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

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.

In Vitro and In Vivo Antitumor Activity of a Novel Alkylating Agent, Melphalan-Flufenamide, against Multiple Myeloma Cells

“…Multiple myeloma cell growth is associated with angiogenesis (44,45). As noted earlier, ANPEP expression/ activity is associated with malignant phenotype, including angiogenesis (46); and importantly, mel-flufen is ANPEP-activated prodrug of melphalan.…”

“…We examined the effect of mel-flufen on multiple myeloma cell migration and angiogenesis using Transwell insert systems and in vitro tubule formation assays. VEGF is elevated in the multiple myeloma bone marrow microenvironment, and prior studies showed that VEGF triggers growth, migration, and angiogenesis in multiple myeloma (44,45,47). We first examined whether mel-flufen affects VEGFinduced multiple myeloma cell migration.…”

Increased serum levels of vascular endothelial growth factor predict risk of progression in early B‐cell chronic lymphocytic leukaemia

“…In keeping with results obtained in solid tumours, our results qualify S-VEGF as a marker suitable for predicting the clinical outcome of disease also in haemopoietic neoplasias such as CLL. As a matter of fact in MM an increased angiogenesis was implicated in the mechanisms of progression from MGUS or non-active MM to active MM (Vacca et al, 1999).…”

“…Despite these ®ndings suggesting a central role of angiogenesis in the mechanisms of progression of many solid tumours, little is known regarding angiogenesis in response to haematological malignancies. Increased micovessel density has been reported in BM of active multiple myeloma (MM) in comparison with nonactive MM and monoclonal gammopathy of undetermined signi®cance (MGUS) patients (Vacca et al, 1999).…”

Novel therapeutic strategies targeting growth factor signalling cascades in multiple myeloma

“…Specifically, increased microvessel density (MVD) in MM patient BM specimens parallels disease progression and correlates with poor prognosis (Vacca et al, 1999), and BM MVD at diagnosis is an important prognostic factor for survival of patients who undergo autologous transplantation as frontline therapy. Bone marrow angiogenesis is partly sustained by VEGF, basic fibroblast growth factor (bFGF), and matrix metalloproteinases (MMPs) secreted by MM cells (Vacca et al, 1999). Conversely, BMECs secrete growth factors, including VEGF and bFGF, promoting MM cell growth in the BM milieu ( Fig 1).…”