| Objective: Recombinant Vascular Basement Membrane Derived Multifunctional Peptide (rVBMDMP) was designed and produced by integration of two tumor therapeutic strategies: inhibition of tumor angiogenesis and inhibition of tumor cell proliferation. Our former research confirmed that rVBMDMP inhibits proliferation of certain tumor cells and endothelial cells. The main aim of this study was to further explore mechanism of action of rVBMDMP inhibiting angiogenesis by interaction with endothelial cells.Methods: Human umbilical vein endothelial cell line (HUVE-12) cells were cultured; plate colony formation assay was used to detect inhibitory effect of rVBMDMP on growth of serum-stimulated HUVE-12 cells; AO and EB duel staining under fluorescence microscope and DNA agarose gel electrophoresis were employed to detect apoptosis induction of rVBMDMP on serum-activated HUVE-12 cells. The matrigel coated-96 well plate model and murine matrigel plug model were employed to evaluate inhibitory effect of rVBMDMP on VEGF stimulated- HUVE-12 cell tube structure formation in vitro and in vivo. High throughput cDNA microarray of extracellular matrix and adhesion molecules was used to identify gene expression at the transcriptional level, involved in rVBMDMP mediated serum-stimulated HUVE-12 cell angiogenesis balance alteration; real-time PCR was used to confirm the results of cDNA microarray. Results: Plate colony formation assay indicated that rVBMDMP potently inhibited growth of serum stimulated-HUVE-12 cells in a concentration dependent manner. AO and EB dual staining under fluorescence microscope demonstrated that by rVBMDMP at 0.1, 1.0, 10.0μM concentrations treatment, the number of serum-stimulated HUVE-12 cells with morphological feature of apoptosis including chromatin condensation and nuclei fragmentation increased; there was highly statistical significance (P<0.05) in the apoptotic index between rVBMDMP at 0.1, 1.0, 10.0μM concentrations treatment (11.8%±5.8%, 51.9%±10.6%, and 75.5%±11.6%, respectively) and the control (0.2%±0.8%). DNA agarose gel electrophoresis demonstrated that two lanes loaded total DNA of HUVE-12 cells by 1.0μM rVBMDMP treatment for 24h and 48h shew typical DNA ladder, respectively. In vitro matrigel coated 96 wells plate endothelial cell tube structure formation assay indicated that the number of endothelial cell tube structure by 1.0 and 10.0μM rVBMDMP for 48h (4.1±1.3 and 3.4±0.9 per well, respectively), compared with the control (36.1±3.9 per well), significantly decreased (P<0.01). Murine matrigel plug endothelial cell tubular structure formation assay indicated that after H.E staining under light microscope (×40 magnification), the number of endothelial cell tubular structure by 1.0 and 10.0μM rVBMDMP treatment for 7d (9.0±2.4 and 8.5±2.1 per 5 visual fields, respectively), compared with the control (48±6.9 per 5 visual fields), significantly decreased (P<0.01); DAPI staining under fluorescence microscope (×40 magnification), the number of endothelial cell tubular structure by 1.0 and 10.0μM rVBMDMP treatment for 7d (8.0±2.1 and 7.0±2.6 per 5 visual fields, respectively), compared with the control (37.0±5.2 per 5 visual fields), significantly decreased (P<0.01). cDNA microassay indicated that the transcriptional expression of extracellular matrix molecules (including Collagen 4α2, Collagen 6α2, Collagen 7α1, Collagen 15α1 and Collagen 27α1) on HUVE-12 cells by 1.0μM concentration rVBMDMP treatment, compared with the control, increased more than 2 times; the transcriptional expression of extracellular matrix molecules (including IntegrinαV,Integrinβ7,Integrinβ8,Integrinα7,Integrinβ2, MMP12, MMP1, MMP11 and MMP7) decreased less than 50%. Real time PCR revealed that when compared with the control and corrected by internal control gene, the transcriptional expression of collagen7α1 increased by rVBMDMP at 0.1, 1.0 and 10.0μM concentrations treatment; the transcriptional expression of integrinαv, integrinβ3, MMP-10 and MMP-12 decreased by rVBMDMP at 0.1, 1.0 and 10.0μM concentrations treatment.Conclusion:1. rVBMDMP induces HUVE-12 cell growth inhibition and apoptosis.2. rVBMDMP inhibits formation of HUVE-12 cell tube structure in vitro and in vivo.3. The possible mechanism of action of rVBMDMP inhibiting angiogenesis is that rVBMDMP induces HUVE-12 cell apoptosis, mediates endothelial cell angiogenesis balance destruction by regulating the transcriptional expression of extracellular matrix molecules and MMPs on endothelial cells, by altering microenvironment of angiogenesis, by favoring the advancement of angiogenesis inhibition.
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