Unique homologous siRNA blocks hypoxia-induced VEGF upregulation in human corneal cells and inhibits and regresses murine corneal neovascularization.
OBJECTIVE
To determine whether RNA interference (RNAi) could block hypoxia-induced upregulation of vascular endothelial growth factor (VEGF) in human corneal epithelial cells in vitro and inhibit and regress injury-induced murine corneal neovascularization in vivo.


METHODS
siRNA selected on the basis of target sequence homology between mouse and human VEGF was placed into expression cassettes and transfected into human corneal epithelial cells. Hypoxia-induced VEGF synthesis was assayed. Also, the effect of a plasmid capable of directing the expression of an siRNA against VEGF when injected into mouse corneas 8 hours before alkali-mechanical trauma was studied. Leukocyte count, VEGF protein levels, and degree of neovascularization in corneas were compared with that of a control siRNA plasmid. Plasmids were injected 1 week after injury to assess the ability of RNAi to regress corneal neovascularization.


RESULTS
Hypoxia-induced VEGF mRNA synthesis and protein secretion by human corneal epithelial cells was efficiently suppressed by an siRNA targeted against a sequence uniquely identical for the mouse and human VEGF genes. Intrastromal delivery of a plasmid expressing this siRNA before murine corneal injury suppressed corneal VEGF by 55.7% versus control (P = 0.014), leukocyte infiltration by 69.5% (P < 0.001), and neovascularization 1 week after injury by 72.3% (P = 0.001). At the regression time point, treated corneas had 72.8% less neovascularization (P < 0.001).


CONCLUSIONS
RNAi significantly suppresses expression of VEGF induced by hypoxia in human corneal epithelial cells in vitro. In vivo, intrastromal delivery of a plasmid expressing siRNA against VEGF suppresses injury-induced VEGF expression, leukocyte infiltration, and angiogenesis and was able to regress corneal neovascularization.
Singh
Nirbhai
N
Department of Ophthalmology, Medical College of Georgia, Augusta, GA, USA.
Higgins
Eric
E
Amin
Shivan
S
Jani
Pooja
P
Richter
Elizabeth
E
Patel
Anup
A
Kaur
Rajwinder
R
Wang
Jin
J
Ambati
Jayakrishna
J
Dong
Zheng
Z
Ambati
Balamurali K
BK
D000818:Animals; D018929:Cell Culture Techniques; D016510:Corneal Neovascularization / Q000378:metabolism / Q000517:prevention & control*; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000378:metabolism*; D020868:Gene Silencing; D018390:Gene Targeting; D006801:Humans; D000860:Hypoxia / Q000378:metabolism / Q000517:prevention & control*; D007958:Leukocyte Count; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse