Effects Of Brain-Derived Neurotrophic Factor On Multiple Myeloma Pathogenesis

Multiple Myeloma (MM) is a plasma cell malignancy. Plasma cells are important component of the acquired immune system; the highly heterogeneous of malignant plasma cells were accounted in fatal immune deficiency and progressive bone destruction. As medical science advances, more and more valuable interventions were introduced into clinical practice and its effect has been verified. However, myeloma cells are prone to resistant to the traditional or high-dose chemotherapy, and low survival rate of bone marrow transplantation as an effective treatment, therefore, new and effective remedy still to be developed.Accumulating clinical observations and bench side evidences suggest that some abnormal in bone marrow microenvironment were involved in the development of multiple myeloma. Our previous studies showed that brain-derived neurotrophic factor (BDNF) expression were abnormally high in peripheral blood plasma and in bone marrow of MM patients. We further examined samples of peripheral blood plasma and bone marrow plasma from 125 cases of newly diagnosed patients with multiple myeloma and found that BDNF expression was significantly higher than that of healthy controls。Based on this result, we further investigated how BDNF involved in angiogenesis and bone destruction. Applying cell proliferation assay, tube formation assay and cell migration assay, we found that BDNF significantly enhanced bone marrow endothelial cells proliferation, tube formation and migration activity; with osteoclast formation assay and bone resorption assay as well as detection of signaling pathways related to BDNF in osteoclasts, we identified BDNF as a promoter to induce the differentiation from mononuclear cells to osteoclasts and directly unregulated osteoclasts activity, can lead to significantly increased bone resorption, thus contributing to the development and progress of multiple myeloma bone disease. Our study suggests that BDNF involved in the development and progress of angiogenesis and bone destruction in multiple myeloma, and its antagonist can inhibit the angiogenesis in multiple myeloma, and delay the progress of bone destruction. Thus, BDNF is expected to be a new target in the treatment of multiple myeloma.PARTⅠThe Effects of Brain-derived Neurotrophic Factor in Angiogenesis in Multiple MyelomaObject: To study the effect of abnormal expression of brain-derived neurotrophic factor in multiple myeloma on bone marrow angiogenesis. Methods: 71 cases of multiple myeloma patients and 63 cases of non-malignancies hematological patients peripheral blood of patients with non-blood plasma levels of BDNF; cultured human bone marrow endothelial cells (HBMEC); RT-PCR Detection of bone marrow endothelial cell mRNA of BDNF receptor TrkB expression level; flow cytometry of bone marrow endothelial cells of the protein expression level of TrkB; rhBDNF using MTT assay on bone marrow endothelial cell proliferation activity; modified Boyden chamber assay of bone marrow endothelial cells rhBDNF migration capacity; tubules in vitro Experimental Detection of the formation of bone marrow rhBDNF differentiation of endothelial cells. Results: The results of ELISA show that BDNF in MM patients with high expression in peripheral blood plasma, the expression level was significantly higher than that (4.39±0.67ng/ml vs 1.96±0.39ng/ml, P <.05); RT-PCR experiments show that bone marrow endothelial cell Expression of TrkB mRNA; flow cytometry showed that expression of TrkB protein; MTT cell proliferation rhBDNF in vitro experiments show that bone marrow endothelial cells on a clear role in promoting the proliferation, the proliferation of bone marrow endothelial cell culture medium degree of correlation rhBDNF concentration, 160ng / ml rhBDNF the role of bone marrow endothelial cells, after 48h the cells of the control group was 1.57±0.10-fold (P <.05); rhBDNF a dose-dependent manner to promote migration of endothelial cells in bone marrow, and 100ng/ml rhBDNF bone marrow endothelial cells to reach the the largest migration activity. The experimental group compared with the control group, respectively, the migration index: 25ng/ml rhBDNF group 1.40±0.11,50 ng / ml rhBDNF group 1.64±0.16,100 ng / ml rhBDNF group 2.06±0.25,25 ng / ml VEGF group was 2.18±0.21. tubule formation in vitro experiments show that the size of the formation of tubular structures in culture medium and the concentration of a positive correlation rhBDNF. No control group, tubules form a complete structure of the gel matrix bone marrow endothelial cells loose irregular distribution. 100ng/ml rhBDNF group showed complete bone marrow endothelial cells of the small tube structure, and size and diameter were significantly higher (P <.05). 25ng/ml VEGF group comparison, 100ng/ml rhBDNF group and they form a lumen area of the difference was not significant. Conclusion: peripheral blood of patients with multiple myeloma in the presence of abnormal plasma high expression of BDNF, the high bone marrow endothelial cells to express its high affinity receptor TrkB. rhBDNF in vitro can significantly promote the proliferation of bone marrow endothelial cells, migration and tubule formation, and its ability to promote angiogenesis and extensive research to promote angiogenesis factor VEGF considerable. This study confirmed that BDNF has a significant role in promoting angiogenesis in bone marrow, myeloma cells may be induced angiogenesis plays an important role in multiple myeloma is expected to become anti-angiogenesis new target for treatment. PARTⅡThe Effects of Brain-derived Neurotrophic Factor in Bone Destruction in Multiple MyelomaMM is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In the present study, we investigated whether BDNF was possibly involved in MM cell-induced osteolysis. We showed that BDNF was elevated in MM patients and the bone marrow plasma levels of BDNF positively correlated with extent of bone disease. In osteoclast formation assay, bone marrow plasma from patients with MM increased osteoclast formation and the effect was significantly blocked by neutralizing antibody to BDNF, suggesting a critical role for BDNF in osteoclast activation. Furthermore, the direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF, demonstrating that BDNF utilized TrkB for its effects on osteoclast. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein (MIP)-1αand receptor activator of nuclear factor-κB ligand (RANKL), two major osteoclast stimulatory factors in MM. These results support an important role for BDNF in the development of myeloma bone disease.