Novel Synthetic Peptide Promotes Vascular Angiogenesis In Vitro and In Vivo in a VEGF-Independent Pathway.
Authors: Hardy Britta , Raiter Annat , Weiss Chana , Battler Alexander 
Published: October 2019

Abstract
Angiogenesis is a fundamental process of generating new blood vessels by sprouting of endothelial cells from pre-existing vessels. Angiogenesis plays a crucial role in embryonic organ development and in adult vascular diseases. Adult tissues respond to hypoxia with compensatory mechanisms in which they can survive. Up-regulation of growth factors such as VEGF stimulate growth of vascular endothelial cells. Insufficient angiogenesis is relevant to many diseases such as coronary artery disease and delayed wound healing. Therapeutic angiogenesis aims to treat local hypovascularity by stimulating or inducing neovascularization for the treatment of ischemic vascular disease under the influence of an appropriate angiogenic molecule. We have identified a novel synthetic peptide, RoY, selected from a phage display peptide library by screening against endothelial cells under different physiological conditions. RoY peptide induces angiogenesis in vitro in a VEGF- independent mechanism and promotes collateral vessel growth and blood perfusion in vivo. In vitro assays demonstrated specific binding of peptide to endothelial cells and not to peripheral blood lymphocytes. Proliferation with RoY peptide at different dosages induces significant proliferation reaching 1.6 to 1.8-fold increase over control at 10ng/ml. The ability of RoY peptide to induce migration of endothelial cells was measured at different concentrations and under normoxic and hypoxic conditions using a Boyden cell migration chamber. RoY at 50 ng/ml induced a significant two-fold increase in migration compared to control and similar to VEGF. However, under hypoxia, RoY at 20 ng/ml induced a significant 2.5 fold increase in migration, significantly more migration than a similar dose under normoxia. RoY induced tube formation on Matrigel in both human umbilical vascular and dermal microvascular endothelial cells.
A mouse ear angiogenesis model and a mouse ischemic hind limb model were used to evaluate the in vivo effects of RoY peptide. Angiogenesis in a mouse ear was induced by injection of RoY and was compared to the number of blood vessels in the control ear. RoY induced a 50% increase in the number of blood vessels in the treated ear. Ligation of the femoral artery in C57/ Bl mice induces ischemia in the operated leg. Injection of 1–10μg of RoY peptide to the operated ischemic legs resulted in a 100 percent perfusion in the operated leg versus the control non-operated leg. This increase was maintained and significant 21 days post operation while similar treatment with VEGF was maximal 14 days post operation and at 21 days the percent perfusion decreased. A possible angiogenic mechanisms of the synthetic novel peptide will be discussed. However, we have found that endothelial cells incubated with RoY peptide at different concentrations and times did not induce VEGF A secretion, nor was there an increase in VEGF receptors Flt-1 and Kdr, as revealed by FACS analysis. Gene expression of VEGF-A, Flt-1 and Kdr were not induced by RoY peptide, and the levels determined by Real Time PCR were not significantly different compared to controls. We have evaluated the angiogenic activity of a novel synthetic proangiogenic peptide. Characterization of the nature of endothelial cell signaling by this peptide will provide the basis for the development of targeted angiogenic therapy.