Expression of Vascular Endothelial Growth Factor and Its Receptors in Hematopoietic Malignancies¹

For solid tumors to grow beyond 2 mm³ in size, there is a strict requirement for angiogenesis (1, 2). Tumor angiogenesis is influenced by a number of regulatory factors and although a wide variety of growth factors with known angiogenic activity could have been evaluated, we chose to focus initially on the expression of VEGF³ and bFGF, two growth factors that appear to play key roles in angiogenesis (1, 3, 4). Because of its effects on endothelial cell growth and microvascular permeability, VEGF is believed to be an important mediator of angiogenesis (5). A variety of malignant human tumors, including breast, lung, and prostate carcinomas, are known to secrete VEGF (6, 7, 8, 9). In general, the levels of VEGF mRNA and protein expression in nonhematopoietic human tumors often correlate positively with malignant progression (7, 10). Clinical studies have demonstrated that both the number and density of microvessels in several human solid cancers may be directly correlated with invasion and metastasis and often predicts an unfavorable prognosis in many cases (11, 12, 13, 14, 15). A central role of VEGF in tumor growth in vivo was demonstrated by Kim et al. (16), who showed that the addition of neutralizing antibodies to VEGF resulted in a marked suppression of tumor growth. In addition to its expression in neoplastic tissues, VEGF is expressed in normal cells including activated macrophages (17, 18), renal glomerular epithelial and mesangial cells (19, 20), platelets (21), and keratinocytes (22).

Although the importance of angiogenesis in solid tumors is well established, its role in hematopoietic tumors is not. In a study of 88 patients with B-cell non-Hodgkin’s lymphoma, Ribatti et al. (23) found an increase in the microvessel density in lymph nodes that correlated with the severity of the disease. A similar study of childhood acute lymphocytic leukemia also revealed an increase in the microvessel density, as assessed by factor VIII-related antigen staining, in the bone marrow of leukemic patients compared with that found in normal controls (24). An increase in bone marrow microvessel density was also observed in a series of patients diagnosed with multiple myeloma compared with patients with monoclonal gammopathies of unknown significance (25). Such findings, although not conclusive, are suggestive of a role for angiogenic growth factors in hematopoietic malignancies.

Although its expression was demonstrated previously in the HL-60 promyelocytic leukemia cell line (26), it is not known whether VEGF or other angiogenic factors play a role in human hematopoietic malignancies. The present studies were undertaken to identify whether VEGF and its receptors were expressed in other hematopoietic malignancies in general and in multiple myeloma in particular.

Human tumor cell lines were obtained from the American Type Culture Collection (Rockville, MD). All tumor cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 1% (v/v) penicillin (100 units/ml), streptomycin (100 μg/ml), and 1% (v/v) l-glutamine (all from Grand Island Biological Supply Co, Grand Island, NY) and maintained at 37°C in 5% CO₂-95% air atmosphere. The HUVEC line was obtained from Dr. S. Williams (University of Arizona) and was maintained in endothelial growth medium-MV medium (Clonetics, San Diego, CA).

Total cellular RNA was isolated from the cell lines using guanidinium isothiocyanate and cesium chloride gradient centrifugation according to the method of Chirgwin et al. (27). RNA (5 μg) was denatured at 65°C for 10 min in 50% formamide, 6.5% formaldehyde, 40 mm 3-(N-morpholino)propanesulfonic acid, and subjected to electrophoresis on a 1% formaldehyde-agarose gel. The RNA was transferred by capillary action to a nylon membrane (Nytran; Schleicher & Schuell, Keene, NH) and immobilized by exposure to UV light. Membranes were prehybridized in Rapid-hyb buffer (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions. Hybridization was carried out at 42°C for 24 h with ³²P-labeled cDNA probes generated by RT-PCR. Following high stringency washes, the filters were exposed to X-ray film (Kodak; XAR-5) at −80°C with an intensifying screen.

Total RNA isolated from exponentially growing cells was treated with an enzyme reverse transcriptase (SuperScript II; Gibco BRL, Gaithersburg, MD) to generate cDNA; the cDNA was then amplified by the PCR using Taq DNA polymerase (Life Technologies, Inc., Gaithersburg, MD). The primers were designed to span intron-exon borders to distinguish amplified cDNA from genomic DNA. PCR primers used to detect VEGF were: 5′-GAA GTG GTG AAG TTC ATG GAT GTC (forward) and 5′-CGA TCG TTC TGT ATC AGT CTT TCC (reverse); flt-1, 5′- GAG AAT TCA CTA TGG AAG ATC TGA TTT CTT ACAGT-3′ (forward) and 5′- GAG CAT GCG GTA AAA TAC ACA TGT GCT TCT AG-3′ (reverse); and KDR, 5′-CAA-CAA-AGT-CGG-GAG-AGG-AG-3′ (forward) and 5′-ATG-ACG-ATG-GAC-AAG-TAG-CC-3′ (reverse; Ref. 28). The VEGF primers detect the four spliced RNA transcripts. The primers used for bFGF were: 5′- GTG TGT GCT AAC CGT TAC CT (forward) and 5′-GCT CTT AGC AGA CAT TGG AAG (reverse; Ref. 7). Amplification of the “housekeeping” gene, glyceraldehyde-3-phosphate dehydrogenase was used to verify mRNA isolation and RT-PCR techniques. PCR-amplified products were electrophoresed in 1.0% agarose gel and stained with ethidium bromide.

Supernatants were obtained from in vitro tissue culture studies and were examined for expression of VEGF using a quantitative solid-phase ELISA assay (R&D Systems, Minneapolis, MN). Samples (200 μl) or standards (200 μl) were added to a coated microtiter plate and incubated for 2 h at room temperature. The plates were then rinsed, and 200 μl of recombinant anti-VEGF₁₆₅ polyclonal antibody conjugated to horseradish peroxidase were added to the wells. This ELISA assay will detect both the M_r 165,000 and M_r 121,000 VEGF isoforms. After an additional 2- incubation, the wells were again rinsed, and 200 μl of hydrogen peroxide and tetramethylbenzidine were added. The reaction was stopped by the addition of 50 μl of 2 n sulfuric acid. The absorbance of each well was measured spectrophotometrically at 450 nm and plotted against a standard curve with VEGF levels expressed as ng/ml. The lower detection limit of the ELISA assay is 5.0 pg/ml. Each cell line was analyzed in triplicate.

HUVECs were plated into T-75 tissue culture flasks and allowed to grow to ∼60% confluence. The cells were then exposed to 10 ng/ml VEGF for 72 h. A parallel flask of HUVEC cells was set up without VEGF exposure and maintained under identical culture conditions as a control. After VEGF exposure, total cellular RNA was isolated, and an RNase protection assay was carried out using the Riboquant system according to the manufacturer’s instructions (PharMingen, Inc., San Diego, CA). The human cytokine/chemokine set hCK-4, which included templates for the following human cytokines: hIL-3, hIL-7, human GM-CSF, human granulocyte-CSF, human macrophage-CSF, hIL-6, leukemia inhibitory factor, human stem cell factor, and human oncostatin M was used for probe generation. Probe synthesis was carried out with a reaction mixture containing [α-³²P]UTP, GACU nucleotide pool, DTT, transcription buffer, RNase protection assay template, and T7 DNA polymerase. The mixture was incubated at 37°C for 1 h and purified using phenol/chloroform extraction. Five μg of total cellular RNA isolated from either the VEGF-exposed HUVEC cells or from unexposed control HUVEC cells were pipetted into a microfuge tube and dried in a vacuum concentrator. The samples were then reconstituted in hybridization buffer. Concomitantly, the probe was diluted with the same hybridization buffer to yield a concentration of 2.9 × 10⁴ cpm/μl, with 2 μl added to the tube containing the RNA. Mineral oil was added to each tube to prevent evaporation. Samples were then denatured at 90°C and hybridized for 16 h at 56°C. After hybridization, the samples were placed at 37°C, and an RNase cocktail was added to each tube. The RNase treated samples were purified using phenol/chloroform extraction and resolved on a nondenaturing acrylamide gel. After electrophoresis, the gel was carefully removed from the glass plate and dried at 80°C for ∼1 h. The dried gel was then placed on X-ray film (Kodak X-AR), and autoradiography was carried out. To control for differences in sample loading, two housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase and leukemia inhibitory factor were included as controls.

