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Originally published as JCO Early Release 10.1200/JCO.2005.03.723 on June 6 2005 © 2005 American Society of Clinical Oncology. Thalidomide Downregulates Angiogenic Genes in Bone Marrow Endothelial Cells of Patients With Active Multiple Myeloma
From the Department of Internal Medicine and Clinical Oncology, and Department of Human Anatomy and Histology, University of Bari Medical School, I-70124 Bari; Hematology Operative Unit 2, Ospedale Maggiore, I-20122; Department of Hematology and Bone Marrow Transplantation, Istituto Nazionale dei Tumori, I-20133 Milan; and the Department of Chemical Engineering Processes, University of Padua, I-35131 Padua, Italy Address reprint requests to Angelo Vacca, MD, Text: 4024, Department of Internal Medicine and Clinical Oncology, Policlinico—Piazza Giulio Cesare, 11 I-70124 BARI, Italy; e-mail: a.vacca{at}dimo.uniba.it.
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.
Thalidomide is effective in patients with multiple myeloma (MM), but its mechanisms are unclear. It may produce an antiproliferative effect on plasma cells and/or bone marrow stromal cells,1,2 reduce expression of interleukin (IL) -6, IL-1ß, tumor necrosis factor alpha (TNF-  ), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF),3 and cell surface adhesion molecules in stromal cells,4 immunomodulate T cells,5 enhance anti-plasma cell cytotoxicity of natural killer (NK) cells,6 inhibit COX-2,7 and induce an antiangiogenic effect that is probably accompanied by sizeable or major antitumor activity.8 It has, in fact, been shown to inhibit bFGF- and VEGF-induced angiogenesis in rabbit cornea and chick embryo chorioallantoic membrane in vivo assays,9,10 and the proliferation of cultured endothelial cells (ECs).10 It decreases microvessel density in a mouse model,11 and impacts the expression of bFGF and VEGF in human lung carcinoma cells.12 In contrast with the earlier evidence,13 MM patients responsive to thalidomide display a significant decrease in bone marrow microvessel density, whereas nonresponders do not.14 The plasma and bone marrow levels of bFGF, VEGF, and hepatocyte growth factor (HGF), which parallel MM activity,15 also decrease in responders.16 We found that thalidomide at 180 to 300 mg/d inhibits proliferation and capillarogenesis of bone marrow ECs from patients with MM (MMECs).17 Despite these findings, thalidomide's antiangiogenic activity is still the subject of investigation. This article describes its effects on the expression in MMECs of genes involved in autocrine and paracrine loops of neovascularization: VEGF, bFGF, HGF, insulin-like growth factor-1 (IGF-1), insulin-like growth factor binding protein-3 (IGFBP-3), angiopoietin 1 (Ang1), and Ang2. VEGF, bFGF and HGF are powerful stimulators of angiogenesis.18 Their expression in ECs increases with the angiogenic switch and persists throughout angiogenesis, except during vessel stabilization when inhibitors prevail.18 IGF-1 is angiogenic through activation of the cognate IGF receptor-1 and upregulation of VEGF gene expression.19 IGFBP-3 is the major binding protein of IGF-1, and reinforces its mitogenic effect by conveying it into the IGF receptor-1.20 It regulates physiologic, inflammation-associated, and tumor angiogenesis.20 Ang1 and Ang2 are mandatory for vessel sprouting and remodeling.18 They behave as survival factors by preventing apoptosis. Ang1 promotes sprouting in the presence of VEGF and stabilizes the perivascular EC interactions. Ang2 exerts a vessel destabilizing effect that allows VEGF-mediated vascular reorganization. Bone marrow ECs from patients with monoclonal gammopathies unattributed/unassociated (MG[u]), or with diffuse large B-cell lymphoma (NHL-ECs), a tumor not responsive to thalidomide,21 a cell line from Kaposi's sarcoma (KS), a tumor sensitive to thalidomide,22 and healthy human umbilical vein ECs (HUVECs) were compared with MMECs.
