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1 Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, University of Bristol, Southwell Street, Bristol, United Kingdom; 2 Section of Pharmacology, Department of Molecular Biology, University of Siena, Siena, Italy; 3 Institute of Nephrology, First Teaching Hospital, University of Beijing, Beijing, People’s Republic of China; and 4 Bristol Urology Institute and 5 International Blood Group Reference Laboratories, National Blood Transfusion Centre, Southmead Hospital, Westbury on Trym, Bristol, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The multiple isoforms of conventional vascular endothelial growth factor-A result from differential splicing of pre-mRNA from eight exons resulting in at least six mRNA and (presumed) peptide species identified by the exon composition and amino acid length of the final proteins (ref. 5
; Fig. 1
). All conventional splice variants studied, including the most common isoform, VEGF165, have been shown to be proangiogenic, and they exert this effect by stimulating endothelial cell migration, proliferation, and lumen formation and are potent proangiogenic vasodilators and mediators of increased microvascular permeability (6
, 7)
. These effects are mediated principally by activation of VEGF receptor 2 (VEGFR-2)(KDR/flk1; refs. 3
, 8
). Endothelial cells also express VEGFR-1; however, the specific role of this receptor in endothelial physiology is less well defined (3
, 8)
.
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VEGF was originally described as a growth and survival factor for endothelial cells. It is, however, becoming increasingly apparent that VEGF is also an important factor in the function, physiology, and pathology of cancer cells themselves, as well as a wide variety of nonneoplastic cell types, including the migration and growth of neurones (14 , 15) and migration of monocytes (16) . For instance, it has been shown to act as an autocrine survival factor for breast and prostate cancer cells (17 , 18) but also for hematopoietic stem cells (19) and podocytes (20) .
Considerable interest therefore results from the realization that VEGF expression is highly regulated at both mRNA and protein levels in tissues that are not normally angiogenic, e.g., cerebellum (21)
, prostate (22)
, pancreatic islets (23)
, and glomeruli (24)
. While investigating this paradox, we recently discovered mRNA encoding a novel isoform, VEGF165b, which did not appear to stimulate endothelial cell proliferation or migration and was down-regulated in renal cell carcinoma (25)
. This isoform, formed by distal splice site selection in the terminal exon of VEGF, predicts an open reading frame encoding an alternate COOH-terminal sequence but the same number of amino acids in the mature protein (Fig. 1B)
. This predicted the translation of a protein of the same length as VEGF165, but with a different sequence and hence possibly a different mechanism of action. VEGF165b was so named because it also contains 165 amino acids. However, the COOH-terminal six amino acids usually coded for by exon 8 (CDKPRR) would be replaced by six different amino acids (SLTRKD) coded for by 18 bases of mRNA spliced 66 bases downstream of the usual acceptor splice site for exon 8, if this isoform was translated. We initially termed this new open reading frame exon 9, although there is no true intron between the two reading frames, and the alternative splicing should perhaps more correctly be referred to as exon 8 proximal and distal splicing sites. This isoform was identified serendipitously, and the high degree of homology between VEGF165b and VEGF165 (96%) perhaps explains the elusiveness of this isoform until now. VEGF165b and VEGF165 would not be distinguished in most nucleic acid and protein assays but would be assumed to be the same product.
It has been previously shown that the COOH terminus of VEGF is necessary for determining mitogenic potency (26) . We therefore speculated that the novel COOH terminus would influence function and subsequently showed that conditioned media from cells expressing synthetic, recombinant VEGF165b inhibited VEGF165-mediated endothelial cell proliferation and migration in vitro and vasodilatation ex vivo (25) . The mechanisms by which this occurred, however, and whether VEGF165b was a true endogenous inhibitor (i.e., it was produced as an endogenous protein and could block angiogenesis in vivo) were not shown. Moreover, the discovery of the exon 8 distal splicing site predicted but did not demonstrate the existence of an entire family of sister isoforms (VEGFxxxb), all with inhibitory potential. We have therefore carried out experiments to determine (a) whether VEGF165b competitively inhibits VEGF165-mediated activity by binding to the same receptor (VEGF-R2) but inhibiting receptor phosphorylation and downstream intracellular signaling, (b) whether exon 8 distal splicing site isoforms are expressed in human tissues and plasma, e.g., VEGF189b, VEGF165b, and VEGF121b, (c) whether VEGF165b inhibits VEGF165-mediated angiogenesis in vivo; and (d) whether the different family of isoforms have differing effects on tumor growth.
