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Expression of Vascular Endothelial Growth Factor and its Receptors in Canine Lymphoma
ARTICLE IN PRESS J. Comp. Path. 2007,Vol. 137, 30^40
www.elsevier.com/locate/jcpa
Expression of Vascular Endothelial Growth Factor and its Receptors in Canine Lymphoma B. Wolfesberger, A. Guija de Arespacohagay, M. Willmann, W. Gernerz, I. Millery, I. Schwendenweiny, M. Kleiter, M. EgerbacherO, J. G. Thalhammer, L. Muellauer#, M. Skalickyz and I. WalterO Clinic of Internal Medicine and Infectious Diseases, yCentral Laboratory, zInstitute of Clinical Immunology, yInstitute of Medical
Chemistry, OInstitute of Histology and Embryology, zInstitute of Pathophysiology, University of Veterinary Medicine,Vienna, and # Clinical Pathology, Medical University of Vienna,Vienna, Austria
Summary Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and has a pivotal role in tumour angiogenesis. The expression of VEGFand its receptors VEGFR-1 and VEGFR-2 was examined immunohistochemically in 43 specimens of canine lymphoma and in six normal lymph nodes.Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detectVEGF protein and mRNA, respectively. VEGF protein was expressed by 60% of the tumours with di¡use cytoplasmic labelling of the neoplastic cells. Endothelial cells, macrophages and plasma cells were also immunolabelled. VEGFR-1 was expressed by variable numbers of neoplastic cells in 54% of lymphoma specimens. VEGFR-1 was also expressed by macrophages, plasma cells, reticulum cells, and vascular endothelial cells. Macrophages and lymphocytes in germinal centres of normal lymph nodes were also immunoreactive with anti-VEGFand VEGFR-1. Most tumours did not express VEGFR-2 but in 7% of sections there was focal labelling of neoplastic and endothelial cells, with a cytoplasmic and perinuclear pattern. The observed variability in expression of VEGF and its receptors probably relates to the fact that lymphoma is a heterogenous lymphoproliferative tumour. Individual di¡erences inVEGFand VEGFR expression must be taken into account whenVEGFandVEGFR-targeted approaches for anti-angiogenic therapy are considered in dogs. r 2007 Elsevier Ltd. All rights reserved. Keywords: vascular endothelial growth factor; vascular endothelial growth factor receptor; dog; lymphoma
Introduction The hypothesis that the growth of tumours is dependent on angiogenesis was ¢rst proposed by Ide and colleagues (1939) who also postulated that tumours may produce a blood vessel growth-stimulating factor. One of the most important angiogenic factors described to date is vascular endothelial growth factor (VEGF, also known as VEGF-A).VEGF is a homodimeric heparin-binding protein (Leung et al.,1989) and has several di¡erent isoforms generated by alternative splicing. VEGF is essential for Correspondence to: B. Wolfesberger, Clinic of Internal Medicine and Infectious Diseases, University of Veterinary Medicine,Vienna, Austria (e-mail: [email protected]). 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2007.03.003
the formation of blood vessels in embryos (Ferrara et al., 1996), induces vascular permeability (Senger et al.,1983), stimulates endothelial cell proliferation (Connolly et al., 1989), and has been shown to be a tumour angiogenesis factor in vivo (Kim et al.,1993).VEGFactivates endothelial cells via its receptors. Two major forms of endothelial VEGF receptor exist, VEGFR-1 (also known as £t-1) and VEGFR-2 (also known as £k-1 or KDR) (Quinn et al., 1993). The precise function of VEGFR-1 is still debated, but it appears to have both negative inhibiting and positive promoting roles in angiogenesis (Ferrara et al., 2003; Rahimi, 2006). By contrast,VEGFR-2 stimulates mitosis and motility of endothelial cells after bindingVEGF (Waltenberger et al.,1994). r 2007 Elsevier Ltd. All rights reserved.
ARTICLE IN PRESS Growth Factor Expression in Canine Lymphoma
Numerous studies have been performed to demonstrate expression of VEGF and its receptors in malignant and non-malignant tissues. VEGF mRNA was detected in the brain, pituitary gland, kidney and corpus luteum of healthy adult rats (Ferrara et al., 1992), and in normal guinea-pig and human tissues including lung, kidney, liver, heart, gastric mucosa and adrenal gland (Berse et al., 1992). VEGF is expressed in human tumours including breast carcinoma (Brown et al., 1995), angiosarcoma (Hashimoto et al., 1995) and lymphoma (Ho et al., 2002). To date, little is known about VEGFand VEGFR expression in normal or diseased canine tissue. Scheidegger and colleagues (1999) demonstrated that canine and human VEGF are structurally almost identical, with the same biological and cell-binding properties, and that the canine VEGF receptors closely resemble their human counterparts. VEGF expression was increased in canine mammary carcinoma, and was most often associated with poorly di¡erentiated neoplastic cells (Restucci et al., 2002). VEGF expression and micro-vessel density were also increased in seminomas compared with normal testicular tissue of dogs. Canine cutaneous squamous cell carcinomas also showed VEGFexpression (Restucci et al., 2003), but VEGF was not expressed within the majority of canine basal cell tumours (Maiolino et al., 2000). The aim of the present study was to investigate the expression of VEGF and its receptors in normal canine lymph nodes and in specimens of canine lymphoma. Lymphoma is the most common canine haemopoietic tumour and it has been suggested that the prevalence of this neoplasm is increasing (Moulton and Harvey,1990).
Materials and Methods Tissue Specimens
Samples from 43 canine lymphomas and six normal canine lymph nodes were ¢xed in 4% neutral bu¡ered formalin and embedded in para⁄n-wax. Sections (4 mm) were stained by haematoxylin and eosin (HE). The lymphomas were classi¢ed according to theWorking Formulation Classi¢cation (Carter et al., 1986) and immunohistochemistry was performed to distinguish between B- and T-cell lymphoma. Antibody against CD3 (A452; Dako, Glostrup, Denmark) at1in100 dilution was used to identify T-cells and antibody against CD79a (M7051, clone HM57; Dako) at 1in 100 dilution was used for the identi¢cation of B-cells. Samples for western blotting and RT-PCR were frozen at 80 1C. Immunohistochemistry (IHC)
Immunohistochemical labelling was performed on sections (4 mm) of formalin-¢xed and para⁄n-wax
31
qembedded tissue. Sections were ¢rst de-waxed and treated with H2O2 0.6% in methanol for 15 min to block endogenous peroxidase activity. Antigen retrieval was achieved by microwave heating (800 W) in citrate bu¡er (pH 6.0, 0.01M) for four cycles of 5 min. Primary antibodies directed against VEGF (A-20, sc152, a rabbit polyclonal antibody; Santa Cruz Biotechnology, CA, USA), and VEGFR-1 (Flt-1, C-17, sc-316, rabbit polyclonal antibody; Santa Cruz Biotechnology, CA, USA) at 1 in 100 dilution, and VEGFR-2 (Flk-1, A-3, sc-6251, mouse monoclonal antibody; Santa Cruz Biotechnology) at 1 in 50 dilution in phosphate buffered saline (PBS) were applied overnight at 4 1C. Peroxidase-labelled anti-mouse and rabbit PowerVisions secondary system (ImmunoVisionTechnologies, CA, USA) was employed for antibody detection using Histogreen (Linaris, Wertheim, Germany) as the coloured substrate. Nuclei were counterstained with nuclear fast-red sulphate (Chroma, Germany). The primary antibody was omitted for negative control slides. To demonstrate antibody speci¢city, the VEGF antibody (at 1 in 100 dilution) was pre-incubated with a blocking peptide (10 mg VEGF A-20 in 100 ml PBS; Santa Cruz Biotechnology), and this resulted in extinction of VEGFexpression.VEGFandVEGFreceptor expression were scored independently by two observers (BW and IW) as 0 for absent, 1 for less than 10% positively labelled cells, 2 for 10^50% positively labelled cells, and 3 for more than 50% positively labelled cells at 400 magni¢cation. For vimentin labelling, antigen retrieval was achieved by microwave heating (800 W) in citrate buffer (pH 6.0, 0.01M) for two cycles of 5 min. Sections were incubated with anti-mouse vimentin antibody (M 0725 clone V9; Dako) at 1 in 100 dilution. The secondary system was peroxidase-labelled anti-mouse PowerVisions (ImmunoVisionTechnologies), followed by diaminobenzidine (DAB) (Sigma,Vienna, Austria) for ‘‘visualization’’of the reaction. Western Blotting
