Doxycycline enhances the inhibitory effects of bevacizumab on corneal neovascularization and prevents its side effects.
OBJECTIVE
To investigate the combination therapeutic effects of topical doxycycline temperature-sensitive hydrogel (DTSH) and bevacizumab on corneal neovascularization (CNV) and corneal wound healing (CWH) and to explore the underlying mechanisms of doxycycline on CNV and CWH.


METHODS
Rats were treated with a saline solution, topical DTSH (0.1%), topical bevacizumab (2.5 mg/0.1 mL), or a DTSH and bevacizumab combination. For the bFGF-induced CNV model (n = 15/group), the length and area of CNV were measured on day 7. In the alkali burn model (n = 33/group), the length and area of CNV were determined on days 3, 7, 14, and 21 after alkali burn. The activity of matrix metalloproteinase (MMP)-2 and MMP-9 was determined by a fluorogenic peptide substrate. Western blot, real-time PCR, and ELISA were used to analyze the expression of induced nitric oxide synthase (iNOS), VEGF, VEGFRS, MMP-2, MMP-9, and IL-1β.


RESULTS
Combination therapy more effectively inhibited CNV than therapy with topical bevacizumab or DTSH alone. DTSH combined with bevacizumab significantly accelerated delayed CWH caused by topical bevacizumab in the alkali burn model (P = 0.018). Combination therapy showed better inhibitory effects on MMP expression and phosphorylated VEGFR1 and VEGFR2. With DTSH treatment, doxycycline inhibited the activity and expression of MMPs, the expression of VEGF and of phosphorylated VEGFR1 and VEGFR2, and the production of iNOS and IL-1β in local cornea.


CONCLUSIONS
Doxycycline enhances the inhibitory effects of bevacizumab on CNV and prevents its side effects on CWH, possibly by inhibiting the expression and activity of MMPs, the expression of VEGF and of phosphorylated VEGFR1 and VEGFR2, and the production of iNOS and IL-1β.
State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 XianlieSouth Road, Guangzhou, China.
Wenru
W
Su
Zhanrong
Z
Li
Yongping
Y
Li
Miaoli
M
Lin
Lin
L
Yao
Yi
Y
Liu
Zixin
Z
He
Chuanbin
C
Wu
Dan
D
Liang
D000287:Administration, Topical; D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D061067:Antibodies, Monoclonal, Humanized / Q000494:pharmacology*; D000068258:Bevacizumab; D015153:Blotting, Western; D002057:Burns, Chemical / Q000188:drug therapy; D016510:Corneal Neovascularization / Q000188:drug therapy* / Q000201:enzymology; D004195:Disease Models, Animal; D004318:Doxycycline / Q000494:pharmacology*; D004357:Drug Synergism; D004797:Enzyme-Linked Immunosorbent Assay; D005126:Eye Burns / Q000139:chemically induced; D005260:Female; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D040262:Receptors, Vascular Endothelial Growth Factor / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects
D020533:Angiogenesis Inhibitors; D000900:Anti-Bacterial Agents; D061067:Antibodies, Monoclonal, Humanized; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D000068258:Bevacizumab; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D040262:Receptors, Vascular Endothelial Growth Factor; D020778:Matrix Metalloproteinase 2; C522363:Mmp2 protein, rat; D020780:Matrix Metalloproteinase 9; D004318:Doxycycline