Immunohistochemical analysis was carried out on formalin-fixed, paraffin-embedded bone marrow clots or cores as described previously (29). Slides were stained for expression of factor VIII-related antigen (Dako-Patts, Santa Barbara, CA), VEGF, KDR, or Flt-1 (Santa Cruz Biotechnology, Santa Cruz, CA). The anti-VEGF antibody used in these studies has been demonstrated to be specific for VEGF and does not cross-react with other known VEGF/placenta growth factor family members. Its specificity has been established through the use of VEGF-specific blocking peptides. Both the KDR and Flt-1 antibodies are also specific and do not cross-react with each other or with other protein tyrosine kinase membrane receptors. All reactions were performed using an automated immunostainer (GenII; VMS, Tucson, AZ; Ref. 29). Detection of bound antibody was assessed through the use of immunoperoxidase methodologies with diaminobenzidine serving as the color substrate or by alkaline phosphatase methodologies using a biotinylated goat-anti-rabbit antibody (Dako-Patts) in conjunction with alkaline phosphatase-conjugated streptavidin followed by NBT/5-bromo-4-chloro-3-indolyl phosphate as the color substrate. The VMS antibody diluent was used as a negative control. Nuclei were counterstained with methyl green or hematoxylin, and sections were evaluated by light microscopy. Endogenous peroxidase was inhibited with methanol containing 0.01% H₂O₂.

Paraffin-embedded bone marrows (clots or cores) were sectioned 3 μm thick and placed on glass slides with a “sausage” control section containing placenta, liver, spleen, colon, and pancreas. The slides were baked 1 h at 60°C and then deparaffinized in two changes of xylene for 10 min each, two changes of 100% ethanol for 2 min each, followed by a graded series of alcohols (95%, 80%, and 70%), and finally, two changes of diethylpyrocarbamate-treated H₂O. They were then placed in APK wash (VMS). All additional steps were carried out using an automated in situ hybridization instrument (Gen II; VMS). The details of this procedure have been published previously (29, 30, 31). A 24-mer VEGF-specific oligonucleotide probe was designed based on published sequences and was synthesized with six biotins at the 3′end (Research Genetics, Inc., Huntsville, AL). Before the addition to the probe, slides were treated with Protease 1 (VMS) for 4 min. A total of 100 μl of probe, diluted to a concentration of 1 ng/μl in a hybridization solution composed of 35% formamide, 5× Denhardt’s, 10% dextran sulfate, 100 μg/ml of salmon sperm DNA, and 4× SSC (0.6 M NaCl; 0.06 M Na₃ citrate, pH 7.0), was manually added to each slide. For a negative control, the hybridization solution alone was applied. The slides were then denatured at 65°C for 4 min, followed by hybridization at 45°C for 60 min. After hybridization, three stringency washes were performed for 4 min each at 50°C as follows: 1× SSC, 0.5× SSC, and 0.1× SSC to remove unbound probe. Detection was carried out at 40°C by incubating the slides in streptavidin-alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN), followed by NBT and 5-bromo-4-chloro-3-indolyl phosphate substrate. The slides were counterstained off the instrument with contrast red (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Hybridization with a d(T)₃₀ oligonucleotide probe confirmed the integrity of the RNA (not shown). To insure that the oligonucleotide probe was recognizing mRNA and not genomic DNA, a subset of slides was incubated in either DNase or RNase before the addition of the probe.

Studies were undertaken to examine the expression of angiogenic growth factors and their receptors in a series of human hematopoietic tumor cell lines representing multiple cell lineages (Table 1).

Our data revealed the expression of either VEGF, bFGF, or both by all of the cell lines in our panel (Table 2). As shown in Fig. 1, RT-PCR analysis revealed transcripts corresponding to the 121 and the 165 isoforms of VEGF in the cell lines examined. Fig. 2 shows RT-PCR data demonstrating the expression of bFGF transcripts. In contrast to VEGF, bFGF was found in 50% of the cell lines examined. These findings suggest that, although different angioregulatory molecules may be operating in a given tumor, VEGF appears to be expressed in all hematopoietic tumors examined and therefore is likely playing an important role in their pathogenesis (Table 2). Expression of VEGF was confirmed using Northern blot analysis. As shown in Fig. 3, we observed a 4.4-kb transcript in all cell lines examined and an additional transcript of ∼3.7 (not shown).

On the basis of these findings, additional studies were initiated to examine the expression of the two receptors for VEGF, KDR and Flt-1. Using RT-PCR and Northern blot analysis, we found Flt-1 to be expressed not only in the HUVEC cell line as expected but also in several of the hematopoietic tumor cell lines examined (Table 2 and Fig. 4). This was an unexpected finding in light of the literature reports of KDR and Flt-1 expression being restricted to vascular endothelial cells. Interestingly, the finding of VEGF receptor expression in tumor cells may not be unique to hematopoietic cell lines because we have observed expression of KDR, but not Flt-1, in a series of small cell and non-small cell lung cancers.⁴ The finding of Flt-1 in these cell lines suggests the possibility of an autocrine pathway in which the tumor cells may stimulate their own growth after VEGF exposure. Studies are presently under way to examine this possibility.