Western blotting was performed semi-quantitatively to compareVEGFexpression in canine lymphomas and normal lymph nodes. Samples (n ¼ 7) were homogenized in low salt extraction bu¡er. Protein content in cell lysates was determined by a Coomassie Brilliant Blue G-250 binding assay (Bradford, 1976), performed in microplate format (Microplate Reader Sunrise; Tecan, Groedig, Austria) with a bovine serum albumin calibration curve. Equal amounts of protein (82 mg) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions on 12.5% polyacrylamide gels. SDS-PAGE was performed according to Laemmli (1970) using a Hoefer Mighty Small II
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electrophoresis unit (Hoefer Scienti¢c Instruments, San Francisco, CA, USA). Kaleidoscope pre-stained standard (Bio Rad, Hercules, CA, USA) and Magic Markers XP Western standard (Invitrogen, Lofer, Austria) were used for molecular weight calibration and gels were stained with Coomassie blue. Following separation by SDS-PAGE, the proteins were transferred onto a PVDF-membrane (Amersham Life Science, Buckinghamshire, UK) in a semi-dry blotting device (Semi-Phor; Hoefer Scienti¢c Instruments). Dried milk powder (5%) was used as a blocking solution to minimize non-speci¢c binding of the primary antibody.The membrane was incubated with primaryVEGF antibody (A-20 as above) at 1 in 400 dilution in PBS/ Tween overnight at 4 1C. The ECL-secondary system (enhanced chemiluminescence; Amersham-Biosciences, Vienna, Austria) was used to detect binding. Human VEGF (58MS-1467-PCL; Labvision, CA, USA) and recombinant canine VEGF (1603-CV; R&D Systems Europe, Oxon, UK) were used as positive controls. Reverse Transcriptase Polymerase Chain Reaction (RTPCR)
Samples of canine lymphoma (n ¼ 1), feline lymphoma (n ¼ 1), normal canine lymph node (n ¼ 1), canine ovary (n ¼ 1) and uterus (n ¼ 1) were immersed in RNAlaters (Ambion Europe Ltd., Cambridge, UK) until total RNA extraction with TRIzols (Invitrogen GmbH, Lofer, Austria). ReverseTranscription was performed using a SuperScript One-Step RT-PCR kit with Platinum Taq (Invitrogen GmbH) according to the manufacturer’s instructions. Canine-speci¢cVEGF primers (Scheidegger et al., 1999) were used: 50 -ATGAACTTTCTGCTCTCTTGG-30; 50 -TCACCGCC TCGGCTTGTC-30 (Sigma Genosys, Su¡olk, UK). Samples were ampli¢ed by 35 cycles of 95 1C for 30 s, followed by 60 1C for 30 s and 1min at 72 1C. PCR products were separated on 1.5% agarose gels containing ethidium bromide and images were captured during examination under UV-light. Statistical Analysis
Pearson’s correlation coe⁄cient was used to investigate whether the scores of VEGF, VEGFR-1 and VEGFR-2 immunohistochemical labelling correlated with histology, tumour grade and classi¢cation as B- versus T-cell lymphoma. Correlation was considered signi¢cant if the P-value was o0.05.
Results Immunohistochemistry
The expression of VEGF, VEGFR-1 and VEGFR-2 in canine lymphoma is summarized inTable 1. There was
no signi¢cant correlation between tumour grade, histology, di¡erentiation to B- or T-cell lymphoma and the expression of VEGF,VEGFR-1 orVEGFR-2. Expression of VEGF. Sixty per cent of lymphomas had high expression of VEGF with 50% or more of the neoplastic cells showing immunoreactivity (score 3). Twelve per cent of samples had a score of 2,14% a score of 1, and 14% had no VEGF expression. The pattern of labelling was di¡use and cytoplasmic (Fig. 1b). Endothelial cells, macrophages, plasma cells, and reticulum cells were also positively labelled (Fig. 1a, Fig. 2c), whereas residual normal lymphoid follicles were always negative. In six normal lymph nodes, there was strong expression of VEGF by macrophages and plasma cells located mainly in the medullary sinuses and the medullary cords (Fig. 2a). Lymphocytes in the germinal centre of follicles were positively labelled but were surrounded by unlabelled cells (Fig. 1c, d). Endothelial cells were variably labelled. Expression of VEGFR-1. Forty six per cent of the lymphoma samples did not expressVEGFR-1; a score of 1was recorded in 12%, a score of 2 in 19%, and a score of 3 in 23% of samples. The pattern of labelling was primarily di¡use and cytoplasmic, and in one case perinuclear. Residual normal lymphoid follicles were unlabelled. Macrophages showed strong expression of VEGFR-1, as did plasma cells and reticulum cells. Positively labelled macrophages were aggregated in medullary cords or occasionally with perivascular distribution. Approximately 50% of blood vessels had endothelial expression of VEGFR-1 (Fig. 3a, c, d). Very intensive labelling of numerous macrophages was seen throughout the normal lymph nodes (Fig. 3b). Germinal centre lymphocytes were positively labelled and a few reticulum cells also showed reactivity. Only a proportion of vessels had positively labelled endothelial cells. Expression of VEGFR-2. Seventy two per cent of lymphoma samples did not expressVEGFR-2; a score of 1was recorded in14%, a score of 2 in 7% and a score of 3 in 7% of samples in which there was intense focal labelling with a cytoplasmic or perinuclear pattern. Smooth muscle and endothelial cells were sometimes positively labelled (Fig. 4a, b). Of the six normal lymph nodes, two were completely negative for VEGFR-2 expression, another three showed perinuclear labelling of very few single lymphocytes (o1%), and one sample also had positive expression in smooth muscle cells of vessels. Only one lymph node was scored as 1, and this sample had perinuclear expression of VEGFR-2 by cortical lymphocytes (Fig. 4c, d).
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Growth Factor Expression in Canine Lymphoma Table 1 Expression of VEGF,VEGFR-1 and VEGFR-2 in canine lymphoma Patient.
Sex
Age (Y)
Breed
Grade
Histology
B/T Cell
VEGF
VEGFR-1
VEGFR-2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
M Fn M M F Mn M M M Mn M M M Mn F M F F F Mn Fn Mn M F M F F M M Fn Fn M M M Fn Fn Fn M Mn M Fn F F
10 13 9 6 12 13 3 10 10 4 6 8 8 4 12 10 5 3 10 4 5 8 9 9 4 5 6 10 9 12 14 11 4 7 4 7 4 12 4 8 7 4 11
German hunting terrier Siberian husky Mixed Yorkshire terrier Mixed Mixed Labrador retriever Labrador retriever German shepherd Rottweiler Rhodesian ridgeback Mixed Mixed Mixed German shepherd Golden retriever German shepherd Golden retriever Irish setter Mixed Akita inu Afghan hound German shepherd Mixed Gordon setter Boxer Dogo Argentino Irish setter Mixed Bordeaux masti¡ West-Highland terrier Mixed Dogo Argentino Bull terrier Mixed Mixed Sta¡ordshire German shepherd Mixed Bordeaux masti¡ Pinscher Mixed Mixed
L L L L I I I I I I I I I I I I I I I I I I I I I I I I I I I H H H H H H H H H H H H
DSLL DSLL DSLL DSLL MMCL MMCL DMCL DMCL DMCL DMCL DMCL DMCL DMCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL LCIBL LCIBL LCIBL LCIBL LCIBL LCIBL LCIBL LBL LBL LBL SNCCL SNCCL
0 0 T B B B 0 0 0 B T T B 0 0 B T B B T B B B B/T B/T B/T B B B B B B/T B B B B B 0 B/T B B/T B/T B
1 3 2 3 3 3 3 3 2 0 1 3 3 2 3 2 3 0 0 3 3 3 3 1 0 1 3 3 1 0 2 3 3 3 0 3 3 1 3 3 3 3 3
0 2 0 0 3 0 1 2 3 0 1 3 3 2 3 0 0 0 0 2 2 3 0 0 3 1 2 3 1 0 2 1 3 2 0 3 0 0 0 0 0 0 0
0 0 1 0 0 2 0 0 1 0 0 0 2 1 0 0 0 1 0 3 0 3 0 0 0 0 0 0 0 0 0 1 0 0 0 3 0 1 0 0 0 2 0
Fn, female neutered; Mn, male neutered; L, low-grade lymphoma; I, intermediate-grade lymphoma; H, high-grade lymphoma; DSLL, di¡use small lymphocytic lymphoma; MMCL, macro-nucleated medium-sized cell lymphoma; DMCL, di¡use mixed cell lymphoma; DLCL, di¡use large cell lymphoma; LCIBL, large cell immunoblastic lymphoma; LBL, lymphoblastic lymphoma; SNCCL, small non-cleaved cell lymphoma; 0 not classi¢able; B, B-cell lymphoma;T,T-cell lymphoma; B/T, B andT-cell lymphoma;VEGF, vascular endothelial growth factor;VEGFR-1, vascular endothelial growth factor receptor 1; VEGFR-2, vascular endothelial growth factor receptor 2; score 0, no labelling; score 1, o10% labelling; score 2, 10^50% labelling; score 3, 450% positively labelled lymphoma cells.
