Investigation Of Anti-angiogenic Activity And Comparison Of Phosphorylation Of Signaling Proteins By RVBMDMP

Background Vascular basement membrane-derived multifunctional peptide (VBMDMP) sequence (fusion gene of human IgG3 upper hinge region and two tumstatin-derived fragments) obtained by artificial synthesis was cloned into Vector pUC19, was introduced into an expression Vector pGEX-4T-l to construct a prokaryotic expression Vector pGEX-4T-1-VBMDMP. VBMDMP was produced as a recombinant molecule in Escherichia coli. rVBMDMP has been proved to have significant biological activity of anti-tumor growth and anti-metastasis in Lewis lung carcinoma transplanted into mouse C57BL/6. In this study, we investigate the inhibition of proliferation and induction of apoptosis activities of Recombinant Vascular Basement-derived Multifunctional Peptide (rVBMDMP) in human umbilical vein endothelial (HUVEC-12) cell. In addition, phosphorylation changes of signaling molecules in human umbilical vein endothelial (HUVEC-12) cell were detected in order to initially elucidate the molecular mechanism of anti-angiogenic of vascular basement-derived multifunctional peptide.Methods The effect of rVBMDMP on the proliferation of vascular endothelial cell was measured by MTT assay. The influence of rVBMDMP on the growth of vascular endothelial cell was determined by cell counting method in trypan blue staining. The effect of rVBMDMP on the formation of endothelial tube was surveyed by endothelial tube assay. We adopt flow cytometry to detect vascular endothelial cell apoptotic rate and acridine orange staining method to observe the morphological changes of vascular endothelial cell after treatment with rVBMDMP. The effect of rVBMDMP on the migration of vascular endothelial cell was viewed by cell scratch method. HUVEC—12 cells cultured in vitro were tread with rVBMDMP(1.0μM) or vehicle (sterile PBS) as control, followed by preparation of whole cell extracts. Signal transduction antibodyarray ^TM(Hypromatrix) was employed for screening protein tyrosine (serine and threonine) phosphorylation.ResultsThe results of MTT assay and cell counting method in trypan blue staining showed that rVBMDMP inhibit the proliferation and growth of vascular endothelial cell in a concentration-and time-dependent manner. After treatment with rVBMDMP at 1.0μM for 96h, humerus of viable cells is only 50％of control group. Experimental results indicated that rVBMDMP distinctly reduce the number of endothelial tube (17.67±3.055 vs 50.33±3.055). Flow cytometric analysis exhibited that vascular endothelial cell apoptotic rate was increased accompanied by the raise of rVBMDMP's concentration. The apoptotic rate is 14.41％after treatment with rVBMDMP at 1.0μM for 72h. We can observe typical apoptotic cells with fluorescence microscope. Cell scratch method showed rVBMDMP has no effect on the migration of vascular endothelial cell.Level of phosphorylation of signaling proteins were elevated in rVBMDMP-treated compared with control, including cell integrin protein av,β1 andβ3 subunit,cell adhesion molecule (PECAM—1, ICAM—1); apoptosis perform molecule Caspase 3, cell frame and tight junction protein (Connexin 43, Ezrin, Paxillin, ZO—1, PAR—3); non-receptor tyrosine kinase Axl, Csk and transcription factor ATF—3, p—ATF—3; death receptor including DR3, DR4, DR5, receptor interaction protein (RIP), interferon relative factor (IRF1, IRF2). In contrast, level of phosphorylation of signaling proteins were descended, including cell cycle regulation protein (Cyclin A, Cyclin B, CyclinE, CDK 2); epithelium growth factor receptor (EGFR, p-EGFR), vascular endothelial growth factor receptor VEGFR1, SURVIV, earlier period growth response gene Egr—1, the signal transducer and activator of transcription(STAT 1) .Conclusion1. rVBMDMP possesses the effect of inhibiting proliferation of human vascular endothelial cell. 2. rVBMDMP significantly induces apoptosis of human vascular endothelial cell.3. rVBMDMP possesses the inhibitory effect of endothelial tube formation of human vascular endothelial cell.4. The possible molecular mechanisms of rVBMDMP's anti-angiogenic activity as following:(1) rVBMDMP displays the effect of inhibiting proliferation and inducing apoptosis of endothelial cell by interacting with integrin protein av,β1,β3 subunit, then, increasing the level of phosphorylation of them, altering them conformation and activity and impacting complex and multieffect signal transduction pathway of integrin protein a1β1 and avβ3.(2) rVBMDMP can degrade the level and activity of phosphorylation of epithelium growth factor receptor and vascular endothelial growth factor receptor and exhibit the effect of inhibiting proliferation and inducing apoptosis of endothelial cell via signal transduction two-way flow model between signal transduction of integrin protein and receptor tyrosine kinase.(3) rVBMDMP promotes apoptosis of endothelial cell through cross talk of integrin protein signal transduction and SURVIV signal transduction, then, degrading the level and activity of phosphorylation of SURVIV.