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MATERIALS AND METHODS |
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Receptor Binding Assays
Immulon II HB Flat well 96-well plates were incubated with 100 µL of 1 µg·mL-1 VEGFR-2-Fc Chimera (R&D Systems) overnight, and washed three times with 1x PBS (pH 7.4), supplemented with 0.05% Tween 20 (PBS-T; Sigma-Aldrich, Poole, Dorset, United Kingdom). For cell assays human umbilical vascular endothelial cells were grown to confluency in 96-well plates. The plate was blocked with 800 µL of 3% BSA for 2 hours and again washed twice with PBS-T. A total of 10 ng·mL-1 125I-VEGF165 and 100 µCi·mL-1 was mixed with increasing concentrations of VEGF165b or VEGF165, and 100 µL added to the ELISA plate. This was incubated for 4 h at room temperature, then gently washed with PBS three times. 10% SDS was then added, and the solution transferred to a gamma counter for assays. Counts were expressed as the percent of the counts of wells incubated with 125I-VEGF165 alone.
Cell Culture
Terminally differentiated conditionally immortalized glomerular visceral epithelial cells (podocytes, a kind gift of Moin A. Saleem, Children’s Academic Renal Unit, Southmead Hospital, University of Bristol, Bristol, United Kingdom) were cultured as described previously (27)
. Chinese hamster ovary (CHO) cells were transfected with either an empty vector (PEQ176) or VEGFR-2 containing expression vector. Cells were routinely passaged in nutrient mixture DMEM:Ham’s F-12 (CHO) or RPMI (podocytes) with L-glutamine (Invitrogen Corp.) supplemented with 10% fetal calf serum (Invitrogen Corp.), 1% penicillin-streptomycin (Invitrogen Corp.), and 1% of the selection agent, Geneticin (Invitrogen Corp). Transfected CHO cells were incubated with serum-free media for 18 to 24 hours followed by incubation with media or media containing 40 ng·mL-1 VEGF165, 40 ng·mL-1 VEGF165b, or 40 ng·mL-1 VEGF165, and 40 ng·mL-1 VEGF165b for 20 minutes at 37°C. Cells were washed and collected with PBS followed by centrifugation at 3000 rpm for 5 minutes and protein extracted as described below.
Tissue and Blood Samples
Blood was collected from normal healthy volunteers ages 21 to 40. Blood was taken into EDTA vacutainers and centrifuged at 4000 x g for 10 minutes. Plasma was removed from the erythrocytes and stored at –80°C until defrosted and used for ELISA. Prostate chips were obtained from patients undergoing transurethral resection of the prostate for lower urinary tract symptoms. Samples were frozen at –80°C immediately until processed. Patients with benign prostatic hyperplasia and advanced prostate cancer (stage T3NxM0–1; UICC2002) were included. Chips were defrosted on ice and manually sliced with a sterile blade. The mass of each tissue was recorded, and samples were homogenized on ice for 10 minutes and protein extracted as described below. Rat lungs were removed from animals that were humanely killed by cervical dislocation. An equivalent volume of PBS containing either saline, 1 nmol/L VEGF165, 1 nmol/L VEGF165b, or 1 nmol/L VEGF165 and 1 nmol/L VEGF165b was added to each sample. The samples were chopped up crudely on ice with a sterile scalpel blade and then incubated for 20 minutes at 37°C in a shaking incubator. After this they were snap-frozen in liquid nitrogen.
Protein Extraction
Lysis buffer (1 µL/mg tissue; 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Va3N04, 1.0 µg/mL leupeptin, 1 µg/mL aprotonin, and 1 µg/mL pepstatin, in radioimmunoprecipitation assay buffer) was added to cells and prostate tissue and homogenized on ice for 10 minutes, placed at 4°C on an agitating rocker plate for 20 minutes, removed and placed in ice for an additional 60 minutes, and agitated every 10 to 15 minutes.
Samples were centrifuged at 4°C for 15 minutes at 13,000 rpm, and the supernatant collected and stored at –20°C. Protein concentrations were determined by photospectrometry. For rat lung tissue, after thawing, the samples were mixed gently, excess liquid was removed, and protein extracted by adding an equal volume of lysis buffer (140 mmol/L NaCl, 3 mmol/L KCl, 10 mmol/L Na2HPO4, 2.7 mmol/L KH2PO4, 1% NP40/IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L sodium orthovanadate, 20 µg·mL-1 aprotinin, 10 µg·mL-1 leupeptin, and 10 µg·mL-1 pepstatin) and homogenizing on ice for 10 minutes. Homogenates were left at 4°C on a rocker for 1 hour and then spun down in a benchtop centrifuge for 15 minutes at 13,000 rpm at 4°C to separate the protein from cell debris.
Production of Monoclonal Antibody to VEGF165b
Peptide Fragments and Immunization.
Synthetic peptide fragments of the nine amino acid COOH-terminal sequence of VEGF165b were coupled to keyhole limpet hemocyanin (Department of Biochemistry, University of Bristol) serving as carrier molecules and were then used to immunize 6 to 8-week-old female BALB/c mice. The animals received s.c. injections of 100 µg of peptide-keyhole limpet hemocyanin conjugates in Freund’s Complete Adjuvant on days 1, 21, and 42 and boosted by i.p injections at 63, 64, and 65 days. Mice were killed humanely the next day and spleens collected. Splenocytes were fused to the NS0 mouse myeloma cell line with polyethylene glycol. Fused cells were cultured in 96-well plates for 2 weeks. Cells from positive wells determined by ELISA screening were serially diluted in 96-well plates and cultured in 10% DMEM and hybridoma-cloning enhancing factor. The same procedure was repeated until 100% positivity from each plate was achieved three consecutive times. Screening was done in Immulon II HB Flat well 96-well plates (Thermo Life Sciences Ltd.), coated with goat antihuman VEGF antibody (0.8 µg/mL in PBS; R&D Systems). After washing with PBS-T, 100 µL of 2 ng/mL VEGF165b (from the conditioned medium of transfected cells) or recombinant VEGF165 (R&D systems) were added to the wells and incubated for 15 minutes at 37°C with shaking. After washing, 100 µL of conditioned medium from hybridoma cells were added and incubated for 15 minutes at 37°C with shaking. After washing, 100 µL of horseradish peroxidase (HRP)-conjugated goat antimouse immunoglobulins (1:1000 in 1% BSA/PBS; DAKO, Carpinteria, CA) were added and incubated for 15 minutes at 37°C with shaking. After final washing, O-phenylenediamine dihydrochloride substrate (Sigma Chemical Co., St. Louis, MO) was added, and the absorbance at 492 nm was measured using a plate reader. To purify and concentrate the monoclonal antibodies, the selected clones of hybridoma cells were cultured in DMEM (Sigma Chemical Co.) containing 10% bovine IgG-depleted FCS (Hyclone, Logan, UT) with 100 units of penicillin, 100 µg of streptomycin, and 2 mmol/L L-glutamine. Monoclonal antibodies were purified on protein-G Sepharose 4 Fast Flow columns (Amersham Biosciences). The antibodies were concentrated with vivaspin 20 (Vivascience AG, Hannover, Germany) and finally dissolved in PBS.