Western Blotting
A rabbit anti-human antibody was used to detect VEGF in canine tissues (Fig. 5). HumanVEGF protein, the positive control, resulted in a distinct band of approximately 20 kD molecular mass. Recombinant canine VEGF resulted in a very broad band, proving that the anti-human antibody recognizes canine
VEGF. Bands of 20 kD molecular mass, but of di¡erent intensity, were obtained with extracts from canine lymphomas and normal lymph node (Fig. 5, lane 4). Lanes 5^8 of Fig. 5 represent VEGF expression in samples from patients with intermediategrade lymphoma. The more intense bands in lanes 9 and 10 of Fig. 5 derive from dogs with high-grade lymphoma.
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Fig. 1A^D. (A) Section of canine lymphoma showing positive labelling of scattered macrophages (black arrows), reticulum cells and weak labelling of two vessels (open arrows). There is no positive labelling of tumour cells (score 0). (B) Section of canine lymphoma showing cytoplasmic expression of VEGF by 450% of the tumour cells (score 3). (C) Section of normal dog lymph node showing VEGF immunoreactivity in the germinal centre, medullary sinus and medullary cords. (D) Higher magni¢cation of (C). IHC. (A) 250; (B) 250; (C) 40; (D) 250.
Reverse Transcriptase Polymerase Chain Reaction (RTPCR)
The presence of three distinct VEGF mRNA isoforms (VEGF 120, 164, 188) was demonstrated in canine and feline lymphoma tissue as well as in normal lymph node tissue. Canine ovarian and uterine tissue provided positive controls for this assay (Fig. 6).
Discussion Lymphomas are a heterogenous group of lymphoproliferative tumours which occur in humans and dogs. A number of studies have assessed the expression of the major angiogenic cytokine VEGF in human lymphoma, but to our knowledge such expression has not been studied in the canine disease. In the present study 60% of the 43 samples of canine lymphoma showed a high level of expression of VEGF by the neoplastic cells. In studies of human lymphoma, VEGF was shown to be expressed by lymphoma cell lines (Bellamy et al., 1999), in 100% of 39 tumour sam-
ples (Kadowaki et al., 2005), in all samples from 24 patients with angioimmunoblastic T-cell lymphoma (Zhao et al., 2004), in 70.6% of 58 cases of classical Hodgkin’s lymphoma (Doussis-Anagnostopoulou et al., 2002), in 41.2% of 17 cases of mantle cell lymphoma (Potti et al., 2002), in 33.8% of 71cases of non-Hodgkin’s lymphomas (Hazar et al., 2003), and in three of nine lymph nodes taken from lymphoma patients (Ho et al., 2003). In contrast to the results of these studies, Foss and coworkers (1997) found no VEGF transcription in nonHodgkin’s and Hodgkin’s lymphoma (except in one case in which there was a single labelled tumour cell). Reasons for these vastly diverse results could be accounted for by di¡erent subtypes of the lymphoma in each study, the use of variousVEGFantibodies, and different experimental methods including immunohistochemistry and in situ hybridization. In the present study,VEGFexpression in tissue from canine lymphoma was also found in endothelial cells, macrophages, reticulum cells and plasma cells. Peripheral blood T lymphocytes and lymphocytes in¢ltrating
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Fig. 2A^D. (A) Section of normal canine lymph node showing expression of VEGF by plasma cells and macrophages. (B) Demonstration of immunolabelling of B lymphocytes and plasma cells by CD79a antibody in a normal lymph node. (C) VEGF positive reticulum cells in a section of canine lymphoma. (D) Vimentin staining of reticulum cells in a section of canine lymphoma. IHC. (A) 1000; (B) 1000; (C) 400; (D) 400.
human prostatic and bladder tumours synthesize VEGF protein (Freeman et al., 1995). VEGF has also been found in plasma cells of patients with multiple myeloma (Kimlinger et al., 2006) and in plasma cells within nasal polyps (Ito et al.,1995). In one study of Hodgkin’s lymphoma in human patients, endothelial cells failed to express VEGF except in three cases where there was positive labelling in small vessels, but plasma in the blood vessels reacted positively in all samples (Doussis-Anagnostopoulou et al., 2002). In cases of the present study where plasma was present in vessels, we also found VEGF immunoreactivity in the lumens of the vessels. A possible explanation for this ¢nding is that blood platelets contain VEGF which is released during platelet aggregation (Maloney et al., 1998). Doussis-Anagnostopoulou et al. (2002) also reported variable amounts of strong extracellular VEGF staining in stromal tissue, which was also detected in the present study. These authors suggested that the VEGF-positive extracellular matrix may have an important role as storage area for this protein, providing ready access of the tissue to angiogenic factors.
VEGF-positive macrophages were documented in canine mammary tumours (Restucci et al., 2002), and Doussis-Anagnostopoulou et al. (2002) also recognized VEGF immunolabelling in macrophages in samples from patients with Hodgkin’s lymphoma. It is reported that macrophages may stimulate the growth of ¢brosarcomas in mice (Evans, 1977). The present study also documentsVEGF-positive macrophages in canine lymphoma; however we also detected intense immunolabelling of macrophages in normal lymph nodes, a ¢nding which undermines this interesting theory. Plasma cells, lymphocytes (especially those in the germinal centre), and variable numbers of blood vessels also showed expression of VEGF in normal canine lymph nodes. The presence of VEGF protein was con¢rmed by western blotting and three VEGF mRNA isoforms were detected by RT-PCR in neoplastic and normal canine lymph nodes. These observations raise the question of the role of VEGF in normal lymph nodes. As neoangiogenesis does not take place under physiological conditions in adults (except in the ovary and uterus), VEGF expression by cells as plasma cells, lymphocytes, macrophages and endothelial cells could
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Fig. 3A^D. (A) Section of canine lymphoma showing expression of VEGFR-1 by macrophages. (B) Section of normal canine lymph node showing expression of VEGFR-1 by macrophages. (C) Section of canine lymphoma showing expression of VEGFR-1 by blood vessels. (D) VEGFR-1immunolabelling of the cytoplasm of reticulum cells in canine lymphoma. IHC. (A) 250; (B) 250; (C) 250; (D) 400.
