizumab eyedrops have inhibitory effects on corneal
epithelial wound healing and NGF expression in rats.
Necessity
 Studies have indicated that VEGF plays a role in mediating
corneal nerve repair11 and that anti–VEGF delays corneal
epithelial wound healing in rabbits. (Invest Ophthalmol Vis Sci.
2009;50:4653-4659.)
 However, implications for anti–VEGF therapy for corneal
wound healing by VEGF inhibition leading to NGF expression
have not been explored.
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120 Sprague-Dawley male rats (250–300 g) were divided into two
groups of 60 rats each..
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Bevacizumab Group: instilled anti-VEGF(5 mg/ml) and antibiotics (Cravit®:
0.5% levofloxacin) four times daily
Control Group : antibiotics eyedrops only four times daily
Corneal wound healing was evaluated by fluorescein staining at 0, 24, 48,
and 72 hours after 5 mm-sized epithelial debridement.
Nerve growth factor (NGF) and vascular endothelial growth factor (VEGF)
protein were measured in rat corneas by ELISA.
Immunofluorescent staining for NGF and VEGF were performed in rat
corneas.
NGF mRNA and VEGF mRNA were measured in rat corneas by real time
PCR.
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Wound healing in the bevacizumab
group was lower than that in the
control group at 24, 48, and 72 hours
after debridement, respectively (*P
< 0.05; n = 20 per group; MannWhitney U test).
20
Healed area (mm2)
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15
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*
10
Bevacizumab
5
Control
0
24 Hours 48 Hours
72 Hours
Time after debridement (Hours)
Bevacizumab
0 Hour
Control
24 Hours
48 Hours
72 Hours
Bevacizumab
0 Hour
24 Hours
48 Hours
72 Hours
Control
•Photographs in the bevacizumab group show subepithelial haze at 24
hours, subepithelial fibrosis at 48 hours, and slight stromal thinning at 72
hours after debridement.
•In the control group, however, epithelial defects were healed without
any haze.
The cornea was trephined with a 4.0-mm-diameter trephine before epithelial debridement
and 24, 48, and 72 hours after wounding
VEGF(pg/ml)
500
400
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70
60
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300
Bevacizumab
200
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50
Control
40
Bevacizumab
30
20
100
Control
*
10
0
24 Hours
A
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NGF(pg/ml)
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48 Hours
72 Hours
0
24 Hours
Time after debridement (Hours)
B
48 Hours
72 Hours
Time after debridement (Hours)
Corneal VEGF and NGF concentrations in the bevacizumab group were decreased
compared to that in the control group at 24, 48, and 72 hours after debridement,
respectively (*P < 0.05; n = 20 per group; Mann-Whitney U test).
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At 48 hours, immunofluorescent stains of VEGF and NGF were most strong in
control corneas(DAPI counter stain )
Bevacizumab
Control
VEGF
NGF
DAPI was used as nuclear staining (blue). Original magnification, 200X.
At 48 hours, immunofluorescent stains of VEGF and NGF were most strong
in control cornea, but no stain in bevacizumab cornea.
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Expression ratio
0.8
0.6
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VEGF
0.4
NGF
0.2
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0
24 Hours
48 Hours
72 Hours
Time after debridement (Hours)
The control VEGF and NGF is regarded as
a standard control (RQ = 1)
VEGF and NGF mRNA expressions in
bevacizumab group were significantly decreased
compared with the control group at 24, 48, and 72
hours after debridement, respectively (*P < 0.05; n
= 20 per group; Mann-Whitney U test)
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Corneal epithelial healing was delayed in Anti-VEGF group
VEGF and NGF was expressed after corneal damage.
VEGF and NGF was less expressed in Anti-VEGF group
VEGF and NGF was more expressed in control group
Anti-VEGF (Bevacizumab) may negatively affect corneal
wound healing.
Anti-VEGF (Bevacizumab) may downregulate NGF
expression.
Caution should be applied in damaged and thin cornea.