SDS-PAGE
All protein samples were resuspended in SDS sample buffer, heated at 100°C for 5 minutes, and subsequently resolved by SDS-PAGE and transferred to polyvinylidene difluoride membrane. The membranes were blocked with PBS, 10% Marvel, and 0.05% Tween 20 for 1 hour at room temperature, followed by overnight incubation at 4°C with the primary antibody (see below).
Western Blot and Immunodetection
VEGF165b Protein.
Membranes containing recombinant VEGF165 and/or VEGF165b protein (100 ng of each) and protein samples extracted from cells and transurethral resection of the prostate chips (100 to 150 µg of each) were probed with mouse anti-VEGF165b IgG1 described above (Fig. 2A)
. The membranes were then probed with HRP-conjugated stabilized goat antimouse IgG for 1 hour at room temperature [1:2000, all dilutions in PBS, 5% dried milk, 0.05% Tween 20 (5% Marvel–PBS-T); Santa Cruz Biotechnology, Santa Cruz, CA]. Immunodetection of recombinant protein was done with a BM Chemiluminescence Blotting Substrate (POD) kit (Roche). Immunodetection of other proteins (e.g., from tissues and cells) was assessed using SuperSignal West Femto Maximum Sensitivity Substrate Kit (Pierce, Rockford, IL). Membranes were stripped with enhanced chemiluminescence stripping buffer and probed with a pan VEGF primary antibody [VEGF-(C-1); Santa Cruz Biotechnology] 1:100 dilution in 5% Marvel–PBS-T for recombinant proteins, and 1:200 dilution for transurethral resection of the prostate chip proteins) overnight at 4°C. Membranes were then incubated with the HRP-conjugated goat antimouse IgG used above (1:2000), and immunodetection was done.
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/AKT1 (PkB-175, 1:400; Sigma) washed and incubated for 1 hour with HRP-conjugated goat antimouse antibody, as above. Immunoreactive bands were visualized by chemiluminescence with a compact X4 developer from X-ograph imaging systems. Densitometry, measured using the NIH image software, was used to determine the mean intensity of the immunoreactive bands.
Immunohistochemistry
Immunohistochemistry was done on formalin-fixed, paraffin-embedded tissue derived from the normal pole of nephrectomy specimens. Five-micrometer thick sections were cut and mounted onto poly-L-lysine–coated glass slides. Sections were dewaxed, rehydrated, washed in distilled water, and then rinsed in 0.01 mol/L Tris-buffered saline (PBS, pH 7.2). Sections were microwave heated in 0.1 mmol/L Tris-HCl/2 mmol/L EDTA pH buffer (pH 9.0) at 650 watts for two cycles of 8 minutes, washed twice with distilled water for 5 minutes, then treated with 0.015% trypsin (Invitrogen Corp.) diluted in PBS for 15 minutes at 37°C. Sections were washed twice, incubated with 3% hydrogen peroxide solution, washed twice with PBS, blocked with 3% BSA (A4378; Sigma) in PBS and then with 1.5% normal horse serum (S-2000; Vector Lab, Peterborough, United Kingdom) in PBS. Sections were then incubated with 2 µg/mL mouse monoclonal anti-VEGF165b IgG or a normal mouse IgG (I8765; Sigma) as a negative control diluted in 1.5% normal horse serum in PBS overnight at +4°C in a humid chamber. Sections were washed twice in 0.05% Tween Tris-buffered saline [PBS/Tween (pH7.2)] for 5 minutes and were treated with both the nonspecific blocking solution, as above, then incubated with biotinylated antimouse-IgG (BA2000; Vector Lab) at a 1:750 dilution in 1.5% normal horse serum in PBS at room temperature for 30 minutes in a humid chamber. Sections were washed twice in PBS/Tween, 5 minutes per wash, and then incubated with Vectastain ABC solution (PK4000; Vector Lab) for 30 minutes at room temperature followed by two additional washes in PBS/Tween. Sections were treated with 3,3'-diaminobenzidine peroxidase substrate solution (SK4100; Vector Lab) until color was visualized. Rinsing in distilled water stopped the reaction. Sections were washed twice in distilled water for 5 minutes, then counterstained in hematoxylin for 5 minutes, washed, dehydrated, cleared in xylene, mounted with DPX, and glass coverslipped. Sections were examined under oil immersion with a x100 objective on a Nikon Eclipse E-400 microscope, and images were captured using a Coolpix 995 digital camera and a DN-100 digital imaging system (Nikon Instruments, Surrey, United Kingdom).