be necessary to sustain the numerous mature blood vessels in normal lymph nodes. It is also possible that the expression of VEGF, which is also called vascular permeability factor (VPF; Ferrara et al., 2003), may play a role in maintaining the baseline permeability of the normal microcirculation (Berse et al.,1992). VEGF has been identi¢ed in many normal human tissues including lung, kidney, liver, gastric mucosa, breast and in activated macrophages, but it was not detected in resident peritoneal and alveolar macrophages (Berse et al.,1992). Turley and colleagues (1998) also examined normal human tissues and observed consistently strong reactivity in the ovary, uterus, salivary gland, pancreatic islet cells and plasma.Variable numbers of plasma cells, ¢broblasts and lymphocytes were labelled in the stroma of all tissues, whereas most of the endothelial cells were negative. Little is known about VEGF expression in non-reactive lymph nodes. In a study byTurley et al. (1998), three normal human lymph nodes were examined and weak labelling of lymphocytes was described. One of ¢ve non-reactive human lymph nodes showed weak expression of VEGF (Kadowaki et al., 2005). Unfortunately,
no details were given concerning which type of cells showed this reactivity. No statement about VEGF expression in macrophages, plasma cells and endothelial cells in human lymph nodes has been published. Approximately one half of our canine lymphoma samples showed VEGFR-1 immunoreactivity in neoplastic cells and 50% of the blood vessels in these samples were also positively labelled. Zhao and colleagues (2004) identi¢ed VEGFR-1 in neoplastic lymphocytes and endothelial cells in human angioimmunoblastic T-cell lymphoma by immunohistochemistry, but no expression was found in mantle cell lymphoma or follicular lymphoma (Ho et al., 2003). The present study reports detection of VEGFR-1 protein in plasma cells, reticulum cells and macrophages in samples of canine lymphoma as well as in normal lymph nodes. In human tissue VEGFR-1 was expressed by monocytes, macrophages, endothelial cells, haemopoietic stem cells, pericytes, smooth muscle cells, osteoblasts and colorectal tumour cells, but normal lymph node was not examined (Byrne et al., 2005). Interestingly, human monocytes, the precursor cells of macrophages, were found to express the
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Fig. 4A^D. (A) Section of canine lymphoma showing expression of VEGFR-2 by endothelial and smooth muscle cells of an artery and its arterioles. (B) Section of canine lymphoma showing cytoplasmic or perinuclear expression of VEGFR-2 by neoplastic cells. (C) Section of normal canine lymph node showing no expression of VEGFR-2. (D) Section of normal canine lymph node showing perinuclear expression of VEGFR-2 by o10% of lymphocytes (score 1). IHC. (A) 100; (B) 400; (C) 400; (D) 400.
Fig. 6. RT-PCR gel electrophoresis demonstrating expression of VEGF m-RNA isoforms in canine tissue. 1kB DNA ladder (lane 1), feline lymphoma extract (lane 2), normal canine lymph node (lane 3), canine lymphoma (lane 4), canine ovary (lane 5), canine uterus (lane 6). Fig. 5. Western blot analysis of VEGF expression in canine tissue. Molecular weight standard (lane 1), humanVEGFas a positive control (lane 2), canineVEGFas a positive control (lane 3), normal canine lymph node extract expressing VEGF (lane 4), di¡erent canine lymphoma extracts (lanes 5^10). The position of theVEGF band is indicated.
VEGFR-1 gene and it has been suggested that VEGF may be able to induce a migration signal in monocytes viaVEGFR-1 (Barleon et al.,1996).The precise function of VEGFR-1 remains unclear. On the one hand, VEGFR-1 has great a⁄nity forVEGF, but on the other it has only very weak tyrosine kinase activity (Hiratsuka et al.,1998). Findings suggest that VEGFR-1 behaves
more like a decoy than a signal-transducing receptor, preventing VEGF from binding to VEGFR-2 and thereby decreasing the biological activity of VEGF (Park et al., 1994). Therefore, at this time it is not clear if the presence of VEGFR-1 is an advantage or disadvantage in the course of the disease. No VEGFR-2 expression was found in about three quarters of the canine lymphomas examined in this study. Only three patients (one with high-grade and two with intermediate-grade lymphoma) showed focal intense labelling of neoplastic cells. In these locations endothelial cells were also highly immunoreactive. No
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expression of VEGFR-2 by cancer cells in angioimmunoblastic T-cell lymphoma was described by Zhao and colleagues (2004), whereas in¢ltrating cells in two of nine lymphomas showed slight labelling for VEGFR-2 (Ho et al., 2003). In contrast, 74% of lymphomas were positive for phosphorylated VEGFR-2 expression in a study performed by Pillai and co-workers (2005). We did not ¢nd any VEGFR-2 immunoreactivity in macrophages as described by Stewart et al. (2003), although northern blot analysis showed that monocytes do not express the VEGFR-2 gene (Barleon et al., 1996). The positive labelling of vascular smooth muscle cells noted in the present study corresponds with the ¢ndings of other studies (Byrne et al., 2005). To date, much e¡ort has been expended in order to ¢nd new drugs blockingVEGFor its receptors in cancer, in order to decrease endothelial and tumour cell proliferation and ultimately to improve survival. E¡ects of antibodies against VEGF and inhibitors of the VEGFR tyrosine kinase pathway in human cancer are currently being evaluated in clinical trials in which such agents are administered either as a single drug therapy or in combination with conservative chemotherapy. No studies assessing the potential bene¢t of anti-angiogenic drugs have been performed in dogs.We hypothesize that some dogs with lymphoma may be potential candidates for the use of angiogenesis inhibitors; in particular the two animals of the present study that had a high amount of VEGF protein in western blots or the three dogs demonstrating strong VEGFR-2 immunoreactivity on immunohistochemistry. However, considering the observed variability in expression of VEGF and its receptors in our tumour samples it is not possible to suggest a general anti-angiogenic therapy for all canine lymphoma patients. On the contrary, these wide di¡erences suggest that each patient’s tumour should be individually assessed forVEGFandVEGFRexpression, if such targeted therapy is considered in the future.
Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Magdalena Helmreich Mag, Waltraud Tschulenk, Christine Bayer, Sonja Dolezal and Doris Rosenfellner. This work was supported by a grant from the Veterinary University of Vienna (Pro¢llinie I Oncology).
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Maloney, J. P., Silliman, C. C., Ambruso, D. R., Wang, J., Tuder, R. M. and Voelkl, N. F. (1998). In vitro release of vascular endothelial growth factor during platelet aggregation. AmericanJournal of Physiology, 275,1054^1061. Moulton, J. E. and Harvey, J. W. (1990). Tumors of lymphoid and hematopoietic tissue. In: Tumors of Domestic Animals, 3rd Edit., J.E. Moulton, Ed., University of California Press, Berkeley, pp. 150^196. Park, J. E., Chen, H. H.,Winer, J., Houck, K. A. and Ferrara, N. (1994). Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high a⁄nity binding to Flt-1 but not to FLK-1/KDR. Journal of Biological Chemistry, 269, 25646^25654. Pillai, G., Cook, N.,Turley, H., Leek, R. D., Blasquez, C., Pezella, F., Harris, A. L. and Gatter, K. C. (2005). The expression and cellular localization of phosphorylated VEGFR2 in lymphoma and non-neoplastic lymphadenopathy: an immunohistochemical study. Histopathology, 46, 209^216. Potti, A., Kishor, G. A., Kargas, S. and Koch, M. (2002). Immunohistochemical detection of c-kit (CD117) and vascular endothelial growth factor (VEGF) overexpression in mantle cell lymphoma. Anticancer Research, 22, 2899^2902. Quinn,T. P., Peters, K. G., DeVries, C., Ferrara, N. andWilliams, L. T. (1993). Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium. Proceedings of the National Academy of Sciences, 90,7533^7537. Rahimi, N. (2006).VEGFR-1 and VEGFR-2: two non-identical twins with a unique physiognomy. Frontiers in Bioscience, 11, 818^829. Restucci, B., Maiolino, P., Paciello, O., Martano, M., De Vico, G. and Papparella, S. (2003). Evaluation of angiogenesis in canine seminomas by quantitative immunohistochemistry. Journal of Comparative Pathology, 128, 252^259. Restucci, B., Paparella, S., Maiolino, P. and De Vico, G. (2002). Expression of vascular endothelial growth factor in canine mammary tumors. Veterinary Pathology, 39, 488^493. Scheidegger, P., Weiglhofer, W., Suarez, S., Kaser-Hotz, B., Steiner, R., Ballmer-Hofer, K. andJaussi, R. (1999).Vascular endothelial growth factor (VEGF) and its receptors in tumor-bearing dogs. Biological Chemistry, 380,1449^1454. Senger, D. R., Galli, S. J., Dvorak, A. M., Peruzzi, C. A., Harvey,V. S. and Dvorak, H. F. (1983).Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites £uid. Science, 219, 983^985. Stewart, M., Turley, H., Cook, N., Pezzella, F., Pillai, G., Ogilvie, D., Cartlidge, S., Paterson, D., Copley, C., Kendrew, J., Barnes, C., Harris, A. L. and Gatter, K. C. (2003). The angiogenic receptor KDR is widely distributed in human tissues and tumours and relocates intracellularly on phosphorylation. An immunohistochemical study. Histopathology, 43, 33^39. Turley, H., Scott, P. A., Watts,V. M., Bicknell, R., Harris, A. L. and Gatter, K. C. (1998). Expression of VEGF in routinely ¢xed material using a new monoclonal antibodyVG1. Journal of Pathology, 186, 313^318.
