title: Doxycycline-mediated inhibition of corneal angiogenesis: an MMP-independent mechanism.
abstract: OBJECTIVE
  We explored the underlying mechanisms of doxycycline inhibition of corneal angiogenesis.
  
  
  METHODS
  Human umbilical vein endothelial cells (HUVECs) were cultured in vitro in the presence or absence of VEGF, doxycycline, or broad-spectrum matrix metalloproteinase (MMP) inhibitors (1,10-phenanthroline [1PT] and batimastat). HUVEC proliferation was determined using Cell Counting Kit-8. Rats with VEGF-induced corneal neovascularization (CNV) were treated with saline solution, 0.1% doxycycline, 0.1% 1PT, or 50 μM batimastat (n = 7/group). The length and area of CNV were measured on day 7. The activity of MMP-2 and MMP-9 was determined by a fluorogenic peptide substrate. Western blotting and ELISA were used to analyze the expression of phosphorylated eNOS and Akt, and PI3K activity.
  
  
  RESULTS
  Our results showed that doxycycline inhibited HUVEC proliferation induced by VEGF in a dose-dependent manner in vitro, and decreased CNV induced by VEGF in vivo in terms of vessel length and area. 1PT and batimastat showed similar MMP inhibitory functions with doxycycline in vitro and in vivo, but they had no effects on HUVEC proliferation, and only partially mimicked the inhibitory properties of doxycycline (∼45%) on angiogenesis induced by VEGF. In addition, although doxycycline is capable of modulating the PI3K/Akt-eNOS pathway in vitro and in vivo in an MMP-independent manner, 1PT and batimastat were not.
  
  
  CONCLUSIONS
  The mechanism of doxycycline-mediated inhibition of angiogenesis occurs through MMP inhibitory activity and modulation of the PI3K/Akt-eNOS pathway, an MMP-independent mechanism.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China. suwr3@mail.sysu.edu.cn.
authors.forename: Wenru
authors.initials: W
authors.lastname: Su
authors.forename: Zhanrong
authors.initials: Z
authors.lastname: Li
authors.forename: Fan
authors.initials: F
authors.lastname: Li
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Chen
authors.forename: Qian
authors.initials: Q
authors.lastname: Wan
authors.forename: Dan
authors.initials: D
authors.lastname: Liang
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D016510:Corneal Neovascularization / Q000175:diagnosis / Q000378:metabolism / Q000517:prevention & control*; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004318:Doxycycline / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D042461:Vascular Endothelial Growth Factor A / Q000633:toxicity
chemical_list: D020533:Angiogenesis Inhibitors; D011480:Protease Inhibitors; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt; D020782:Matrix Metalloproteinases; D004318:Doxycycline