The Effect And Mechanism Of Novel Peptide CW-703 On Ocular Neovascularization Inhibition

Purpose:To synthesize a novel peptide CW-703 derived from functionally active site of insulin-like growth factor-2(IGF-2).To preliminarily study and discuss the efficacy and safety of the peptide in ocular neovascularization inhibition and its possible mechanism in vitro and in vivo.Methods:1.Screening?synthesis and identification of peptide: A sequence of amino acids that mimic the active site of IGF-2 were screened by protein sequence alignment and crystal threedimensional structure analysis;the novel peptide CW-703 was synthesized through in vitro solid-phase synthesis technology and was identified using high performance liquid chromatography and mass spectrometry analysis.2.Efficacy and safety of CW-703 in ocular neovascularization inhibition: In vitro study covering 4 classical models: endothelial cell proliferation assay,cell migration assay including scratch wound assay and transwell assay,and tubule formation assay aimed to confirm the efficacy of CW-703 in inhibiting neovascularization.The safety of the peptide was evaluated through MTS and TUNEL test to confirm whether CW-703 leads to the inhibition of cell activity and apoptosis in endothelial cells.Two animal models: chick chorioallantoic membrane(CAM)and oxygen-induced retinopathy(OIR)models were built to study the efficacy of CW-703 in inhibition of neovascularization in vivo,meanwhile,visual electrophysiological technique and light microscopy were performed to evaluate the effect of CW-703 on retinal structure and physiological function.3.Mechanism of CW-703 in ocular neovascularization regulation: In OIR animal model,mRNA and protein levels of VEGF,PEDF,IGF-1,IGF-2 and IGF-1R in retina were measured using real-time PCR,Western blot and ELISA test to determine the effects which CW-703 exerts on the cytokines and receptor above.The protein expression of PI3K,Akt and ERK,key signal messengers related to angiogenesis,was measured using Western blot to preliminarily study the downstream signal transduction pathway in CW-703's inhibiting ocular neovascularization.Results:1.Screening by bioinformatics methods,peptide CW-703 containing 12 amino acids was synthesized through in vitro solidphase synthesis technology,with sequence: LCGGELVDTLQF,MW:1294.49 Da.2.In vitro experiments,CW-703 in different concentrations can all significantly inhibit VEGF-induced chemotaxis of human umbilical vein endothelial cells(HUVECs);in scratch wound assay,when the peptide concentration reached 1?M or above,the uncovered wound area was much bigger in CW-703 treated group than in VEGF group;when the concentration reached 100 n M or above,VEGF-induced tubule formation in HUVECs was significantly suppressed,and CW-703 have inhibitory effect on VEGF-induced proliferation of HUVECs when the concentration reached 100?M.The results of MTS and TUNEL test showed that there was no significant difference in cell activity and apoptosis between CW-703 treated group and other control group.In vivo experiments,compared with hyperoxia +PBS group,hyperoxia +CW-703 group had enlarged avascular area in CAM model;in OIR model,foveal avascular zone and peripheral neovascularization area in retina were significantly reduced among mice receiving intravitreal injection of CW-703,compared with those receiving PBS.For safety evaluation,Wistar rats received intravitreal injection of CW-703 and PBS,the b-wave amplitude in electroretinogram(ERG)did not differ obviously between CW-703-treated group and control group.3.In OIR animal model,compared with hyperoxia + PBS group,the mRNA and protein expression of VEGF decreased,while the mRNA and protein expression of PEDF and IGF-1 increased in hyperoxia +CW-703 group.The protein level of IGF-2 decreased obviously when treated with CW-703,while its mRNA level remained unchanged.There was no significant difference in mRNA and protein expression of IGF-1R between the two groups,while the protein level of phosphorylated IGF-1R decreased in by CW-703.The total protein and mRNA expression of PI3 K,Akt and ERK remained unchanged,while phosphorylated PI3 K,Akt and ERK were down-regulated by CW-703 in OIR.Conclusions:In vitro,CW-703 significantly inhibits VEGF-induced cell proliferation,migration and tubule formation with almost no side effects on cell activity and apoptosis.In vivo,CW-703 also leads to a significant neovascularization inhibition in both CAM and OIR animal models without any toxic effects on normal endothelial cell,and any abnormal on retinal structure and function.The possible mechanism of CW-703 acting on neovascularization inhibition may be related to the down-regulation of phosphorylated IGF-1R level in retina and blocking PI3K/Akt and MAPK/ERK activation in downstream signaling pathway.Meanwhile,the up-regulated level of VEGF is accompanied with down-regulated level of PEDF,the rebalance of angiogenic stimulators and inhibitors may also contribute to this novel peptide's neovascularization inhibition effect.