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Waltenberger, J., Claesson-Welsh, L., Siegbahn, A., Shibuya, M. and Heldin, C. H. (1994). Di¡erent signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. Journal of Biological Chemistry, 269, 26988^26995. Zhao, W. L., Mourah, S., Mounier, N., Leboeuf, C., Danesphouy, M. E., Legres, L., Meignin,V., Oksenhendler, E., Maignin, C. L., Calvo, F., Briere, J., Gisselbrecht, C. and Janin, A. (2004). Vascular endothelial growth factor-A is
expressed both on lymphoma cells and endothelial cells in angioimmunoblastic T-cell lymphoma and related to lymphoma progression. Laboratory Investigation, 84, 1512^1519.
Received, August 23rd, 2006 Accepted, March 13th, 2007
www.elsevier.com/locate/jcpa
Expression of Vascular Endothelial Growth Factor and its Receptors in Canine Lymphoma B. Wolfesberger, A. Guija de Arespacohagay, M. Willmann, W. Gernerz, I. Millery, I. Schwendenweiny, M. Kleiter, M. EgerbacherO, J. G. Thalhammer, L. Muellauer#, M. Skalickyz and I. WalterO Clinic of Internal Medicine and Infectious Diseases, yCentral Laboratory, zInstitute of Clinical Immunology, yInstitute of Medical
Chemistry, OInstitute of Histology and Embryology, zInstitute of Pathophysiology, University of Veterinary Medicine,Vienna, and # Clinical Pathology, Medical University of Vienna,Vienna, Austria
Summary Vascular endothelial growth factor (VEGF) stimulates endothelial cell proliferation and has a pivotal role in tumour angiogenesis. The expression of VEGFand its receptors VEGFR-1 and VEGFR-2 was examined immunohistochemically in 43 specimens of canine lymphoma and in six normal lymph nodes.Western blotting and reverse transcriptase polymerase chain reaction (RT-PCR) were performed to detectVEGF protein and mRNA, respectively. VEGF protein was expressed by 60% of the tumours with di¡use cytoplasmic labelling of the neoplastic cells. Endothelial cells, macrophages and plasma cells were also immunolabelled. VEGFR-1 was expressed by variable numbers of neoplastic cells in 54% of lymphoma specimens. VEGFR-1 was also expressed by macrophages, plasma cells, reticulum cells, and vascular endothelial cells. Macrophages and lymphocytes in germinal centres of normal lymph nodes were also immunoreactive with anti-VEGFand VEGFR-1. Most tumours did not express VEGFR-2 but in 7% of sections there was focal labelling of neoplastic and endothelial cells, with a cytoplasmic and perinuclear pattern. The observed variability in expression of VEGF and its receptors probably relates to the fact that lymphoma is a heterogenous lymphoproliferative tumour. Individual di¡erences inVEGFand VEGFR expression must be taken into account whenVEGFandVEGFR-targeted approaches for anti-angiogenic therapy are considered in dogs. r 2007 Elsevier Ltd. All rights reserved. Keywords: vascular endothelial growth factor; vascular endothelial growth factor receptor; dog; lymphoma
Introduction The hypothesis that the growth of tumours is dependent on angiogenesis was ¢rst proposed by Ide and colleagues (1939) who also postulated that tumours may produce a blood vessel growth-stimulating factor. One of the most important angiogenic factors described to date is vascular endothelial growth factor (VEGF, also known as VEGF-A).VEGF is a homodimeric heparin-binding protein (Leung et al.,1989) and has several di¡erent isoforms generated by alternative splicing. VEGF is essential for Correspondence to: B. Wolfesberger, Clinic of Internal Medicine and Infectious Diseases, University of Veterinary Medicine,Vienna, Austria (e-mail: [email protected]). 0021-9975/$ - see front matter
doi:10.1016/j.jcpa.2007.03.003
the formation of blood vessels in embryos (Ferrara et al., 1996), induces vascular permeability (Senger et al.,1983), stimulates endothelial cell proliferation (Connolly et al., 1989), and has been shown to be a tumour angiogenesis factor in vivo (Kim et al.,1993).VEGFactivates endothelial cells via its receptors. Two major forms of endothelial VEGF receptor exist, VEGFR-1 (also known as £t-1) and VEGFR-2 (also known as £k-1 or KDR) (Quinn et al., 1993). The precise function of VEGFR-1 is still debated, but it appears to have both negative inhibiting and positive promoting roles in angiogenesis (Ferrara et al., 2003; Rahimi, 2006). By contrast,VEGFR-2 stimulates mitosis and motility of endothelial cells after bindingVEGF (Waltenberger et al.,1994). r 2007 Elsevier Ltd. All rights reserved.
ARTICLE IN PRESS Growth Factor Expression in Canine Lymphoma
Numerous studies have been performed to demonstrate expression of VEGF and its receptors in malignant and non-malignant tissues. VEGF mRNA was detected in the brain, pituitary gland, kidney and corpus luteum of healthy adult rats (Ferrara et al., 1992), and in normal guinea-pig and human tissues including lung, kidney, liver, heart, gastric mucosa and adrenal gland (Berse et al., 1992). VEGF is expressed in human tumours including breast carcinoma (Brown et al., 1995), angiosarcoma (Hashimoto et al., 1995) and lymphoma (Ho et al., 2002). To date, little is known about VEGFand VEGFR expression in normal or diseased canine tissue. Scheidegger and colleagues (1999) demonstrated that canine and human VEGF are structurally almost identical, with the same biological and cell-binding properties, and that the canine VEGF receptors closely resemble their human counterparts. VEGF expression was increased in canine mammary carcinoma, and was most often associated with poorly di¡erentiated neoplastic cells (Restucci et al., 2002). VEGF expression and micro-vessel density were also increased in seminomas compared with normal testicular tissue of dogs. Canine cutaneous squamous cell carcinomas also showed VEGFexpression (Restucci et al., 2003), but VEGF was not expressed within the majority of canine basal cell tumours (Maiolino et al., 2000). The aim of the present study was to investigate the expression of VEGF and its receptors in normal canine lymph nodes and in specimens of canine lymphoma. Lymphoma is the most common canine haemopoietic tumour and it has been suggested that the prevalence of this neoplasm is increasing (Moulton and Harvey,1990).