To assess whether the VEGF protein was being secreted, cell lines were seeded into T-25 flasks at a concentration of 1 × 10⁶ cells/flask in RPMI 1640 with 1% FCS. After 48 h, the supernatants were collected for VEGF determination by ELISA assay. Secreted VEGF was detected in the conditioned medium from all cell lines examined, thereby indicating that these cells are capable of synthesizing and exporting VEGF (Fig. 5). The H-460 human large cell lung carcinoma cell line was included for reference to compare the expression observed in the hematopoietic tumor cell lines with that of a known producer of VEGF. Values obtained from hematopoietic tumor cell lines in the ELISA assay ranged from a high of 2.2 ng/ml per 10⁶ cells in the Hut 78 cell line to a low of 0.1 ng/ml in the ARH-77 cell line and compared with 2.8 ng/ml observed in the H-460 cells.

We next examined whether exposure of human endothelial cells to VEGF could stimulate the expression of known hematopoietic cytokines. After exposure of HUVEC cells to VEGF for 72 h, an increase in the message for several hematopoietic growth factors was observed (Fig. 6). mRNAs for human macrophage-CSF, human granulocyte-CSF, human IL-6, human stem cell factor, and human oncostatin M were observed in both the VEGF-stimulated and control HUVEC cells. However, there was clearly an increase in these messages in response to VEGF exposure. We have repeated this experiment three times with similar results.

Expression of VEGF and its receptors was examined in clinical bone marrow samples from patients diagnosed with multiple myeloma. To date, a total of 16 myeloma patients have been assessed with similar findings observed in all. Immunohistochemical analysis was carried out on formalin-fixed, paraffin-embedded bone marrow clots or cores as described (29). In a series of 16 cases diagnosed with multiple myeloma, plasma cell expression of VEGF was observed in the bone marrows from 12 of the patients (Table 3). An additional patient demonstrated a heterogeneous pattern of plasma cell expression. VEGF expression in plasma cells revealed a diffuse cytoplasmic pattern of staining that varied in intensity among the individual patients. Although a gradient of VEGF expression is usually observed in solid tumors, with the strongest expression being in areas of hypoxia, we did not observe such a finding in the bone marrow core samples. This observation may reflect a relative lack of hypoxic areas in the samples chosen for analysis. Our findings in myeloma patients contrasted with those from normal bone marrow specimens, where a low level of plasma cell VEGF expression was observed in only one of six samples. In both normal and myeloma samples, strong VEGF staining was observed in myeloid and monocytic as well as in megakaryocytic cells. Such a pattern of expression is consistent with the known expression of VEGF in macrophages and platelets. Staining in erythroblastic or lymphoblastic cells was not observed in any of the specimens examined. Occasional staining of polymorphonuclear cells was also observed in both normal and myeloma samples. Fig. 7,A demonstrates cytoplasmic expression of VEGF, as evidenced by brown staining, in the neoplastic plasma cells growing in the bone marrow of one of these patients. Fig. 7,B presents the same tumor mass stained with an antibody against factor VIII-related antigen, revealing the expression of positive cells within the tumor mass, which represent the neovasculature associated with an angiogenic response. In Fig. 7, panels C and D display the expression of the Flt-1 and KDR receptors, respectively. Although Flt-1 and KDR were not examined in all patient samples, neither of these two receptors were found to be expressed in the tumor cells; however, the normal bone marrow cells, predominantly myeloid and monocytic cells, revealed expression of both of these receptors, supporting a possible paracrine role of VEGF in this disease. Expression of VEGF in the malignant plasma cells was confirmed using in situ hybridization (Fig. 8).

Although it is well established that growth in solid tumors is dependent on the formation of neovasculature, the role of angiogenesis in hematopoietic neoplasms has not been determined. VEGF, a key angiogenic molecule, is a multifunctional cytokine that acts both as a potent inducer of vascular permeability and as a specific endothelial cell mitogen (5, 6). Because of its effects on endothelial cell growth and microvascular permeability, VEGF is believed to be an important mediator of tumor angiogenesis.

