Human multiple myeloma cell line RPMI8226 activates brain derived neurotrophic factor autocrine loop of co-cultured endothelial cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of multiple myeloma cells on normal endothelial cells in co-culture system.</p><p><b>METHODS</b>Human multiple myeloma cell line RPMI8226 was co-cultured with human umbilical vein endothelial cells (HUVECs). HUVECs cultured alone were used as control. The expression of brain derived neurotrophic factor (BDNF) and its specific 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 (ELISA). After transferring the co-culture, the effects RPMI8226 on HUVECs angiogenesis were studied by modified transwell migration assay and net-like formation assay.</p><p><b>RESULTS</b>The median BDNF concentration in culture supernatant was increased in co-cultured HUVECs compared with that in HUVECs cultured alone [(31.6 +/- 7.2) ng/ml vs (12.4 +/- 5.1) ng/ml, P < 0.05]. The expression of BDNF transcript demonstrated by RT-PCR did the same in the two culture systems (1.7 fold increase, P < 0.05). TrkB mRNA was hardly detected in culture of HUVECs alone but was increased in co-cultured HUVECs (4.4- fold increase, P < 0.05). The BDNF and TrkB protein expressions determined by Western blot were similar to that of their mRNAs. On the other hand, the RPMI8226 activated HUVECs showed enhanced migration and net-like formation, being increased by 99% and 72% , respectively. Addition of anti-human BDNF antibody to the culture medium partly reduced these effects.</p><p><b>CONCLUSION</b>Multiple myeloma cells activated BDNF/TrkB autocrine loops in co-cultured endothelial cells and resulted in endothelial self-activating angiogenesis.</p>

Human multiple myeloma cells stimulate differentiation of endothelial cells to form capillary-like networks in two different co-culture systems / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of multiple myeloma cells on differentiation of endothelial cells to form capillary-like networks and the role of brain derived neurotrophic factor (BDNF) in this process.</p><p><b>METHODS</b>Human multiple myeloma cell line RPMI8226 or fresh myeloma cells were co-cultured with human umbilical vein endothelial cells (HUVECs) in two different systems: the contact and the non-contact systems. The HUVECs cultured alone were used as control. The effects of soluble cytokine and adhesion molecule on angiogenesis in HUVECs co-cultured with MM cell were studied by modified Matrigel capillary-like networks formation assay and BDNF levels in co-culture system supernatant by enzyme-linked immunosorbent assay. In the contact co-culture system, the formation of capillary-like networks and the secretion of BDNF were detected again after MM cell was preincubated and cultured with anti-CD29 and anti-CD18 monoclonal antibodies.</p><p><b>RESULTS</b>In RPMI8226 co-culture system, the number of capillary-like structure was increased in non-contact HUVECs system compared with that in monoculture, but the increase was lower than that of contact HUVECs system (75% vs. 113%). In fresh myeloma cells co-culture system, the numbers of capillary-like structure were 138% and 188% for the non-contact and the contact co-culture systems, respectively, above that in HUVECs cultured alone. The median BDNF concentrations in culture supernatants of mono-cultured, contact co-cultured with RPMI8226, non-contact co-cultured with RPMI8226, contact co-cultured with fresh myeloma cells and non-contact co-cultured with fresh myeloma cells were (12.4 +/- 5.1) ng/ ml, (38.5 +/- 8.2) ng/ml, (31.6 +/- 7.2) ng/ml, (37.1 +/- 8.7) ng/ml and(27.9 +/- 7.6) ng/ml, respectively. Either of the two adhesion molecule monoclonal antibodies reduced the capillary-like networks formation and the BDNF secretion in the contact co-culture system.</p><p><b>CONCLUSION</b>Human multiple myeloma cells stimulate differentiation of endothelial cells to form capillary-like networks in two different co-culture systems. BDNF takes part in this progress and is modulated by soluble cytokine and adhesion molecule expressed by both kinds of cells.</p>

Involvement of AKT/eNOS in brain derived neurotrophic factor-induced angiogenesis / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the signaling pathways involved in brain derived neurotrophic factor (BDNF) -induced angiogenesis and to provide a novel pathway to anti-angiogenesis in multiple myeloma.</p><p><b>METHODS</b>The phosphorylation of AKT and endothelial NO synthase (eNOS) in human umbilical venous epithelial cells (HUVEC) were detected by Western blot. The angiogenic activity in vitro was evaluated by transwell migration assay and tube 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.</p><p><b>RESULTS</b>BDNF activated the phosphatidylinositol-3-kinase (PI3K)/AKT/eNOS pathway in HUVEC in a time- and dose-dependent manner. BDNF-stimulated NO production was blocked by LY294002, a PI3K inhibitor. In vitro, BDNF induced HUVEC migration and tube formation on Matrigel, which could be significantly blocked by LY294002 and N(G)-nitro-L-arginine methyl ester (L-NAME) respectively; but BDNF induced HUVEC apoptosis could be blocked only by LY294002. In vivo, BDNF increased capillary ingrowth into subcutaneously implanted Matrigel plugs in mice, which could be significantly reduced in L-NAME treated mice.</p><p><b>CONCLUSION</b>BDNF induces angiogenesis through the AKT/eNOS signaling kinase pathway. It may be a novel target for the anti-angiogenesis therapy for multiple myeloma.</p>

