Background
VEGF plays a crucial role in tumour expansion by initiating permeabilization of blood vessels, by extravasation of plasma proteins, by invasion of stromal cells, and by causing the sprouting of new blood vessels that supply the tumour with oxygen and nutrients [
1]. As a result of alternative splicing, 6 VEGF isoforms of 121, 145, 165, 183, 189 and 206 amino acids are produced from a single gene [
2]. Due to differential incorporation of basic residues encoded by exon 6 and 7, VEGF isoforms differ in their heparin-binding properties, membrane association, and secretion [
3]. VEGF
121, which lacks the basic residues of both exons, does not bind heparin-containing cell surface proteoglycan [
4], and is freely soluble. VEGF
165 is also secreted. However, cationic residues in exon 7 enable VEGF
165 to bind heparin, thus, some remains bound to the cell surface or to extracellular matrix. VEGF
189 which retain both exons, has the highest affinity for heparin and therefore, remains tightly cell associated.
Detection of circulating VEGF has been investigated as a potential serum diagnostic marker for malignant disease and for inflammation [
5]. Increased serum concentrations of free VEGF have been measured in various types of cancer, including brain, lung, gastrointestinal, hepatobiliary, renal, and ovarian cancers [
6]. However, the relationship between the pattern of the production of VEGF protein isoforms in tumour tissues and their concentration in the circulation is still unclear.
A number of studies have shown that expression of certain VEGF transcripts are correlated with tumour progression. Increased mRNA expression of VEGF
189 is correlated with poor prognosis in osteosarcoma [
7] and non-small cell lung cancer [
8,
9], whereas expression of VEGF
121 was correlated with lymph node metastasis in primary lung tumours [
10]. Although increases of certain VEGF transcripts have been demonstrated to correlate with the progression of various tumours, the actual protein levels of the different VEGF isoforms and their significance during cellular transformation are unknown. Moreover, it has been suggested that elevated protein expression in tumour tissues was mediated by both enhanced transcription [
11] and translation [
12]. Thus, in order to understand the role of VEGF in tumour progression, it is important to investigate expression of different VEGF isoforms at the protein level during tumourigenesis. To our knowledge, no studies focusing on the VEGF isoform pattern at the protein level and their relationship with respect to total VEGF in the circulation have been reported.
Therefore, the aims of this study were to determine: 1) The protein expression pattern of VEGF isoforms in colorectal and lung tumours in comparison to the corresponding adjacent normal tissues in order to understand whether specific VEGF protein isoforms play an important role during tumourigenesis. 2) The relationship between the expression pattern of VEGF and the level of total circulating VEGF in the blood.
Methods
Selection of patients and sample
Between April 2002 and June 2004, samples were collected from cancer patients at Maharaj Nakorn Chiang Mai Hospital, which comprised 76 colorectal tumours (averaged age was 59 ± 15.2 (mean ± SD), 46 females and 30 males) and 18 non-small cell lung tumours (averaged age was 55 ± 14.6, 10 females and 8 males, 9 adenocarcinomas and 9 squamous cell carcinomas). In each case, adjacent normal tissue was collected. These specimens were immediately placed in vials, frozen in embedded medium for the preservation of cell integrity, and stored at -80°C until analyzed. Samples were graded by a pathologist according to the pathological features of the tumours, which included tumour size in maximal diameter, histological grading, lymph node metastasis, distant metastasis, and tumour staging (the AJCC TNM classification).
To avoid pre-analytical sample-to-sample variation due to blood collecting procedures, each blood sample was allowed to clot for at least 4 hrs before collecting serum as it has been reported that the release of VEGF during clotting period would have reached a plateau by this time [
13]. Of 94 patients recruited in this study, serums were obtained from 56 cancer patients prior to the operation (38 from colorectal cancer patients, 18 from lung cancer patients). The age range of cancer patients was 58 ± 12.5 years and composed of 32 females and 24 males.
Serums were also collected from 47 healthy volunteers with no history of rheumatoid arthritis or recent pregnancy, trauma, surgery (within 1 month) or menstruation (within 1 week) using the same procedure as for the cancer patients so that a comparison could be made. The age range of healthy volunteers was 51 ± 10.9 (mean ± SD) years, composed of 20 female and 27 males. All serums were stored at -70°C until analyzed. The study was approved by the ethical committee of the Faculty of Medicine, Chiang Mai University (document number 56/2545).