We have observed the expression of either VEGF, bFGF, or both by all of the hematopoietic tumor cell lines in our panel. In contrast to VEGF, which was observed in all of the cell lines, bFGF expression was observed in only 50% of the cell lines. These findings suggest that different angioregulatory molecules may be operating in hematopoietic tumors and that VEGF appears to be expressed in all hematopoietic cell lines examined. Results from our ELISA assay demonstrated that VEGF was secreted into the culture medium at concentrations that are within its range of biological activity. Concentrations of VEGF as low as 0.05 ng/ml have been demonstrated to stimulate endothelial cells in vitro (17). Thus, concentrations produced by the ARH-77 and Jurkat cell lines, the two cell lines with the lowest observable VEGF levels in our study, potentially have the ability to stimulate endothelial cells.

Although the requirement of tumor-stromal interactions is well established in multiple myeloma, the role of angiogenic factors, which act primarily on the stromal elements, is not understood in relation to hematopoietic malignancies. Most cancers may be considered as a two-compartment system in which tumor cells and stromal cells interact in a paracrine fashion. The role of the stromal compartment is well established in multiple myeloma, involving interactions between myeloma cells and the microenvironment of the bone marrow by means of cell-cell contact, adhesion molecules, and cytokines (32, 33, 34, 35, 36). Endothelial cells are found in the stromal layer of long-term bone marrow cultures in close contact with hematopoietic cells and are known to produce IL-6, an important growth factor in multiple myeloma (36, 37). They have also been demonstrated to secrete several CSFs in response to cytokines such as IL-1 (38). Thus, it is possible that endothelial cells may, in response to angiogenic factors, release cytokines capable of sustaining tumor growth. Indeed, porcine brain microvascular endothelial cells have been shown to support the expansion of human progenitor cells in vitro (39). In addition to endothelial cells, macrophages are also believed to play an important role in tumor angiogenesis through their ability to release proteases, growth factors (including VEGF), and other cytokines (18). Although we did not measure the release of growth factors into the cell culture medium, others have reported an increased secretion of GM-CSF from human endothelial cells after VEGF exposure (40). Such findings reveal that VEGF can increase the level of message expression for several growth factors with known stimulatory effects in hematopoietic malignancies including multiple myeloma. Interestingly, GM-CSF and G-CSF have been shown to stimulate human endothelial cells in vitro to migrate and proliferate (41).

Paracrine control mechanisms are increasingly recognized as being important for tumor growth. IL-6 is well established as a paracrine growth factor in myeloma, both in vitro and in vivo (33, 36, 42, 43). In normal bone marrow as well as in patients, the stromal cells appear to be the major producers of IL-6 (42, 44). We have observed that recombinant human VEGF is capable of increasing the expression of mRNA for IL-6 in a human vascular endothelial cell line. This points to the possibility that VEGF may also increase the expression of IL-6 in the bone marrow, a hypothesis we are presently examining.

To date, two high-affinity receptors for VEGF have been identified: KDR (also referred to as flk-1) and Flt-1, both of which are class III receptor tyrosine kinases (45, 46). Although it is generally held that the expression of these two receptors is restricted to endothelial cells, both human uterine smooth muscle cells and retinal pigment cells have been demonstrated to express both KDR and flt-1, whereas murine retinal progenitor cells have also been reported to express the KDR receptor (47, 48, 49). In our studies, the VEGF receptor Flt-1 was found to be expressed at moderate to high levels in 5 of the 12 tumor cell lines examined. This finding alludes to the possibility of an autocrine pathway involving VEGF operating in these cells.

The expression of VEGF in patient samples with multiple myeloma suggests that this growth factor, which has previously been believed to play a role only in solid tumors, may also be playing a role in hematopoietic tumors such as myeloma. In bone marrow from myeloma patients, VEGF expression was observed in the tumor cells both by immunohistochemistry and in situ hybridization, whereas the Flt-1 and KDR receptors were observed to be expressed in the normal marrow myeloid and monocytic cells. These data raise the possibility that VEGF may play a role in the growth of multiple myeloma through a paracrine or an autocrine mechanism. Studies are presently under way in our laboratory to determine whether VEGF is indeed playing a role in the pathophysiology of this disease. By identifying a role for VEGF in multiple myeloma or other hematopoietic malignancies, the possibility of using new treatment strategies in these patients is thus opened. Traditionally, treatment of myeloma has been hampered by the development of drug resistance in this generally incurable disease. By developing treatment strategies that target both the stromal and tumor compartments, drug resistance may be overcome, and the effect on therapeutic outcome enhanced. Although such an approach appears to hold promise for solid tumors (50), it remains to be seen whether it will be effective in hematopoietic neoplasms as well.

We thank Dr. Sydney Salmon for critical reading of the manuscript and thoughtful comments.