Study On The Effect And Mechanism Of Brain Derived Neurotrophic Factor On Angiogenesis In The Bone Marrow Millieu

PRATⅠSilencing of BDNF expression by shRNA inhibits in vivo multiple myeloma growth and angiogenesis in the bone marrow milieuObjective To evaluate the effect of MM derived BDNF on tumor growth and angiogenesis in human multiple myeloma xenograft animal model by using lentiviral based shRNA targeting BDNF expression.Methods RPMI 8226 cells were transfected with lentiviral based shBDNF-pGCSIL-eGFP or control vector. Pure populations of transfected RPMI 8226 cell were isolated via a flow cytometry, and specific BDNF knockdown was verified by western blot. BM stromal cells (BMSCs) were isolated from health donors and their phenotypes were identified with flow cytometer;Confirmation of differentiation of the BMSCs to adipocytes, osteocytes and chondrocytes were performed by staining with alizarin red, oil red O and alcine blue respectively. BDNF shRNA RPMI 8226 cells were inoculated subcutaneously with or without BMSCs in the hind neck of irradiated nude mice; Tumor volume was measured by in-vivo optical imaging and capliper; Microvessel density (MVD) was assessed by immunohistochemical analysis for CD34 expression; BDNF and VEGF expression in tumor were analyzed by western blot.Results The purity of transfectants was determined by flow cytometric analysis of enhanced GFP reporter protein, the selected BDNF shRNA and control shRNA RPMI-8226 cells could reach up to 91.87% and 90.50% when compared with parental RPMI-8226 cells. The silencing capacity of BDNF shRNA was confirmed by western blot;BMSCs, isolated from nine healthy donors, expressed CD44,CD 105 and CD29, but not CD14,CD45 and characterized for adipocytes, osteocytes and chondrocytes differentiation capacity; In human myeloma xenograft model, compared with control group, shBDNF RPMI 8226 group has decreased tumor volume(578.10±138.27 mm3 vs 1100.01±132.38 mm3, P <0.001)and microvessel density(6.40±3.81/HRP vs 12.73±5.89/HRP,P<0.001),and prolonged overall survival; Moreover, the shBDNF RPMI 8226/BMSCs group has dcreased tumor volume(1076.51±161.00mm3 vs 1674.49±174.71 mm3,P<0.001)and microvessel density(22.13±8.56/HRP vs 30.26±12.53/HRP,P<0.001),and prolonged overall survival as well.It also demonstrated that silencing of MM-derived BDNF expression significantly inhibited VEGF expression by tumor immunohistochemistry. Conclusion:Our studies demonstrate that BDNF, as a potential stimulator of angiogenesis, contributes to MM tumorgenesis and may be a new target for MM therapy. PRAT IIMM derived BDNF promotes stroma-derived VEGF secretion in the bone marrow milieuObjective To evaluate the involvement of BDNF/TrkB pathway in MM-BMSCs interaction that mediates bone marrow angiogenesis in MM.Methods BMSCs were isolated from 9 healthy donors,and RPMI 8226 cells were transfected with lentiviral based BDNF/Control shRNA vector. An indirecte co-culture system between RPMI 8226 MM cells and BMSCs was established by using a 0.4μm transwell inserts.ELISA was used to evaluate the effect of BDNF on VEGF secretion from BMSCs. TrkB,STAT3 and p-STAT3 in BMSCs were detected by western blot and DNA-binding of AP-1 was detected by Electrophoretic mobility shift assay (EMSA).Results TrkB was consistently expressed by represent BMSC cultures and induced by BDNF stimulation, whereas TrkB expression was inhibited when BMSCs were co-cultured with BDNF knockdown MM cells compared to control group.Stimulation of BMSCs with exogenous BDNF induced a time-and dose-dependent increase in VEGF secretion, which was completely abolished by K252a, an inhibitor of TrkB.When RPMI-8226 cells were cultured with BMSCs, VEGF secretion was up-regulated 3.1-fold over the sum of basal concentrations in monoculture controls. This up-regulation of VEGF secretion is partially abrogated by K252a and inhibited when BMSCs co-culturing with shBDNF RPMI 8226 cells. Western blot and EMS A determined BDNF induced AP-1 tranloction and STAT3 phosphorylation respectively, and blocking STAT3 activity with AG490 lead to down-regulation of VEGF.Conclusion Our studies demonstrate that BDNF mediates MM-BMSCs interactions via selective activation of specific receptors TrkB and downstream signal transducer STAT3 in regulating VEGF secretion. PARTⅢBrain-derived neurotrophic factor promotes the secretion of MMP-9 in human myeloma cell through modulation of nucleus factor-κBObjective:To explore the mechanism of brain-derived neurotrophic factor (BDNF) promoting human multiple myeloma (MM) cells secreting matrix metalloproteinase-9 (MMP-9). Methods:Gelatin zymograph of culture supernatants was performed to visualize the content of MMPs in myeloma RPMI 8226 cells stimulated by BDNF.NF-κB activity was determined by chemilumiescent electrophoretic mobility shift assay (EMSA). Results: Treatment with 25,50,100 and 200μg/L BDNF for 24 h significantly enhanced the level of MMP-9 secreted by RPMI 8226 cells in a dose-dependent manner (2.03±0.48, 2.99±0.46,4.63±0.62 and 5.62±1.29μg/L, respectively vs 1.00μg/L of the control. P<0.01), while that of MMP-2 was not changed significantly (P>0.05).The BDNF-induced activity on of MMP-9 was inhibited by pretreatment with pyrrolidine dithiocarbamate(PDTC), a NF-κB inhibitor, or K252a, a specific tyrosine inhibitor of TrkB which is the receptor for BDNF.Pretreated with 1 mmol/L PDTC or 500 nmol/L K252a significantly down-regulated MMP-9 secreted by the 100μg/L of BDNF stimulated RPMI 8226 cells (the optical density values were 867.52±101.81 and 727.98±92.05,respectively, vs 1159.01±233.15 of the control).The activity of NF-κB was enhanced by BDNF in a dose-dependent manner, and pretreatment with K252a could significantly inhibit this activation at 1,6,1 2 and 24 h (P<0.05) in a time-dependent manner.Conclusion:BDNF plays an important role in the angiogenesis of MM to promote the up-regulation of MMP-9,which may be induced by enhanced NF-κB activity in MM cells.