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Bone marrow macrophages of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies of undetermined significance (MGUS) and benign anemia (controls) were stimulated for 7 days with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), and analysed for the expression of endothelial cell (EC) markers by reverse transcription (RT)-PCR, real-time RT-PCR, western blot and immunofluorescence. Their vasculogenic ability was investigated in vitro in a Matrigel assay and in vivo on bone marrow biopsies through dual immunofluorescence and confocal laser microscopy. Active MM macrophages exposed to VEGF and bFGF acquired EC markers and formed capillary-like structures mimicking paired bone marrow ECs (multiple myeloma patient-derived endothelial cells, MMECs), with major responsiveness compared to macrophages from nonactive MM, MGUS or controls. Bone marrow biopsies of active MM harbored 'mosaic' vessels, being formed by MMECs, EC-like macrophages and macrophages themselves. These figures were rare in nonactive MM and absent in MGUS or controls. Our data indicate that macrophages contribute to build neovessels in active MM through vasculogenic mimicry, and this ability proceeds parallel to progression of the plasma cell tumors. Macrophages may be a target for the MM antivascular treatment.

Therapeutic doses of zoledronic acid markedly inhibit in vitro proliferation, chemotaxis, and capillarogenesis of bone marrow endothelial cells of patients with multiple myeloma. Zoledronic acid also induces a sizeable reduction of angiogenesis in the in vivo chorioallantoic membrane assay. These effects are partly sustained by gene and protein inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in an autocrine loop. Mevastatin, a specific inhibitor of the mevalonate pathway, reverts the zoledronic acid antiangiogenic effect, indicating that the drug halts this pathway. Our results provide evidence of a direct antiangiogenic activity of zoledronic acid on multiple myeloma patient-derived endothelial cells due to at least four different mechanisms identified either in vitro or in vivo. Tentatively, we suggest that the zoledronic acid antitumoral activity in multiple myeloma is also sustained by antiangiogenesis, which would partly account for its therapeutic efficacy in multiple myeloma.

Vascular endothelial growth factor165 (VEGF165) and semaphorin3A (SEMA3A) elicit pro- and antiangiogenic signals respectively in endothelial cells (ECs) by binding to their receptors VEGFR-2, neuropilin-1 (NRP1), and plexin-A1. Here we show that the VEGF165-driven angiogenic potential of multiple myeloma (MM) ECs is significantly higher than that of monoclonal gammopathy of undetermined significance (MGUS) ECs (MGECs) and human umbilical vein (HUV) ECs. This is probably due to a constitutive imbalance of endogenous VEGF165/SEMA3A ratio, which leans on VEGF165 in MMECs but on SEMA3A in MGECs and HUVECs. Exogenous VEGF165 induces SEMA3A expression in MGECs and HUVECs, but not in MMECs. Moreover, by counteracting VEGF165 activity as efficiently as an anti-VEGFR-2 antibody, exogenous SEMA3A restrains the over-angiogenic potential of MMECs. Our data indicate that loss of endothelial SEMA3A in favor of VEGF165 could be responsible for the angiogenic switch from MGUS to MM.

Purpose: To study the antiangiogenic effect of thalidomide., Patients and Methods: The expression of key angiogenic genes was studied in bone marrow endothelial cells (ECs) of patients with active and nonactive multiple myeloma (MM), monoclonal gammopathies unattributed/unassociated (MG[u]), diffuse large B-cell non-Hodgkin's lymphoma, in a Kaposi's sarcoma (KS) cell line, and in healthy human umbilical vein ECs (HUVECs) following exposure to therapeutic doses of thalidomide., Results: Thalidomide markedly downregulates the genes in a dose-dependent fashion in active MMECs and KS cell line, but upregulates them or is ineffective in nonactive MMECs, MG(u)ECs, NHL-ECs, and in HUVECs. Secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor also diminishes according to the dose in culture conditioned media (CM) of active MMECs and KS, whereas it does not change in the other CM., Conclusion: Inhibition by thalidomide is probably confined to the genes of active MMECs and KS. This would account for its higher efficacy in these diseases.

Endothelial cells (EC) were extracted through a lectin-based method from bone marrow of 57 patients with active multiple myeloma (MM) and compared with their healthy quiescent counterpart, human umbilical vein EC (HUVEC). MMECs exhibit specific antigens that indicate ongoing angiogenesis and embryo vasculogenesis; solid intercellular connections, hence stability of MM neovessels; and frequent interactions with plasma cells, hence tumor dissemination. They show heterogeneous antigen expression, hence existence of subsets. Their main genetic markers are indicative of a vascular phase. They show intrinsic angiogenic ability, because they rapidly form a capillary network in vitro, and extrinsic ability, because they generate numerous new vessels in vivo. They vividly secrete growth and invasive factors for plasma cells. They signal through kinases mandatory for development of neovascularization. Ultrastructurally, they are abnormal and show metabolic activation, like tumor ECs. Thalidomide heavily interferes with their functions. Vasculogenesis and angiogenesis might contribute to the MM vascular tree and progression, in the form of growth, invasion, and dissemination. In view of the heterogeneity of the antigenic phenotype of MMECs, a mixture (or a sequence) of antiangiogenic agents coupled with thalidomide would seem plausible for the biologic management of MM.