Western blotting
Western blotting was performed to evaluate the expression of VEGF in each tissue. Frozen tissues were thawed, cut into small pieces and homogenized in SDS lysis buffer (0.5 M Tris-HCl pH 6.8, 2% SDS (w/v) and 10% glycerol (v/v)) containing a protease inhibitors cocktail (104 mM AEBSF, 0.08 mM aprotinin, 2.2 mM leupeptin, 3.6 mM bestatin, 1.5 mM pepstatin A, 1.4 mM E-64; Sigma, U.S.A). The tissue homogenate was then centrifuged at 10,000 g for 15 minutes at 4°C, after which the supernatant was removed and the protein concentration of the supernatant was estimated using the BCA protein assay kit (PIERCE, U.S.A). Twenty-five micrograms of protein from the tumour tissue and normal tissue from each patient was resolved on a 10% SDS polyacrylamide gel under reducing conditions and electrotransferred onto a nitrocellulose membrane (Biorad, U.S.A). After blocking with 5% non-fat milk in TBS containing 0.05% Tween-20 (TBS-Tween) for 1 hour, the membrane was incubated with anti-VEGF antibodies (Santa Cruz Biotechnology, Inc., USA, Cat. no. SC-152, dilution 1:1000) for 1 hour. After washing with TBS-Tween, the membrane was incubated for 1 hour at RT with horseradish peroxidase-conjugated goat anti-mouse IgG (Dako, U.S.A). After washing with TBS-Tween, immunoreactive protein was visualized with a chemiluminescence-based procedure using the ECL Plus detection kit according to the manufacturer's protocol (Amersham, U.S.A). In order to examine the equality of protein loaded, the amount of total protein loaded into each lane was examined by staining with coomassie blue.
Measurement of total VEGF in serum
For the detection of circulating VEGF in serum, enzyme-linked immunosorbent assay (ELISA) was performed using two different anti-VEGF antibodies purchased from R&D system, USA. Briefly, capture antibodies specific for VEGF (R&D System, cat no. AF293 at concentration 200 ng/ml) was immobilized onto-96-well microtiter plates. Unbound antibody was removed by washing the plate and a blocking reagent was added. Following a wash, recombinant VEGF protein standard (VEGF165) diluted in PBS containing 5% BSA to various concentrations (75–2,500 pg/ml), unknown serum and control serum were then incubated with the solid phase antibodies, which capture VEGF. After washing away unbound molecules, a detection antibody specific for VEGF (R&D System, cat no. MAB293 at concentration 500 ng/ml) was added. After incubation and washing, HRP-conjugated anti-mouse immunoglobulin was added. The plate was washed and a TMB substrate solution (Zymed, USA) was added. After 20 minutes, the color development was stopped and the intensity of color was measured using a microtiter plate reader (450 nm). The color developed in proportion to the amount of bound VEGF. When we measured 20 serum samples twice in two separated assays, the inter-assay variation ranged between 5–10% within the same concentration range. The average recovery of the added recombinant VEGF165 ranged between 85–115%, indicating an acceptable level of specificity of the assay.
Statistical analysis
Total VEGF levels are expressed as mean ± standard deviation. Differences in the circulating VEGF level of two independent groups were evaluated using the Mann-Whitney test. Correlation between VEGF isoform expression and the pathological features were evaluated using chi-square test. All the statistical evaluations were performed by using the SPSS for Window version 10.0 (SPSS, Inc., Chicago, IL, USA).
Discussion
It has become clear that the growth of solid tumours is dependent on the process of angiogenesis and that VEGF is a central positive regulator of this process. Most VEGF-producing cells appear preferentially to express VEGF
121, VEGF
165 and VEGF
189. In this study, we investigated the expression pattern of the VEGF protein isoform in colorectal tumour tissues and in lung tumour tissues and compared them with the expression pattern of normal tissues from each organ, respectively. Three major protein bands with molecular weight 18, 23 and 26 kDa were predominately detected. The 23-kDa protein band is believed to be the VEGF
165 as this band was at the same position as the human recombinant VEGF
165 protein standard (R&D System, USA) used in this study. Expression of VEGF
145 and VEGF
206 is comparatively rare seemingly restricted to cells of placental origin [
14,
15]. Therefore, protein bands with molecular weight of 18 and 26 kDa are assumed to be VEGF
121 [
16,
17] and VEGF
189, respectively.
In colorectal tumours, it was found that VEGF
121 was expressed equally in both tumour and normal tissues, whereas the VEGF
165 and VEGF
189 were only detected at higher level in tumour tissues. However in lung tumour, VEGF
121 appeared to be predominately expressed in normal tissues, whereas VEGF
165 and VEGF
189 were predominately expressed in tumours tissues. Protein expression of VEGF
165 correlated significantly with a smaller tumour size, whereas VEGF
189 correlated significantly with advanced clinical stage and metastasis of the tumours. Although only 18 lung tumours were investigated in this study, the 26-kDa VEGF isoform was also overexpressed significantly in advanced stage of the tumour (Table
1).