Materials and Methods Tissue Specimens
Samples from 43 canine lymphomas and six normal canine lymph nodes were ¢xed in 4% neutral bu¡ered formalin and embedded in para⁄n-wax. Sections (4 mm) were stained by haematoxylin and eosin (HE). The lymphomas were classi¢ed according to theWorking Formulation Classi¢cation (Carter et al., 1986) and immunohistochemistry was performed to distinguish between B- and T-cell lymphoma. Antibody against CD3 (A452; Dako, Glostrup, Denmark) at1in100 dilution was used to identify T-cells and antibody against CD79a (M7051, clone HM57; Dako) at 1in 100 dilution was used for the identi¢cation of B-cells. Samples for western blotting and RT-PCR were frozen at 80 1C. Immunohistochemistry (IHC)
Immunohistochemical labelling was performed on sections (4 mm) of formalin-¢xed and para⁄n-wax
31
qembedded tissue. Sections were ¢rst de-waxed and treated with H2O2 0.6% in methanol for 15 min to block endogenous peroxidase activity. Antigen retrieval was achieved by microwave heating (800 W) in citrate bu¡er (pH 6.0, 0.01M) for four cycles of 5 min. Primary antibodies directed against VEGF (A-20, sc152, a rabbit polyclonal antibody; Santa Cruz Biotechnology, CA, USA), and VEGFR-1 (Flt-1, C-17, sc-316, rabbit polyclonal antibody; Santa Cruz Biotechnology, CA, USA) at 1 in 100 dilution, and VEGFR-2 (Flk-1, A-3, sc-6251, mouse monoclonal antibody; Santa Cruz Biotechnology) at 1 in 50 dilution in phosphate buffered saline (PBS) were applied overnight at 4 1C. Peroxidase-labelled anti-mouse and rabbit PowerVisions secondary system (ImmunoVisionTechnologies, CA, USA) was employed for antibody detection using Histogreen (Linaris, Wertheim, Germany) as the coloured substrate. Nuclei were counterstained with nuclear fast-red sulphate (Chroma, Germany). The primary antibody was omitted for negative control slides. To demonstrate antibody speci¢city, the VEGF antibody (at 1 in 100 dilution) was pre-incubated with a blocking peptide (10 mg VEGF A-20 in 100 ml PBS; Santa Cruz Biotechnology), and this resulted in extinction of VEGFexpression.VEGFandVEGFreceptor expression were scored independently by two observers (BW and IW) as 0 for absent, 1 for less than 10% positively labelled cells, 2 for 10^50% positively labelled cells, and 3 for more than 50% positively labelled cells at 400 magni¢cation. For vimentin labelling, antigen retrieval was achieved by microwave heating (800 W) in citrate buffer (pH 6.0, 0.01M) for two cycles of 5 min. Sections were incubated with anti-mouse vimentin antibody (M 0725 clone V9; Dako) at 1 in 100 dilution. The secondary system was peroxidase-labelled anti-mouse PowerVisions (ImmunoVisionTechnologies), followed by diaminobenzidine (DAB) (Sigma,Vienna, Austria) for ‘‘visualization’’of the reaction. Western Blotting
Western blotting was performed semi-quantitatively to compareVEGFexpression in canine lymphomas and normal lymph nodes. Samples (n ¼ 7) were homogenized in low salt extraction bu¡er. Protein content in cell lysates was determined by a Coomassie Brilliant Blue G-250 binding assay (Bradford, 1976), performed in microplate format (Microplate Reader Sunrise; Tecan, Groedig, Austria) with a bovine serum albumin calibration curve. Equal amounts of protein (82 mg) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions on 12.5% polyacrylamide gels. SDS-PAGE was performed according to Laemmli (1970) using a Hoefer Mighty Small II
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B. Wolfesberger et al.
electrophoresis unit (Hoefer Scienti¢c Instruments, San Francisco, CA, USA). Kaleidoscope pre-stained standard (Bio Rad, Hercules, CA, USA) and Magic Markers XP Western standard (Invitrogen, Lofer, Austria) were used for molecular weight calibration and gels were stained with Coomassie blue. Following separation by SDS-PAGE, the proteins were transferred onto a PVDF-membrane (Amersham Life Science, Buckinghamshire, UK) in a semi-dry blotting device (Semi-Phor; Hoefer Scienti¢c Instruments). Dried milk powder (5%) was used as a blocking solution to minimize non-speci¢c binding of the primary antibody.The membrane was incubated with primaryVEGF antibody (A-20 as above) at 1 in 400 dilution in PBS/ Tween overnight at 4 1C. The ECL-secondary system (enhanced chemiluminescence; Amersham-Biosciences, Vienna, Austria) was used to detect binding. Human VEGF (58MS-1467-PCL; Labvision, CA, USA) and recombinant canine VEGF (1603-CV; R&D Systems Europe, Oxon, UK) were used as positive controls. Reverse Transcriptase Polymerase Chain Reaction (RTPCR)
Samples of canine lymphoma (n ¼ 1), feline lymphoma (n ¼ 1), normal canine lymph node (n ¼ 1), canine ovary (n ¼ 1) and uterus (n ¼ 1) were immersed in RNAlaters (Ambion Europe Ltd., Cambridge, UK) until total RNA extraction with TRIzols (Invitrogen GmbH, Lofer, Austria). ReverseTranscription was performed using a SuperScript One-Step RT-PCR kit with Platinum Taq (Invitrogen GmbH) according to the manufacturer’s instructions. Canine-speci¢cVEGF primers (Scheidegger et al., 1999) were used: 50 -ATGAACTTTCTGCTCTCTTGG-30; 50 -TCACCGCC TCGGCTTGTC-30 (Sigma Genosys, Su¡olk, UK). Samples were ampli¢ed by 35 cycles of 95 1C for 30 s, followed by 60 1C for 30 s and 1min at 72 1C. PCR products were separated on 1.5% agarose gels containing ethidium bromide and images were captured during examination under UV-light. Statistical Analysis
Pearson’s correlation coe⁄cient was used to investigate whether the scores of VEGF, VEGFR-1 and VEGFR-2 immunohistochemical labelling correlated with histology, tumour grade and classi¢cation as B- versus T-cell lymphoma. Correlation was considered signi¢cant if the P-value was o0.05.
Results Immunohistochemistry
The expression of VEGF, VEGFR-1 and VEGFR-2 in canine lymphoma is summarized inTable 1. There was
no signi¢cant correlation between tumour grade, histology, di¡erentiation to B- or T-cell lymphoma and the expression of VEGF,VEGFR-1 orVEGFR-2. Expression of VEGF. Sixty per cent of lymphomas had high expression of VEGF with 50% or more of the neoplastic cells showing immunoreactivity (score 3). Twelve per cent of samples had a score of 2,14% a score of 1, and 14% had no VEGF expression. The pattern of labelling was di¡use and cytoplasmic (Fig. 1b). Endothelial cells, macrophages, plasma cells, and reticulum cells were also positively labelled (Fig. 1a, Fig. 2c), whereas residual normal lymphoid follicles were always negative. In six normal lymph nodes, there was strong expression of VEGF by macrophages and plasma cells located mainly in the medullary sinuses and the medullary cords (Fig. 2a). Lymphocytes in the germinal centre of follicles were positively labelled but were surrounded by unlabelled cells (Fig. 1c, d). Endothelial cells were variably labelled. Expression of VEGFR-1. Forty six per cent of the lymphoma samples did not expressVEGFR-1; a score of 1was recorded in 12%, a score of 2 in 19%, and a score of 3 in 23% of samples. The pattern of labelling was primarily di¡use and cytoplasmic, and in one case perinuclear. Residual normal lymphoid follicles were unlabelled. Macrophages showed strong expression of VEGFR-1, as did plasma cells and reticulum cells. Positively labelled macrophages were aggregated in medullary cords or occasionally with perivascular distribution. Approximately 50% of blood vessels had endothelial expression of VEGFR-1 (Fig. 3a, c, d). Very intensive labelling of numerous macrophages was seen throughout the normal lymph nodes (Fig. 3b). Germinal centre lymphocytes were positively labelled and a few reticulum cells also showed reactivity. Only a proportion of vessels had positively labelled endothelial cells. Expression of VEGFR-2. Seventy two per cent of lymphoma samples did not expressVEGFR-2; a score of 1was recorded in14%, a score of 2 in 7% and a score of 3 in 7% of samples in which there was intense focal labelling with a cytoplasmic or perinuclear pattern. Smooth muscle and endothelial cells were sometimes positively labelled (Fig. 4a, b). Of the six normal lymph nodes, two were completely negative for VEGFR-2 expression, another three showed perinuclear labelling of very few single lymphocytes (o1%), and one sample also had positive expression in smooth muscle cells of vessels. Only one lymph node was scored as 1, and this sample had perinuclear expression of VEGFR-2 by cortical lymphocytes (Fig. 4c, d).
