The Effect Of ERC(N5) Peptide On The Proliferation And Invasion Of Human Umbilical Vein Endothelial Cells (Huvec)

Objectives1. To analyze the binding statues of ERC(N5) and human umbilical vein endothelial cells (HUVEC).2. To investigate the effects of ERC(N5) on the apoptosis and invasion behaviors of HUVEC.Methods1. Design and synthesis of ERC(N5) peptideThe RGD motif with its flanking sequences and C-terminal amino acids of Echistatin (Ech) were conjugated together with the mutation of the fifth amino acid from D to N. The resulted small peptide was designated as ERC(N5) with the sequence of ARGDNMPRNPHK GPAT. It was synthesized by CL.(Xi’an) Bio-Scientific CO.LTD.2. Analysis of binding statues between ERC(N5) and HUVECThe content of integrin αvβ3on cell surface was detected by flow cytometry. The positively staining cells were examined after ERC(N5) or Ech, FITC-conjugated αvβ3monoclonal antibody (LM609) and HUVEC were incubated together. They were used to analyze the competitive binding of ERC(N5)/Ech and LM609.3. Effects of ERC(N5) on apoptosis and invasion behaviors of HUVECThe morphology change of HUVEC treated by ERC(N5) was observed by converted microscope and AO/EB staining. The apoptotic effect of ERC(N5) on HUVEC was examined by Annexin V-FITC/PI double staining. The RNA level expression of caspase3was detected by RT-PCR. The effects of ERC(N5) on migration and invasion behaviors of HUVEC were observed by transwell migration assay, wound healing assay and Matrigel invasion assay. Results1. The content of αvβ3on HUVEC surface was68.3%.2. After HUVECs were incubated with FITC-LM609and different concentration of ERC(N5), the αvβ3positively staining cells decreased with the rising concentration of ERC(N5). And the positively staining cells were1.2%and26.8%when the HUVECs were incubated with16μM Ech and ERC(N5), respectively. This indicates that ERC(N5) could bind to αvβ3stronger than Ech.3. The morphology of HUVECs became round with increasing cell gap and debris as well as orange dying apoptotic cells after incubation with ERC(N5) for24hours. The results of FCM showed that the apoptosis percentage increased5-fold after HUVECs were treated by ERC(N5).4. RT-PCR results showed that the RNA level expression of caspase-3gene increased in a dose-dependent manner compared with the negative control group.5. After treated by ERC(N5), the migration and invasion ability of HUVEC were decreased by78.5%and87%respectively. Wound healing assay showed that the negative control cells moved rapidly and filled the denuded surface after24h, while the wound was still clear in the ERC(N5)-treated cells.Conclusions1. HUVECs could highly express integrin αvβ3, ERC(N5) could bind to HUVEC through competitive binding to αvβ3with LM609.2. ERC(N5) could suppress HUVECs proliferation and induce apoptosis. It could also inhibit the migration and invasion of HUVECs.