title: Blockade of the intermediate-conductance Ca(2+)-activated K+ channel inhibits the angiogenesis induced by epidermal growth factor in the treatment of corneal alkali burn.
abstract: Epidermal growth factor (EGF) is used to treat alkali-burned corneas. However, EGF-induced corneal angiogenesis, which is currently untreatable, is a side effect of this therapy. We therefore explored the role of the intermediate-conductance Ca(2+)-activated K(+) channel (KCa3.1) in EGF-induced angiogenesis and tested whether KCa3.1 blockade can suppress EGF-induced corneal angiogenesis. The proliferation, migration and tube formation of HUVECs (human umbilical vein endothelial cells) in response to EGF, the MEK inhibitor PD98059 and the KCa3.1 inhibitor TRAM-34 were analyzed in vitro via MTT, cell counting, scratch and tube formation assays. The protein and mRNA levels of KCa3.1, phosphorylated-ERK (P-ERK), total-ERK (T-ERK), cyclin-dependent kinase 4 (CDK4), vimentin and MMP-2 were assessed via western blotting and RT-PCR. KCa3.1 and vimentin expression were also detected through immunofluorescence staining. Flow cytometry was performed to examine the cell cycle. Further, an in vivo murine alkali-burned cornea model was developed and treated with EGF and TRAM-34 eye drops to analyze the effect of these treatments on corneal healing and angiogenesis. The corneas were also analyzed by histological staining. The in vitro results showed that EGF induces the upregulation of KCa3.1 and P-ERK in HUVECs and that this upregulation is suppressed by PD98059. EGF stimulates proliferation, migration and tube formation in HUVECs, and this effect can be suppressed by TRAM-34. TRAM-34 also arrests HUVECs in the G1 phase of the cell cycle and downregulates CDK4, vimentin and MMP-2 in these cells. The in vivo results indicated that TRAM-34 suppresses EGF-induced corneal angiogenesis without affecting EGF-induced corneal wound healing. In summary, the upregulation of KCa3.1 may be crucial for EGF-induced angiogenesis through the MAPK/ERK signaling pathway. Thus, KCa3.1 may be a potential target for the treatment of EGF-induced corneal angiogenesis.
authors.affiliation: Department of Anatomy, Harbin Medical University, Harbin 150081, China.
authors.forename: Huike
authors.initials: H
authors.lastname: Yang
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Ma
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Lv
authors.forename: Shu
authors.initials: S
authors.lastname: Zhao
authors.forename: Wen
authors.initials: W
authors.lastname: Lang
authors.forename: Yafang
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002057:Burns, Chemical / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D002465:Cell Movement; D049109:Cell Proliferation; D003315:Cornea / Q000187:drug effects; D016510:Corneal Neovascularization / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D051358:Cyclin-Dependent Kinase 4 / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D004815:Epidermal Growth Factor / Q000633:toxicity*; D005126:Eye Burns / Q000139:chemically induced*; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism / Q000473:pathology; D006801:Humans; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011720:Pyrazoles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012972:Sodium Hydroxide; D015854:Up-Regulation; D014746:Vimentin / Q000235:genetics / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects
chemical_list: D005419:Flavonoids; D051660:Intermediate-Conductance Calcium-Activated Potassium Channels; C496925:Kcnn4 protein, mouse; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D012333:RNA, Messenger; C411671:TRAM 34; D014746:Vimentin; D012972:Sodium Hydroxide; D004815:Epidermal Growth Factor; C495900:CDK4 protein, human; D051358:Cyclin-Dependent Kinase 4; D020928:Mitogen-Activated Protein Kinases; D020778:Matrix Metalloproteinase 2; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one