Patients Thirty-eight patients fulfilling the International Myeloma Working Group diagnostic criteria23 for MM (n = 28) and MG(u) (n = 10) were studied. MM patients were defined as active or nonactive, according to clinical features and M-component level.24 Active patients (n = 16) were those: (1) at diagnosis, with symptomatic disease and an increase in M-component level in the 3 months before analysis (n = 8); (2) at relapse (n = 6); and (3) at leukemic phase (n = 2). They were 10 males and six females ages 38 to 79 years (median age, 66.4 years), and staged24 as IIA (n = 2), IIB (n = 2), IIIA (n = 10) and IIIB (n = 2); the M-component was IgG (n = 12), IgA (n = 3), and k (n = 1). Nonactive patients (n = 12) were those in: (1) post-treatment complete/objective response (n = 6); (2) off-treatment plateau phase (n = 6). They were eight males and four females ages 41 to 84 years (median age, 68.5 years); the M-component was IgG (n = 7), IgA (n = 2),  or  (n = 3). No patient had received thalidomide. The MG(u) patients were six males and four females ages 40 to 88 years (median age, 70.7 years), and their M-component was IgG (n = 6), IgA (n = 2), or (n = 2). Four patients with diffuse large B-cell NHL were also studied at diagnosis. They were three males and one female ages 36 to 81 years (median age, 67.7 years) with bone marrow involvement (stage IV). The study was approved by the local ethics committee, and all patients gave their informed consent.
EC Cultures The purity and viability of cell preparations (more than 95% viable ECs) were assessed by fluorescence-activated cell sorting (FACS, FACScan, Becton Becton Dickinson, San Jose, CA) with double positivity for factor VIII-related antigen (FVIII-RA, a highly specific EC marker), using a MoAb revealed by a secondary fluorescein isothiocyanate (FITC)-conjugated antibody (both from Immunotech), and for CD105 (endoglyn, a molecule selectively expressed by ECs),17 using a phycoerythrin (PE)-conjugated MoAb (Caltag, Burlingame, CA), and negativity for CD14 and CD38 MoAbs followed by reverse transcriptase-polymerase chain reaction (RT-PCR) for mRNA of FVIII-RA, CD38, CD105 and immunoglobulin heavy chain variable diversity joining (IgH VDJ) region, and by trypan blue viable staining.17 RT-PCR (see RT-PCR section for description) was performed with the following primers (Invitrogen, Life Technologies, Carlsbad, CA) for 30 cycles: FVIII-RA, 5'-GTTCGTCCTGGAAGGATCGG-3' and 5'-CACTGACACCTGAGTGAGAC-3'; CD38, 5'-ACCCCGCCTGGAGCCCTATG-3' and 5'-GCTAAAACAACCACAGCGACTGG-3'; and CD105, 5'-TGCCACTGGACACAGGATAA-3' and 5'-GATGAGGACGGCATCGAGAT-3'. The IgH VDJ region was revealed by a semi-nested PCR based on Fr3 protocol, including an upstream primer complementary to the third framework V region (5'-ACACGGC[C/T][G/C]TGTATTACTGT-3'), a downstream primer directed to a conserved sequence of the J region (5'-TGAGGAGACGGTGACC-3', 30 cycles), and in the second round amplification to an inner conserved sequence of the same J region (5'-GTGACCAGGGTNCCTTGGCCCCAG-3', 20 cycles). Paired plasma cells, the U266 myeloma cell line (ATCC, Rockville, MD) and HUVECs were used as controls. The purity of EC preparations was also examined with a Zeiss EM109 transmission electron microscope (Zeiss, Oberkochen, Germany) following conventional embedding and staining.17 KS cells were a non–AIDS-associated immortalized line termed as KSC IMM25 (donated by Adriana Albini, MD, Advanced Biotechology Center, Genoa, Italy), and cultured in their complete medium (Dulbecco's modified Eagle's medium [DMEM] with 10% FCS). HUVECs were obtained from 12 samples following parental consent, grown in their complete medium (M199 medium supplemented with 10% FCS, 0.02% extract of bovine brain, and 0.015% porcine heparin), and studied at the second passage.17 KS cells and HUVECs served as opposite controls: a well-known tumor and normal ECs with which to compare MMECs from active and nonactive patients and MG(u)ECs. In addition, KS cells were studied because KS patients, like those with active MM, respond to thalidomide,13,22 and we also studied patients with diffuse large B-cell NHL, who do not.21 Hence the preferential clinical activity of thalidomide in active MM and KS may be tentatively attributed to its putative inhibitory effects on angiogenic genes of active MMECs and KS cells, but not NHL-ECs.
Treatment With Thalidomide, Enzyme-Linked Immunosorbent Assay of Conditioned Media and Preparation of Total RNA Total RNA was extracted with the Trizol reagent (Invitrogen), and purified using the RNeasy total RNA Isolation Kit (Qiagen, Valencia, CA). RNA integrity was verified with an Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany).
Generation of Gene Expression Profiles Unsupervised sample clustering using the six preselected genes have been applied to highlight sample groups and their associated expression levels. Before clustering, the gene expression values across all samples have been standardized (linearly scaled) to have a mean of 0 and a standard deviation of 1. Hierarchical agglomerative clustering has been carried out using the Pearson correlation coefficient as distance metric and centroid as linkage method.
