Flt-1 intraceptor induces the unfolded protein response, apoptotic factors, and regression of murine injury-induced corneal neovascularization.
OBJECTIVE
To determine whether Flt24K, a recombinant construct of domains 2 to 4 of VEGFR-1 (Flt) coupled with an endoplasmic reticulum retention signal (KDEL) can bind VEGFR-2 and induce apoptosis, unfolded protein response (UPR), and regression of injury-induced corneal neovascularization.


METHODS
Human microvascular endothelial cells were transfected with pCMV.Flt24K and subjected to hypoxia. Cell lysates underwent Western blot analysis with anti-XBP-1 antibody and RT-PCR for CHOP. Human malignant melanoma cells (which express VEGFR-2 but not Flt), were transfected with pCMV.Flt24K, and lysates underwent immunoprecipitation with anti-FLT antibody, and Western blot analysis for VEGF and VEGFR-2. Mouse corneas sustained injury induced by topical NaOH and mechanical scraping and were injected with pCMV.Flt24K 2 weeks later. Corneas were harvested 2 days later for Western blot analysis for XBP-1 and caspase-3 or 1 week later for quantification of neovascularization and TUNEL staining. Saline and empty pCMV vector were used in control experiments.


RESULTS
The mean percentage area of corneal neovascularization in mice 3 weeks after corneal injury and 1 week after intrastromal injection of empty pCMV vector or pCMV.Flt24K was 55.4% +/- 2.7% vs. 19.3% +/- 6.1%, respectively (P < 0.001). Flt24K was found to bind VEGFR-2 and upregulate activated XBP-1 and CHOP in vitro. In vivo, pCMV.Flt24K upregulated activated XBP-1 and caspase-3. Apoptosis was observed in corneal neovascular endothelium in corneas treated with pCMV.Flt24K but not in the control.


CONCLUSIONS
The Flt24K intraceptor can bind VEGFR-2 within cells, induce the unfolded protein response in vitro and in vivo, elicit apoptosis of vascular endothelial cells in vivo, and induce regression of corneal neovascularization in vivo.
Singh
Nirbhai
N
Department of Ophthalmology, Medical College of Georgia, 1120 15th Street, Atlanta, GA 30912, USA.
Jani
Pooja D
PD
Suthar
Tushar
T
Amin
Shivan
S
Ambati
Balamurali K
BK
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D002057:Burns, Chemical / Q000188:drug therapy* / Q000209:etiology / Q000378:metabolism; D053454:Caspase 12 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D003315:Cornea / Q000187:drug effects / Q000378:metabolism; D016510:Corneal Neovascularization / Q000188:drug therapy* / Q000209:etiology / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000473:pathology*; D005126:Eye Burns / Q000139:chemically induced*; D005822:Genetic Vectors; D006801:Humans; D020287:In Situ Nick-End Labeling; D051379:Mice; D008807:Mice, Inbred BALB C; D009687:Nuclear Proteins / Q000378:metabolism*; D018000:Receptors, Peptide / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012972:Sodium Hydroxide / Q000633:toxicity; D014157:Transcription Factors; D014162:Transfection; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000502:physiology*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D000071717:X-Box Binding Protein 1
D004268:DNA-Binding Proteins; C064459:KDEL receptor; D009687:Nuclear Proteins; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C000605544:Xbp1 protein, mouse; D012972:Sodium Hydroxide; C501162:FLT1 protein, human; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2; D053454:Caspase 12; D053148:Caspase 3