Study On Brain Derived Neurotrophic Factor-Induced Angiogenesis In Multiple Myeloma

PARTⅠThe expression of brain derived neurotrophic factor in multiple myeloma patientsObjective: To investigate the expression of brain derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in multiple myeloma patients and analyze the correlation of two cytokine levels to clinical parameters.Method: The plasma concentration of BDNF and VEGF in MM patients and control group were determined by ELISA. The results of patients'clinical examinations were recorded at the same time.Results: The concentration of BDNF were (4. 22±0. 64)×103 pg/ml and (2. 03±0. 38)×103 pg/ml in MM group and control group, respectively ( P <0. 05) . There was also a significant difference between VEGF levels of two groups [(79. 35±13. 25) pg/ml vs. (34. 41±1. 78) pg/ml, P < 0. 01]. The levels of BDNF and VEGF correlated significantly (r = 0. 430, P = 0. 025).Conclusion: The concentrations of BDNF and VEGF in MM patients'peripheral blood were at high level. BDNF is likely tobe a novel angiogenic protein. PARTⅡHuman multiple myeloma cell line RPMI8226 activated brain derived neurotrophic factor autocrine loop in co-cultured endothelial cells Objective To study the influence of multiple myeloma cells on endothelial cells in co-culture system.Methods We co-cultured human multiple myeloma cell line RPMI8226 with human umbilical vein endothelial cells (HUVECs). The HUVECs in monoculture were used as control. The expression of BDNF and its especial acceptor TrkB mRNA and protein in HUVECs were determined by RT-PCR and Western blot, respectively, BDNF levels in culture supernatant by enzyme-linked immunosorbent assay. After transfer co-culture, the effects of HUVECs co-cultured with RPMI8226 on HUVECs angiogenesis were studied by modified Transwell migration assay and net-like formation assay.Results Median BDNF concentration on culture supernatant was increased in co-cultured HUVECs compared with monocultured [(31.6±7.2)×103 pg/ml vs (12.4±5.1)×103 pg/ml, P < 0. 05]. The expression of BDNF transcript in monocultured HUVECs was demonstrated by RT-PCR while that in co-cultured HUVECs was increased (1.7 fold, P < 0. 05). TrkB (BDNF high affinity receptor) mRNA was hardly detected in monocultured HUVECs while that in co-cultured HUVECs was increased (4.4 fold, P < 0. 05), too. The results of BDNF and TrkB protein expression determined by Western blot analysis were similar with that of by RT-PCR. On the other hand, we confirmed that the HUVECs activated by RPMI8226 stimulated the migration and net-like formation of HUVECs in vitro. The migration index and the number of net-like structure were increase in 99% and 72%, respectively, compared with unactivated HUVECs. Addition of anti-human BDNF antibody to the culture medium partly reduced these effects.Conclusion Multiple myeloma cells activated BDNF/ TrkB autocrine loops in co-cultured endothelial cells and it resulted in endothelial self-activating angiogenesis. PARTⅢInvolvement of AKT/eNOS in brain derived neurotrophic factor-induced angiogenesisObjective We analyzed the signaling pathways involved in BDNF-induced angiogenesis and showed a new pathway to anti-angiogenesis in multiple myeloma. Methods The phosphorylation of AKT and endothelial NO synthase (eNOS) in HUVECs were detected by Western Blot. The angiogenic activity in vitro was evaluated by Transwell migration assay and net-like formation assay. BDNF-induced in vivo angiogenic activity was evaluated by Matrigel plug assay. The concentration of NO was detected by nitric acid deoxidizase assay. Cell apoptosis was detected by FITC-Annexin-V/PI double staining and flow cytometry.Results BDNF activated the phosphatidylinositol-3-kinase (PI3K)/Akt/ eNOS pathway in HUVECs in time- and dose-dependent manner. BDNF-stimulated NO production was blocked by LY294002, a PI3K inhibitor. In vitro, BDNF stimulated HUVECs migration and tube formation on Matrigel, which processes were significantly decreased by inhibition of activities of PI3K or eNOS, whereas treatment with LY294002, but not NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, inhibited HUVECs viability. In vivo, BDNF increased capillary ingrowth into subcutaneously implanted Matrigel plugs in mice, and the effect of BDNF was significantly reduced in mice that received L-NAME.Conclusion BDNF induces angiogenesis through the AKT/eNOS signaling kinase pathway. It may be a novel target for the therapy of anti-angiogenesis in multiple myeloma.PARTⅣEstablishment of multiple myeloma mouse models expressing brain derived neurotrophic factorObjective Our previous studies have demonstrated the effects of BDNF on promoting proliferation of MM cells and inducing angiogenesis in MM in vitro. To further explore whether BDNF pathway is a potential therapeutic target in MM, we developed two ways to establish human myeloma xenograft animal model and elucidated their advantages and disadvantages.Methods The models of xenograft tumors were established in the nonobese diabetic /severe combined immunodeficiency (NOD/SCID) mice by subcutaneous (s.c.) injection or intravenous (i.v.) injection of human myeloma cell line RPMI8226. Mice were monitored daily for life state and the volumes of subcutaneous tumors were measured after inoculation. 3 weeks after inoculation, red cell counts, BDNF levels in plasma, humanλlight chain levels and calcium levels in sera of NOD/SCID were detected every two weeks. The histologic and cytologic examinations were performed to observe pathologic features of tumors. Using flow cytometry to observe the expression of human CD38+ cell in murine blood and bone marrow. The change of bone density and skeletal lesions were detected by computer radiography.Results The s.c. injection animal model had high efficacy for growth of RPMI8226 subcutaneous tumors (5/5) and presented several pathologic features of plasmacytomas. There were no obvious increase in light chain levels and sera calcium levels, without spread of human MM cells to murine bone marrow and radiological evidence of skeletal lesions. The i.v. injection animal model had relative low efficacy for growth of tumors (4/7) but MM cells could home to and proliferate in murine bone marrow. The humanλlight chain was detected in sera as early as 3 weeks after inoculation. Myeloma-bearing mice had high levels ofλlight chain, high sera calcium levels and resorption of the murine bone. Furthermore, the concentrations of BDNF were increased with the tumor growth in both models. they were(73±11)pg/ml and(105±18)pg/ml in plasma 9 weeks after inoculation, respectively.Conclusions We establish two appropriate MM xerograft NOD/SCID animal models, which both have high BDNF levels in the plasma. It therefore provides two valuable in vivo systems to explore novel therapeutic target, BDNF/TrkB, in MM.