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Growth Factor Expression in Canine Lymphoma Table 1 Expression of VEGF,VEGFR-1 and VEGFR-2 in canine lymphoma Patient.
Sex
Age (Y)
Breed
Grade
Histology
B/T Cell
VEGF
VEGFR-1
VEGFR-2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
M Fn M M F Mn M M M Mn M M M Mn F M F F F Mn Fn Mn M F M F F M M Fn Fn M M M Fn Fn Fn M Mn M Fn F F
10 13 9 6 12 13 3 10 10 4 6 8 8 4 12 10 5 3 10 4 5 8 9 9 4 5 6 10 9 12 14 11 4 7 4 7 4 12 4 8 7 4 11
German hunting terrier Siberian husky Mixed Yorkshire terrier Mixed Mixed Labrador retriever Labrador retriever German shepherd Rottweiler Rhodesian ridgeback Mixed Mixed Mixed German shepherd Golden retriever German shepherd Golden retriever Irish setter Mixed Akita inu Afghan hound German shepherd Mixed Gordon setter Boxer Dogo Argentino Irish setter Mixed Bordeaux masti¡ West-Highland terrier Mixed Dogo Argentino Bull terrier Mixed Mixed Sta¡ordshire German shepherd Mixed Bordeaux masti¡ Pinscher Mixed Mixed
L L L L I I I I I I I I I I I I I I I I I I I I I I I I I I I H H H H H H H H H H H H
DSLL DSLL DSLL DSLL MMCL MMCL DMCL DMCL DMCL DMCL DMCL DMCL DMCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL DLCL LCIBL LCIBL LCIBL LCIBL LCIBL LCIBL LCIBL LBL LBL LBL SNCCL SNCCL
0 0 T B B B 0 0 0 B T T B 0 0 B T B B T B B B B/T B/T B/T B B B B B B/T B B B B B 0 B/T B B/T B/T B
1 3 2 3 3 3 3 3 2 0 1 3 3 2 3 2 3 0 0 3 3 3 3 1 0 1 3 3 1 0 2 3 3 3 0 3 3 1 3 3 3 3 3
0 2 0 0 3 0 1 2 3 0 1 3 3 2 3 0 0 0 0 2 2 3 0 0 3 1 2 3 1 0 2 1 3 2 0 3 0 0 0 0 0 0 0
0 0 1 0 0 2 0 0 1 0 0 0 2 1 0 0 0 1 0 3 0 3 0 0 0 0 0 0 0 0 0 1 0 0 0 3 0 1 0 0 0 2 0
Fn, female neutered; Mn, male neutered; L, low-grade lymphoma; I, intermediate-grade lymphoma; H, high-grade lymphoma; DSLL, di¡use small lymphocytic lymphoma; MMCL, macro-nucleated medium-sized cell lymphoma; DMCL, di¡use mixed cell lymphoma; DLCL, di¡use large cell lymphoma; LCIBL, large cell immunoblastic lymphoma; LBL, lymphoblastic lymphoma; SNCCL, small non-cleaved cell lymphoma; 0 not classi¢able; B, B-cell lymphoma;T,T-cell lymphoma; B/T, B andT-cell lymphoma;VEGF, vascular endothelial growth factor;VEGFR-1, vascular endothelial growth factor receptor 1; VEGFR-2, vascular endothelial growth factor receptor 2; score 0, no labelling; score 1, o10% labelling; score 2, 10^50% labelling; score 3, 450% positively labelled lymphoma cells.
Western Blotting
A rabbit anti-human antibody was used to detect VEGF in canine tissues (Fig. 5). HumanVEGF protein, the positive control, resulted in a distinct band of approximately 20 kD molecular mass. Recombinant canine VEGF resulted in a very broad band, proving that the anti-human antibody recognizes canine
VEGF. Bands of 20 kD molecular mass, but of di¡erent intensity, were obtained with extracts from canine lymphomas and normal lymph node (Fig. 5, lane 4). Lanes 5^8 of Fig. 5 represent VEGF expression in samples from patients with intermediategrade lymphoma. The more intense bands in lanes 9 and 10 of Fig. 5 derive from dogs with high-grade lymphoma.
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B. Wolfesberger et al.
Fig. 1A^D. (A) Section of canine lymphoma showing positive labelling of scattered macrophages (black arrows), reticulum cells and weak labelling of two vessels (open arrows). There is no positive labelling of tumour cells (score 0). (B) Section of canine lymphoma showing cytoplasmic expression of VEGF by 450% of the tumour cells (score 3). (C) Section of normal dog lymph node showing VEGF immunoreactivity in the germinal centre, medullary sinus and medullary cords. (D) Higher magni¢cation of (C). IHC. (A) 250; (B) 250; (C) 40; (D) 250.
Reverse Transcriptase Polymerase Chain Reaction (RTPCR)
The presence of three distinct VEGF mRNA isoforms (VEGF 120, 164, 188) was demonstrated in canine and feline lymphoma tissue as well as in normal lymph node tissue. Canine ovarian and uterine tissue provided positive controls for this assay (Fig. 6).
Discussion Lymphomas are a heterogenous group of lymphoproliferative tumours which occur in humans and dogs. A number of studies have assessed the expression of the major angiogenic cytokine VEGF in human lymphoma, but to our knowledge such expression has not been studied in the canine disease. In the present study 60% of the 43 samples of canine lymphoma showed a high level of expression of VEGF by the neoplastic cells. In studies of human lymphoma, VEGF was shown to be expressed by lymphoma cell lines (Bellamy et al., 1999), in 100% of 39 tumour sam-
ples (Kadowaki et al., 2005), in all samples from 24 patients with angioimmunoblastic T-cell lymphoma (Zhao et al., 2004), in 70.6% of 58 cases of classical Hodgkin’s lymphoma (Doussis-Anagnostopoulou et al., 2002), in 41.2% of 17 cases of mantle cell lymphoma (Potti et al., 2002), in 33.8% of 71cases of non-Hodgkin’s lymphomas (Hazar et al., 2003), and in three of nine lymph nodes taken from lymphoma patients (Ho et al., 2003). In contrast to the results of these studies, Foss and coworkers (1997) found no VEGF transcription in nonHodgkin’s and Hodgkin’s lymphoma (except in one case in which there was a single labelled tumour cell). Reasons for these vastly diverse results could be accounted for by di¡erent subtypes of the lymphoma in each study, the use of variousVEGFantibodies, and different experimental methods including immunohistochemistry and in situ hybridization. In the present study,VEGFexpression in tissue from canine lymphoma was also found in endothelial cells, macrophages, reticulum cells and plasma cells. Peripheral blood T lymphocytes and lymphocytes in¢ltrating
ARTICLE IN PRESS Growth Factor Expression in Canine Lymphoma
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Fig. 2A^D. (A) Section of normal canine lymph node showing expression of VEGF by plasma cells and macrophages. (B) Demonstration of immunolabelling of B lymphocytes and plasma cells by CD79a antibody in a normal lymph node. (C) VEGF positive reticulum cells in a section of canine lymphoma. (D) Vimentin staining of reticulum cells in a section of canine lymphoma. IHC. (A) 1000; (B) 1000; (C) 400; (D) 400.
human prostatic and bladder tumours synthesize VEGF protein (Freeman et al., 1995). VEGF has also been found in plasma cells of patients with multiple myeloma (Kimlinger et al., 2006) and in plasma cells within nasal polyps (Ito et al.,1995). In one study of Hodgkin’s lymphoma in human patients, endothelial cells failed to express VEGF except in three cases where there was positive labelling in small vessels, but plasma in the blood vessels reacted positively in all samples (Doussis-Anagnostopoulou et al., 2002). In cases of the present study where plasma was present in vessels, we also found VEGF immunoreactivity in the lumens of the vessels. A possible explanation for this ¢nding is that blood platelets contain VEGF which is released during platelet aggregation (Maloney et al., 1998). Doussis-Anagnostopoulou et al. (2002) also reported variable amounts of strong extracellular VEGF staining in stromal tissue, which was also detected in the present study. These authors suggested that the VEGF-positive extracellular matrix may have an important role as storage area for this protein, providing ready access of the tissue to angiogenic factors.