Although the regulation of VEGF expression is becoming well understood, its mode of action, particularly the regulation of expression and distribution of the three primary isoform (VEGF
121, VEGF
165 and VEGF
189), remains unclear. It has been demonstrated that overexpression of smaller isoforms resulted in hemorrhagic events, but the expression of VEGF
189 resulted in increased vessel density [
18]. In this study we found that overexpression of VEGF
189 protein isoform, but not VEGF
121 or VEGF
165, was associated with advanced tumour stage. Our data is consistent with the previous reports where expression of VEGF
189 transcripts was correlated with poor prognosis in non-small cell lung, osteosarcoma, renal, colorectal and esophageal cancer [
7‐
9,
19,
20]. It was also suggested that up-regulation of VEGF
189 might result in increased angiogenesis, tumour growth and metastasis in a colon cancer cell line [
21]. Moreover, VEGF
189 has been demonstrated to be a potent permeability factor in vivo [
22], supporting the role of this isoform in the control of angiogenesis.
VEGF
165 has also been demonstrated to play an important role in tumourigenesis. When different isoforms of VEGF were transfected into the VEGF-null cells in isolation and the transfected cells were implanted into nude mice, it was found that VEGF
165 was the most prominent isoform that can fully rescue expansion of the angiogenesis-deficient tumour, while VEGF
121 and VEGF
189 only partially or failed completely to rescue tumour growth, respectively [
23]. However, these authors suggested that VEGF isoforms work in a coordinated fashion to recruit and expand tumour vasculature. In our study we found that although VEGF
165 was predominately expressed in colorectal tumour tissues, its expression was significantly correlated with smaller tumour size (maximum diameter less than 5 cm.). Although expression of VEGF
121 mRNA has been previously reported to be correlated with lymph node metastasis [
10] of primary lung cancer and the invasiveness of bladder cancer [
24], in our study we found that level of the 18 kDa VEGF protein, which believed to be VEGF
121 [
16], was equally expressed in both normal and tumour tissues of colorectal, and predominately expressed in normal tissues of the lung.
Detection of VEGF has long been known as a potential serum diagnostic marker for malignant diseases. Increased serum VEGF concentrations have been measured in various types of cancer, including, brain, lung, gastrointestinal, hepatobiliary, renal and ovarian cancer [
25]. However, the relationship between the pattern of the production of VEGF protein isoforms in tumours and its concentration in the circulation is still unclear. In this study, we determined the expression pattern of VEGF isoforms in tumour tissues in relation to the level of total VEGF in a patient's serum. The comparison of the VEGF level in serum of cancer patients with that of normal volunteers revealed that cancer patients possessed significantly (p < 0.001) higher levels of VEGF in serum. However, some normal volunteers also possessed quite a high level of VEGF, which may due to the possibility that normal tissues, like lung tissue (Figure
1) can also produce VEGF
121 that is secretable into the circulation. In addition, no significant relationship between level of circulating VEGF and pathologic features was observed.
Conclusion
Our findings indicate that the expression patterns of VEGF isoforms are altered during tumourigenesis as certain isoform overexpression in tumour tissues correlated with tumour progression indicating their important role in tumour development. However, measurement of circulating VEGF in serum may have limited use as a tumour marker. This may be due to the following reasons: 1) The VEGF isoform that appeared to be significantly correlated with tumour progression is VEGF
189, which is the cell-associated isoform, is not soluble. 2) Some normal tissues, i.e. lung (as shown in Figure
1), expressed high-level VEGF isoforms (VEGF
121) that secreted into the circulation. 3) Expression of some secretable VEGF isoforms (VEGF
165) was negatively correlated with the progression of tumour size, thus its level may not positively indicate the stage of the tumour. 4) As has been previously reported, other physiologic and pathologic condition, i.e., pregnancy, RA and cardiovascular diseases can also cause the induction the circulating level of VEGF [
26,
27].
Competing interests
The author(s) declare that they have no competing interest.
Authors' contributions
RC designed and sought funding for the study, initiated coordination, performed statistical analysis and drafted the manuscript. OW recruited healthy volunteers and performed the ELISA and western blot analysis. NL recruited cancer patients and carried out the pathological feature examination. UV participated in design of the study and coordination. All authors read and approved the final manuscript.