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Subrahmanian Tarakkad Krishnaji, Haojie Fu, Amy Birsner, Lauren Bazinet, Robert J D'Amato; Peptides that Modulate Angiogenesis. Invest. Ophthalmol. Vis. Sci. 2018;59(9):234.
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© ARVO (1962-2015); The Authors (2016-present)
We have recently found that Prominin-1 interacts with vascular endothelial growth factor (VEGF) and enhances its activity. We have designed Prominin-1 peptides that modulate angiogenesis using functional endothelial cell assays in vitro, as well as laser-induced choroidal neo-vascularization (CNV) and corneal micropocket assay (CoNV) models in vivo in mice.
We evaluated the effect of two 12mer peptides isolated from Prominin-1 (G8 and S6, and respective scrambled controls G8Scr and S6Scr) on endothelial cell migration, tube formation, and modulation of VEGF receptor (VEGFR2) phosphorylation by western blot and ELISA. We then tested the peptides in CNV and CoNV assays with C57BL/6J mice. For CNV assays, mice received 1µl of peptide drugs in dimethylsulfoxide at 30mg/mL concentration intravitreally 2 days after laser. The animals were euthanized, choroids isolated and immunostained, and CNV area (µm2) was measured on day 7. In the CoNV assay, peptides were incorporated into slow-release sucralfate co-pellets with VEGF at 1:20 ratio of VEGF:Peptide (4 pmol of VEGF per dose). The pellets were surgically implanted into a micropocket made by an incision in the cornea. On day 6, the vessel growth was quantified as vessel area (VA, mm2).
We observed that G8 significantly increased angiogenesis, whereas S6 significantly inhibited it. Western blot analysis showed that G8 treatment increased pVEGFR2 whereas S6 treatment showed decreased pVEGFR2 (Y1175) and reduced total VEGFR2. Correlating with the effects on pVEGFR2, we observed changes in tube formation (G8, P < 0.05 and S6, P < 0.01) and migration (G8, P < 0.05 and S6, P < 0.001) assays. The peptides were potent in promoting (G8, ~35%, P < 0.01) or inhibiting (S6, ~35%, P < 0.001) vessel growth in the CoNV model. Additionally, G8 peptide treatment increased the CNV area significantly in treated mice (41226 ± 6183 µm2) when compared to the control group (22551 ± 4772 µm2, P < 0.05).
Herein we show that Prominin-1 peptides significantly and potently modulated angiogenesis in both CoNV and CNV models, suggesting that they may be useful for the treatment of angiogenesis dependent diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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