VEGF-positive macrophages were documented in canine mammary tumours (Restucci et al., 2002), and Doussis-Anagnostopoulou et al. (2002) also recognized VEGF immunolabelling in macrophages in samples from patients with Hodgkin’s lymphoma. It is reported that macrophages may stimulate the growth of ¢brosarcomas in mice (Evans, 1977). The present study also documentsVEGF-positive macrophages in canine lymphoma; however we also detected intense immunolabelling of macrophages in normal lymph nodes, a ¢nding which undermines this interesting theory. Plasma cells, lymphocytes (especially those in the germinal centre), and variable numbers of blood vessels also showed expression of VEGF in normal canine lymph nodes. The presence of VEGF protein was con¢rmed by western blotting and three VEGF mRNA isoforms were detected by RT-PCR in neoplastic and normal canine lymph nodes. These observations raise the question of the role of VEGF in normal lymph nodes. As neoangiogenesis does not take place under physiological conditions in adults (except in the ovary and uterus), VEGF expression by cells as plasma cells, lymphocytes, macrophages and endothelial cells could
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Fig. 3A^D. (A) Section of canine lymphoma showing expression of VEGFR-1 by macrophages. (B) Section of normal canine lymph node showing expression of VEGFR-1 by macrophages. (C) Section of canine lymphoma showing expression of VEGFR-1 by blood vessels. (D) VEGFR-1immunolabelling of the cytoplasm of reticulum cells in canine lymphoma. IHC. (A) 250; (B) 250; (C) 250; (D) 400.
be necessary to sustain the numerous mature blood vessels in normal lymph nodes. It is also possible that the expression of VEGF, which is also called vascular permeability factor (VPF; Ferrara et al., 2003), may play a role in maintaining the baseline permeability of the normal microcirculation (Berse et al.,1992). VEGF has been identi¢ed in many normal human tissues including lung, kidney, liver, gastric mucosa, breast and in activated macrophages, but it was not detected in resident peritoneal and alveolar macrophages (Berse et al.,1992). Turley and colleagues (1998) also examined normal human tissues and observed consistently strong reactivity in the ovary, uterus, salivary gland, pancreatic islet cells and plasma.Variable numbers of plasma cells, ¢broblasts and lymphocytes were labelled in the stroma of all tissues, whereas most of the endothelial cells were negative. Little is known about VEGF expression in non-reactive lymph nodes. In a study byTurley et al. (1998), three normal human lymph nodes were examined and weak labelling of lymphocytes was described. One of ¢ve non-reactive human lymph nodes showed weak expression of VEGF (Kadowaki et al., 2005). Unfortunately,
no details were given concerning which type of cells showed this reactivity. No statement about VEGF expression in macrophages, plasma cells and endothelial cells in human lymph nodes has been published. Approximately one half of our canine lymphoma samples showed VEGFR-1 immunoreactivity in neoplastic cells and 50% of the blood vessels in these samples were also positively labelled. Zhao and colleagues (2004) identi¢ed VEGFR-1 in neoplastic lymphocytes and endothelial cells in human angioimmunoblastic T-cell lymphoma by immunohistochemistry, but no expression was found in mantle cell lymphoma or follicular lymphoma (Ho et al., 2003). The present study reports detection of VEGFR-1 protein in plasma cells, reticulum cells and macrophages in samples of canine lymphoma as well as in normal lymph nodes. In human tissue VEGFR-1 was expressed by monocytes, macrophages, endothelial cells, haemopoietic stem cells, pericytes, smooth muscle cells, osteoblasts and colorectal tumour cells, but normal lymph node was not examined (Byrne et al., 2005). Interestingly, human monocytes, the precursor cells of macrophages, were found to express the
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Fig. 4A^D. (A) Section of canine lymphoma showing expression of VEGFR-2 by endothelial and smooth muscle cells of an artery and its arterioles. (B) Section of canine lymphoma showing cytoplasmic or perinuclear expression of VEGFR-2 by neoplastic cells. (C) Section of normal canine lymph node showing no expression of VEGFR-2. (D) Section of normal canine lymph node showing perinuclear expression of VEGFR-2 by o10% of lymphocytes (score 1). IHC. (A) 100; (B) 400; (C) 400; (D) 400.
Fig. 6. RT-PCR gel electrophoresis demonstrating expression of VEGF m-RNA isoforms in canine tissue. 1kB DNA ladder (lane 1), feline lymphoma extract (lane 2), normal canine lymph node (lane 3), canine lymphoma (lane 4), canine ovary (lane 5), canine uterus (lane 6). Fig. 5. Western blot analysis of VEGF expression in canine tissue. Molecular weight standard (lane 1), humanVEGFas a positive control (lane 2), canineVEGFas a positive control (lane 3), normal canine lymph node extract expressing VEGF (lane 4), di¡erent canine lymphoma extracts (lanes 5^10). The position of theVEGF band is indicated.
VEGFR-1 gene and it has been suggested that VEGF may be able to induce a migration signal in monocytes viaVEGFR-1 (Barleon et al.,1996).The precise function of VEGFR-1 remains unclear. On the one hand, VEGFR-1 has great a⁄nity forVEGF, but on the other it has only very weak tyrosine kinase activity (Hiratsuka et al.,1998). Findings suggest that VEGFR-1 behaves
more like a decoy than a signal-transducing receptor, preventing VEGF from binding to VEGFR-2 and thereby decreasing the biological activity of VEGF (Park et al., 1994). Therefore, at this time it is not clear if the presence of VEGFR-1 is an advantage or disadvantage in the course of the disease. No VEGFR-2 expression was found in about three quarters of the canine lymphomas examined in this study. Only three patients (one with high-grade and two with intermediate-grade lymphoma) showed focal intense labelling of neoplastic cells. In these locations endothelial cells were also highly immunoreactive. No
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expression of VEGFR-2 by cancer cells in angioimmunoblastic T-cell lymphoma was described by Zhao and colleagues (2004), whereas in¢ltrating cells in two of nine lymphomas showed slight labelling for VEGFR-2 (Ho et al., 2003). In contrast, 74% of lymphomas were positive for phosphorylated VEGFR-2 expression in a study performed by Pillai and co-workers (2005). We did not ¢nd any VEGFR-2 immunoreactivity in macrophages as described by Stewart et al. (2003), although northern blot analysis showed that monocytes do not express the VEGFR-2 gene (Barleon et al., 1996). The positive labelling of vascular smooth muscle cells noted in the present study corresponds with the ¢ndings of other studies (Byrne et al., 2005). To date, much e¡ort has been expended in order to ¢nd new drugs blockingVEGFor its receptors in cancer, in order to decrease endothelial and tumour cell proliferation and ultimately to improve survival. E¡ects of antibodies against VEGF and inhibitors of the VEGFR tyrosine kinase pathway in human cancer are currently being evaluated in clinical trials in which such agents are administered either as a single drug therapy or in combination with conservative chemotherapy. No studies assessing the potential bene¢t of anti-angiogenic drugs have been performed in dogs.We hypothesize that some dogs with lymphoma may be potential candidates for the use of angiogenesis inhibitors; in particular the two animals of the present study that had a high amount of VEGF protein in western blots or the three dogs demonstrating strong VEGFR-2 immunoreactivity on immunohistochemistry. However, considering the observed variability in expression of VEGF and its receptors in our tumour samples it is not possible to suggest a general anti-angiogenic therapy for all canine lymphoma patients. On the contrary, these wide di¡erences suggest that each patient’s tumour should be individually assessed forVEGFandVEGFRexpression, if such targeted therapy is considered in the future.
Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Magdalena Helmreich Mag, Waltraud Tschulenk, Christine Bayer, Sonja Dolezal and Doris Rosenfellner. This work was supported by a grant from the Veterinary University of Vienna (Pro¢llinie I Oncology).
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Received, August 23rd, 2006 Accepted, March 13th, 2007