Flt-1 intraceptors inhibit hypoxia-induced VEGF expression in vitro and corneal neovascularization in vivo.
OBJECTIVE
To determine whether subunits of VEGF receptor-1 coupled with an endoplasmic reticulum retention signal can block hypoxia-induced upregulation of VEGF secretion in corneal epithelial cells and block murine corneal angiogenesis induced by corneal injury.


METHODS
Human corneal epithelial cells, transfected with plasmids encoding Flt23K or Flt24K (the VEGF-binding domains of the Flt-1 receptor coupled with the endoplasmic reticulum retention peptide KDEL), were subjected 2 days after transfection to 5% hypoxia for 24 hours. Supernatant was sampled at 24 hours and assayed for VEGF by ELISA. For in vivo models, mouse corneas underwent intrastromal injections of plasmids encoding Flt23K or Flt24K, and 2 days later, sustained injury induced by topical NaOH and mechanical scraping. Corneas were assessed 2 days later for VEGF ELISA and leukocyte counting or 1 week later for quantification of neovascularization.


RESULTS
Hypoxia induced VEGF by human corneal epithelial cells was sequestered by both Flt23K and Flt24K; Flt-1 23K suppressed VEGF secretion as well. Intrastromal delivery of plasmid Flt23K suppressed VEGF by 40.4% (P = 0.009), leukocytes by 49.4% (P < 0.001), and neovascularization by 66.8% (P = 0.001). Flt24K suppressed VEGF expression by 30.8% (P = 0.042), leukocytes by 25.8% (P < 0.001), and neovascularization by 49.5% (P = 0.015).


CONCLUSIONS
Flt-1 intraceptors, which are endoplasmic reticulum retention signal-coupled VEGF receptors, significantly suppress hypoxia-induced VEGF secretion by corneal epithelial cells in vitro. In vivo, delivery of naked plasmids expressing these intraceptors inhibits injury-induced upregulation of VEGF, leukocyte infiltration, and corneal neovascularization.
Singh
Nirbhai
N
Department of Ophthalmology, Medical College of Georgia, 1120 15th Street, Augusta, GA 30912, USA.
Amin
Shivan
S
Richter
Elizabeth
E
Rashid
Saadia
S
Scoglietti
Vincent
V
Jani
Pooja D
PD
Wang
Jin
J
Kaur
Rajwinder
R
Ambati
Jayakrishna
J
Dong
Zheng
Z
Ambati
Balamurali K
BK
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D016510:Corneal Neovascularization / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D004338:Drug Combinations; D004721:Endoplasmic Reticulum; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D005822:Genetic Vectors; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D007958:Leukocyte Count; D051379:Mice; D018995:Myosin Heavy Chains; D024501:Nonmuscle Myosin Type IIB; D010957:Plasmids; D011506:Proteins / Q000502:physiology*; D018000:Receptors, Peptide / Q000502:physiology*; D014162:Transfection; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1
D004338:Drug Combinations; D016326:Extracellular Matrix Proteins; C064459:KDEL receptor; D011506:Proteins; D018000:Receptors, Peptide; D042461:Vascular Endothelial Growth Factor A; C501162:FLT1 protein, human; C501163:Flt1 protein, mouse; D040281:Vascular Endothelial Growth Factor Receptor-1; D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains