Bone Marrow Neovascularization, Plasma Cell Angiogenic Potential, and Matrix Metalloproteinase-2 Secretion Parallel Progression of Human Multiple Myeloma

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Abstract
To assess whether the progression of plasma cell tumors is accompanied by angiogenesis and secretion of matrix-degrading enzymes, bone marrow biopsy specimens from 20 patients with monoclonal gammopathy of undetermined significance (MGUS), 18 patients with nonactive multiple myeloma (MM), and 26 patients with active MM were evaluated for their angiogenic potential and matrix-metalloproteinase (MMP) production. A fivefold increase of the factor VIII+microvessel area was measured by a planimetric method of point counting in the bone marrow of patients with active MM as compared with nonactive MM and MGUS patients (P < .01). When serum-free conditioned media (CM) of plasma cells isolated from the bone marrow of each patient were tested in vivo for their angiogenic activity in the chick embryo chorioallantoic membrane (CAM) assay, the incidence of angiogenic samples was significantly higher (P< .01) in the active MM group (76%) compared with nonactive MM (33%) and MGUS (20%) groups. Moreover, a linear correlation (P < .01) was found between the extent of vascularization of the bone marrow of a given patient and the angiogenic activity exerted in the CAM assay by the plasma cells isolated from the same bone marrow. In vitro, a significantly higher fraction of the plasma cell CM samples from the active MM group stimulated human umbilical vein endothelial cell (HUVEC) proliferation (53%, P < .01), migration (42%, P < .05), and/or monocyte chemotaxis (38%,P < .05) when compared with nonactive MM and MGUS groups (ranging between 5% and 15% of the samples). Also, immunoassay of plasma cell extracts showed significantly higher (P < .01) levels of the angiogenic basic fibroblast growth factor (FGF)-2 in the active MM patients than in nonactive MM and MGUS patients (153 ± 59, 23 ± 17, and 31 ± 18 pg FGF-2/100 μg of protein, respectively). Accordingly, neutralizing anti–FGF-2 antibody caused a significant inhibition (ranging from 54% to 68%) of the biological activity exerted on cultured endothelial cells and in the CAM assay by plasma cell CM samples from active MM patients. Finally, in situ hybridization of bone marrow plasma cells and gelatin-zymography of their CM showed that active MM patients express significantly higher (P < .01) levels of MMP-2 mRNA and protein when compared with nonactive MM and MGUS patients, whereas MMP-9 expression was similar in all groups. Taken together, these findings indicate that the progression of plasma cell tumors is accompanied by an increase of bone marrow neovascularization. This is paralleled by an increased angiogenic and invasive potential of bone marrow plasma cells, which is dependent, at least in part, by FGF-2 and MMP-2 production. Induction of angiogenesis and secretion of MMPs by plasma cells in active disease may play a role in their medullary and extramedullary dissemination, raising the hypothesis that angiostatic/anti-MMP agents may be used for therapy of MM.