| Angiogenesis, the growth of new blood vessels, is essential during fetal development, female reproductive cycle, and tissue repair. While, uncontrolled angiogenesis contributes greatly to the malignant growth and metastasis of cancer cells. Inhibition of tumor angiogenesis as an attractive anticancer strategy has gained extensive support from scientists and clinicians. The anti-angiogenesis strategy has several theoretic advantages, including broad-spectrum anti-tumor activities, lower incidence of drug resistance, and the avoidance of pharmacokinetic problems.One of the most specific and critical regulators of angiogenesis is vascular endothelial growth factor (VEGF, also known as vascular permeability factor, VPF), which is an endothelial cell-specific mitogen and an angiogenic inducer. There are 6 members in VEGF family: VEGF, PIGF, VEGF-B, VEGF-C, VEGF-D and VEGF-E.VEGF receptors are type III receptor tyrosine kinases, VEGFR1 (Flt-1; fms-like tyrosine kinase) and VEGFR2 (Flk-1/KDR; kinase insert domain containing receptor) are expressed primarily on endothelial cells. Gene targeting studies show that both VEGFR1 and VEGFR2 are essential for development of the embryonic vasculature in mice. VEGFR3 (Flt-4) mainly expressed on lymphatic vessels. Flk-1 is considered the most potent mitogenic receptor and neuropilin aids in the binding of VEGF to Flk-1, thus increasing its mitogenic capability. VEGFR3 binds to VEGF-C and VEGF-D to control the growth and maintenance of lymphatic vessels. VEGFR1 is critical in the recruitment of haematopoietic precursors and monocytes to the pathological pro-inflammatory sites and promoting angiogenesis. The binding of VEGFR to its ligand depends mostly on certain extracellular immunoglobulin-like domain, they are the second and third domain of VEGFR1, the third domain of VEGFR2 and the first and second domain of VEGFR3.Considering the high affinity of VEGFR to VEGF, soluble VEGFR maybe block VEGF more effectively than anti-VEGF antibodies. The success of VEGF trap 12(VEGFR1D2+VEGFR2D3+IgG1Fc) have proved this hypothesis. Based on their practice, we tried to further recombine these key domains to find better traps for block VEGF/VEGF-C. In this study, we1. Cloned full length cDNA of VEGF121, VEGF165, VEGF-C and extracellular domain cDNA of VEGFR-1, VEGFR-2, VEGFR-3 by RT-PCR and chemical synthesis; constructed VEGF121-pCDNA3.1, VEGF165-pCDNA3.1, VEGF-C -pIRES2–EGFP, VEGFR-1/Fc-pCDNA3.1, VEGFR-2/Fc-pCDNA3.1, VEGFR-3/Fc-pCDNA3.1, VEGF Trap12-pCDNA3.1, VEGF Trap1C-pIRES, VEGF Trap3C-pIRES, VEGF Trap1H-pIRES, VEGF Trap1H-pBudCE4.1, VEGF Trap3H-pIRES and VEGF Trap3H-pBudCE4.1 expression vector.2. Constructed stable expression recombinant CHO cell clones of above molecules and purified proteins form cell culture supernatant, these proteins were analysed by SDS-PAGE, Western Blot, and Lc-Ms/Ms. The character of this protein are listed blow:1) the expression level of VEGF121, VEGF165, VEGFR-1/Fc, VEGFR-2/Fc, VEGFR-3/Fc and VEGF Trap12 reach about 2-5μg/106cell·24h; the expression level of VEGF Trap1C, VEGF Trap1H, VEGF Trap3H reach about 50-280 ng/ 106cell·24h and VEGF-C,VEGF Trap3C about 1-5 ng/106cell·24h.2) the molecular weight of VEGF121 was measured at 27, 32, 36.5, 40kD (4 bands) in non-reducing SDS-PAGE and 20, 15, 12 kD (3 bands) in reduced SDS-PAGE; the molecular weight of VEGF165 was measured at about 38, 44kD (2 bands) and 25, 20, 15kD (3 bands) in non-reducing and reduced SDS-PAGE; VEGFR1/Fc about 81kD and 162kD in reduced and non-reducing SDS-PAGE with purity of 91%; VEGFR2/Fc about 96kD and 174kD in reduced and non-reducing SDS-PAGE with purity of 96%; VEGFR3/Fc about 107kD and 190kD in reduced and non-reducing SDS-PAGE with purity of 93%; VEGF trap12 about 68kD and 143kD in reduced and non-reducing SDS-PAGE with purity of 87%; VEGF trap1C about 64kD and 124kD in reduced and non-reducing SDS-PAGE, which is different to our expectation; VEGF trap1H, VEGF trap3H are also out of design。3) the peptide sequence of VEGF121, VEGF165, VEGFR-1/Fc, VEGFR-2/Fc and VEGF Trap12 detected by Lc-Ms/Ms well match the database with sequence coverage of 30-60%.3. Assayed the affinity constant of some VEGF trap, tested its influence on hyperplasia, immigration and tuber formation of endotheliar cells, on CAM angiogenesis and on in vivo angiogenesis and xenograft tumor growth in nude mice, we get:1) the KD value detected by BIAcore of VEGF trap, VEGFR1/Fc, VEGF trap1C to VEGF are 382pM, 265pM and 591pM;VEGF trap 1H was showed no affinity to VEGF; the affinity of VEGFR3/Fc to VEGF-C is higher than that of VEGF trap 3H. 2) the depressive effect to endothelial hyperplasy was obvious of VEGF trap 12, VEGFR1/Fc beyond 200ng/ml, VEGFR2/Fc beyond 2000ng/ml; obvious depression was also seen in cell migration assay when the concentration of VEGFR1/Fc and VEGF trap12 beyond 50ng/ml or VEGFR2/Fc beyond 500ng/ml; there are no obvious inhibition on endotheliar cells tuber formation for all three traps.3) all 3 traps took effect on inhibition of CAM angiogenesis, VEGFR1/Fc and VEGF trap12 displayed concentration-effect relationship between 5pM-25pM; VEGFR2/Fc displayed concentration-effect relationship between 10pM-50pM.4) all 3 traps also obviously inhibited angiogenesis in nude mice, the concentration-effect relationship was seen between 10-10M-10-7M for VEGFR1/Fc and VEGF trap12 and 10-9M-10-6M for VEGFR2/Fc.5) VEGF trap12 inhibited HepG2 cell growth and metastasis obviously at 10mg/kg.In conclusion, we altogether cloned 3 VEGF molecules and 8 VEGF trap molecules, construct 11 stable expression rCHO cell lines and identified the proteins after purification; accurately determined the affinity constant of VEGFR1D1-3, VEGF trap12 to VEGF165 by SPR for the first time and proved that the former hasn't a higher KD value than the later, VEGFR1D1-3, VEGF trap12 and VEGFR2/Fc also showed anti-angiogenesis activity in cultured cells, in chicken embryo and in nude mice. VEGF trap12 has also been proved to inhibited HepG2 cell growth and metastasis in nude mice. |
PDF Full Text Request