RT-PCR
Real-Time RT-PCR
EC Preparations and Gene Expression Profiling Analysis by DNA Microarrays Patients' EC preparations were analyzed by FACS with FVIII-RA and CD105 (EC markers) or FVIII-RA and CD38 (a plasma cell marker) double staining. ECs were more than 97% double stained for FVIII-RA and CD105, whereas their CD38 staining was either absent or insignificant. Figure 1 (panel A) shows an example of MMECs. These findings were confirmed by RT-PCR showing that patients' ECs expressed the FVIII-RA and CD105 mRNA like HUVECs, but not plasma cells, and did not express the CD38 and IgH VDJ mRNA, whereas these were expressed by plasma cells. Panel B shows the MMECs example. No plasma cell contamination was also observed by electron microscopy, which revealed uniform ECs populations, as in the MMECs example in panel C.
Five EC samples from MM patients at diagnosis (active MMECs), five MG(u)ECs, five HUVECs and the KS cell line were examined for the expression levels of the selected genes within the global expression profiles generated by DNA microarrays hybridisation. Raw microarray expression levels of VEGF, bFGF, HGF, IGF-1, IGFBP-3 and Ang1 genes (Ang2 levels were in the background range) showed an overall trend of induction in active MMECs and MG(u)ECs versus HUVECs (Fig 2).
Specifically, VEGF was significantly upregulated in both MMECs (9.4-fold and adjusted P value <.0001) and MG(u)ECs (8.2-fold and adjusted P value < .0001). bFGF, too, was significantly upregulated in MMECs (3.0-fold and adjusted P value < .0001) and MG(u)ECs (3.2-fold and adjusted P value < .01). IGF-1 and Ang1 were significantly upregulated only in MG(u)ECs (5.0- and 3.2-fold and adjusted P value < .0001 and < .017 respectively). IGFBP-3 was induced in MG(u)ECs (1.7-fold and adjusted P value < .022) and showed an induction trend in MMECs (1.2-fold). No modulation of the other transcripts was observed except for a 2.3-fold upregulation of Ang1 in MG(u)ECs versus MMECs (adjusted P value < .04). The KS cell line was not statistically assessable. Even so, it displayed an appreciable expression of VEGF and, like active MMECs, of Ang1.
FACS and RT-PCR Analyses and Thalidomide Treatment
A more in-depth assessment and comparison of the expression was then made using RT-PCR and quantitation of bands for each angiogenic cytokine. The RT-PCR pictures of untreated ECs substantially overlapped the DNA microarray analysis (see "medium" values of Tables 2 and 3): the highest expression levels of VEGF, bFGF and HGF were in active MMECs and values were significantly higher than those of nonactive MMECs and MG(u)ECs (P < .05 or better by Student's t test). IGF-1 and its carrier IGFBP-3 were highly expressed in all patients' ECs (P < .01 or better v HUVECs). Ang1 and Ang2 were sizeably expressed in all ECs. The pronounced expression of VEGF and Ang1 in the KS cells was confirmed.
Exposure to thalidomide at 10 µmol/L and 20 µmol/L produced a significant downregulation of all genes except Ang1. This was only dose-dependent in active MMECs (Table 2). As a whole, the lowering ranged from –20% to –41% at 10 µM, and –30% to –54% at 20 µM. Representative experiments in an active MMECs are shown in Figure 3. The KS cell line behaved like active MMECs for VEGF, bFGF and Ang2, whereas IGF-1 and IGFBP-3 were upregulated dose dependently (Table 3). Representative downregulations are shown in Figure 4. In contrast, significant, non–dose-dependent upregulation of VEGF, bFGF, HGF and IGF-1 was seen in nonactive MMECs (overall range = +12% to +42%, Table 2), MG(u)ECs (+32% to +67%, Table 2); of VEGF, bFGF and HGF in NHL-ECs (+21% to +38%, Table 3); of VEGF and bFGF in HUVECs (+31% to +67%, Table 3), whereas IGFBP-3, Ang1 and Ang2 were unvaried. Representative experiments are shown in Figure 4.
Gene Expression and Thalidomide Treatment Illustrated by Real-Time RT-PCR Changes caused by thalidomide were expressed as percentages of the baseline value (Fig 5). A pronounced dose-dependent downregulation was observed only in active MMECs (average VEGF = –24% and –34% at 10 µmol/L and 20 µmol/L respectively, bFGF = –31% and –45%, HGF = –21% and –36%) and KS (VEGF = –18% and –30%, bFGF = –23% and –42%, HGF = –50% and –79%). In contrast, upregulation by nonactive MMECs, MG(u)ECs, NHL-ECs and HUVECs occurred independent of the dose.