Brain-derived neurotrophic factor inducing angiogenesis through modulation of matrix-degrading proteases / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Recent studies have proved that brain-derived neurotrophic factor (BDNF) possesses angiogenic activity in vitro and in vivo. However, the proangiogenic mechanism of BDNF has not yet been provided with enough information. To explore the proangiogenic mechanism of BDNF, we investigated the effects of BDNF on extracellular proteolytic enzymes, including matrix metalloproteinases (MMPs) and serine proteases, particularly the urokinase-type plasminogen activator (uPA)-plasmin system in human umbilical vein endothelial cells (HUVECs) model.</p><p><b>METHODS</b>Tube formation assay was performed in vitro to evaluate the effects of BDNF on angiogenesis. The HUVECs were treated with various concentrations of BDNF (25 - 400 ng/ml) for different (6 - 48 hours), reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assay MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA in HUVECs, and the conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography, respectively. uPA, plasminogen activator inhibitor (PAI)-1, tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-2 were quantified by western blotting analysis.</p><p><b>RESULTS</b>BDNF elicited robust and elongated angiogeneis in two-dimensional cultures of HUVECs in comparison with control. The stimulation of serum-starved HUVECs with BDNF caused obvious increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation. However, BDNF had no effect on TIMP-1 and TIMP-2 production. BDNF increased uPA and PAI-1 production in a dose-dependent manner. Maximal activation of uPA and PAI-1 expression in HUVECs was induced by 100 ng/ml BDNF, while effects of 200 ng/ml and 400 ng/ml BDNF were slightly reduced in comparison with with those of 100 ng/ml. Protease activity for uPA was also increased by BDNF in a dose-dependent manner. BDNF also stimulated uPA and PAI-1 production beyond that in control cultures in a time-dependent manner from 12 hours to 48 hours after BDNF treatment.</p><p><b>CONCLUSIONS</b>BDNF stimulates MMP and uPA/PAI-1 proteolytic network in HUVECs, which may be important to the acquisition of proangiogenic potential.</p>

PPARalpha agonist--fenofibrate inhibits LPS-induced tissue factor expression in THP-1 cells / 中国实验血液学杂志

This study was aimed to investigate the influence of PPARalpha agonist on the expression of TF (tissue factor) in THP-1 cells. THP-1 cells were pretreated with different concentrations of PPARalpha agonist (fenofibrate) for definite time. Lipopolysaccharide (LPS)-induced TF mRNA and protein levels were detected by RT-PCR and Western blot respectively. The results showed that fenofibrate decreased tissue factor protein and mRNA expression in supernatants of LPS-stimulated human monocytes in a concentration-dependent manner (P < 0.05 - 0.01, n = 5). It is concluded that fenofibrate inhibit TF expression induced by LPS in THP-1 cells, which may be involved in the anti-atherosclerotic effects of PPARalpha agonist.

Effect of brain-derived neurotrophic factor in human myeloma cells on angiogenesis / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of brain-derived neurotrophic factor (BDNF) in multiple myeloma (MM) cells and the correlation between BDNF and MM angiogenesis.</p><p><b>METHODS</b>The expressions of BDNF mRNA transcripts and protein in MM cell lines (RPMI 8226, KM3) were determined by RT-PCR and Western blot, respectively, BDNF levels in culture supernatant by enzyme-linked immunosorbent assay. Proliferation of human umbilical vein endothelial cells (HUVEC) mixed with MM culture medium at different concentrations was examined by MTT assay. The effects of MM culture medium on HUVEC migration and tube formation were studied by modified Boyden chamber assay and tube formation assay, respectively.</p><p><b>RESULTS</b>BDNF was expressed in and secreted by MM cell lines RPMI 8226 and KM3. BDNF concentrations in culture supernatants were within the range of its biological activity. MM culture medium induced a concentration-dependent proliferation of HUVEC. The number of HUVEC at a concentration of 50% KM3 culture medium and at full KM3 culture medium were (1.85 +/- 0.23)-fold and (2.16 +/- 0.29) -fold increase, respectively (P <0.05), compared with that of control. The proliferative activity of HUVEC was reduced on the addition of BDNF antibody to the culture medium. MM culture medium also stimulated the migration and differentiation of HUVEC in vitro, the chemotactic index of HUVEC at a concentration of 50% KM3 culture medium and at full KM3 culture medium were 1.85 +/- 0.23 and 2.16 +/- 0.29, respectively (P < 0.05). Full KM3 culture medium also stimulated capillary-like tube formation in HUVEC (P <0.01), and addition of anti-human BDNF antibody neutralized these effects significantly.</p><p><b>CONCLUSION</b>MM cell lines expressed and secreted biologically active BDNF, which may be involved, at least in part, in MM angiogenesis.</p>