Quantitation of VEGF, bFGF and HGF in the CM by ELISA This showed that unexposed active MMECs secreted 40-100 times higher amounts of the angiogenic factors than HUVECs. Similarly, KS cells secreted 5-10 higher VEGF and bFGF (Fig 6). When cells were exposed to thalidomide, we observed a significant dose-dependent reduction of all factors in active MMECs and KS only, whereas there were no changes in nonactive MMECs, MG(u)ECs, NHL-ECs and HUVECs CM (Fig 6).
We wondered whether ECs from active MM patients (active MMECs) are impacted by thalidomide in VEGF, bFGF, HGF, IGF-1, IGFBP-3, Ang1 and Ang2 genes, which act as the main regulators of angiogenesis.18 Nonactive MMECs, MG(u)ECs, NHL-ECs, healthy ECs (or HUVECs) and a KS cell line were compared. The DNA microarray and RT-PCR analyses showed an overall induction of genes (except Ang1 and Ang2) in active MMECs, nonactive MMECs, MG(u)ECs and NHL-ECs over HUVECs; of VEGF and bFGF in the KS line. In active MMECs, 10 µmol/L and 20 µmol/L thalidomide (corresponding to 180 mg and 300 mg/d in a 70-kg adult)26 downregulates all genes (except Ang1) in a dose-dependent fashion. We showed that both doses inhibit proliferation and capillarogenesis of these cells in vitro.17 In KS cells, a similar downregulation was seen for VEGF, bFGF and Ang2. In contrast, genes were upregulated or unvaried in nonactive MMECs, MG(u)ECs, NHL-ECs and HUVECs. When real time RT-PCR was restricted to VEGF, bFGF and HGF (the growth factors most involved in MM angiogenesis), we confirmed their higher expression in active MMECs, nonactive MMECs, MG(u)ECs and NHL-ECs over HUVECs and their downregulation only in active MMECs and KS cells according to the dose; their upregulation was independent of the dose in the other ECs. Parallel to mRNA, secretion of the translated VEGF, bFGF and HGF proteins progressively fell in the active MMECs CM. Similar behavior was observed for VEGF and bFGF and, to a lesser extent, for HGF in the KS CM. Values varied only marginally or not at all in the other EC CMs. Hence, analogies do exist between active MMECs and KS in terms of overexpression of the genes and their response to thalidomide. Accordingly, we have previously found17 that long-term cultured active MMECs display constitutively: (1) growth advantage over HUVECs because they form a fast-growing and a more closely-knit capillary plexus; (2) five to 70 times higher levels of typical vascular markers (Tie2/Tek, VEGFR-2, bFGFR-2, CD105-endoglin, and VE-cadherin) than HUVECs; (3) ultramicroscopically, enhanced metabolic activation because of hyperplasia of the rough endoplasmic reticulum, wide Golgi complex, numerous mitochondria and lysosome-like structures. Their VEGF/VEGFR-2 loop is operative31 as in KS in which VEGF and bFGF are co-expressed with their cognate receptors and provide autocrine loops of growth.32,33 As shown here, active MMECs also secrete 40 to 100 times higher amounts of VEGF, bFGF and HGF into their CM, in much the same way as KS.17 Overall these findings seem to point to a tumoral phenotype of active MMECs, as shown for ECs in lymphomas.34
These differences in the activity of thalidomide may be attributable to differences in the angiogenic state of ECs leading to dissimilar gene control by transcription factors, such as Sp1 and NF-
NF- In conclusion, here we show that thalidomide exerts a direct antiangiogenic activity on MMECs during the active disease through the downregulation of genes mandatory for autocrine and paracrine life of ECs and generally of other cells in the bone marrow. Antiangiogenesis may explain, at least partly, this drug's therapeutic power in active MM. No antiangiogenic activity may be produced in nonactive MM and MG(u) (as well as in diffuse large B-cell NHL). Response to thalidomide is observed in about one-third of patients with either active (relapsed disease) or smoldering/indolent MM.47,48 However, the median time to response is shorter in active MM (1.3 months v 5 months). Tentatively, we suggest that this can be explained by the divergent response to thalidomide of active MMECs versus nonactive MMECs and MG(u)ECs.
The authors indicated no potential conflicts of interest.
Supported by Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Ministry for Education, the Universities and Research (Project CARSO No. 72/2), and Ministry for Health - Regione Puglia (grant BS2), Rome, Italy. A.V. and C.S. contributed equally to this work. Terms in blue are defined in the glossary, found at the end of this issue and online at www.jco.org. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