mRNA Extraction and Reverse Transcription-PCR
Fifty to 100 mg of transurethral resection of the prostate tissue collected as above was homogenized in Trizol reagent (Life Technologies, Inc., Rockville, MD) and mRNA extracted by using the method of Chomczyinski and Sacchi (28)
. Eight microliters of RNA were treated with RNase free DNase (Promega) according to the manufacturer’s guidelines to prevent genomic DNA contamination. mRNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and poly-d(T). cDNA was then amplified using intron-spanning primers that detect VEGF165b only, even in the presence of 1000x greater concentration of VEGF165 mRNA (25)
. One µmol/L of a primer complementary to exon 4 (5'-GAGATGAGCTTCCTACAGCAC-3') and to exon 9 and the terminal 5 nucleotides of exon 7, incorporating a HindIII restriction site at the 5'-end (exon9/7, 5'-TTAAGCTTTCAGTCTTTCCTGGTGAGAGATCTGCA-3'), together with 1.2 mmol/L MgCl2, 200 µmol/L deoxynucleoside triphosphates, and 1 unit of TaqDNA polymerase (Abgene) were used in reactions which were cycled 35 times, denaturing at 94°C for 30 seconds, annealing at 63°C for 30 seconds, and extending at 72°C for 60 seconds. PCR products were run on 3% agarose gels containing 0.5 µg/mL ethidium bromide and visualized under a UV transilluminator. This reaction consistently resulted in one amplicon at 
220 bp (consistent with VEGF165b). A reverse primer complementary to exon 8 (5'-TCACCGCCTCGGCTTGTCACAT-3') was also used that detects VEGF165 but not VEGF165b. Finally, cDNA was also amplified with primers designed to pick up all of the VEGF isoforms. One µmol/L of primers complementary to exon 2 (5'-GGAGGGCAGAATCATCACGAAG-3') and exon 3 (5'-CACACAGGATGGCTTGAAGATG-3') were used. PCR conditions were as above but annealing at 55°C.
ELISA
A total of 0.8 µg/mL pan-VEGF capture antibody (Duoset VEGF ELISA DY-293; R&D Systems) diluted in 1x PBS (pH 7.4) was adsorbed onto 96-well sterile plates (655161; Greiner Bio-one Ltd., Gloucester, United Kingdom) overnight at room temperature (100 µL/well). The plates were washed three times with 1x PBS-T before and after blocking with 300 µL/well 1% BSA in PBS for 2 hours at 37°C. One hundred µL of duplicate recombinant human VEGF165 or VEGF165b standards (R&D Systems) diluted in wash buffer (ranging from 15 pg/mL to 4 ng/mL) and blank (wash buffer) or sample were added to each well (Fig. 2B)
. After incubation for 1 hour at 37°C and three washes as before, 100 µL of biotinylated goat antihuman VEGF (0.025 µg/mL in wash solution; R&D Systems), or 100 µL of mouse anti-VEGF165b (0.025 µg·mL-1 in wash solution, as described above) was then added to each well, and plates left for an additional hour at 37°C. After washing, 100 µL of streptavidin-HRP (R&D Systems) at 1:200 dilution or HRP-conjugated stabilized goat antimouse IgG (1:200 dilution; Santa Cruz Biotechnology) in PBS were added, and plates left at room temperature for 20 minutes. The plates were washed an additional three times, and 50 µL/well O-phenylenediamine dihydrochloride solution (Substrate reagent pack DY-999; R&D Systems) added, protected from light, and incubated for 20 minutes at room temperature to allow color development. The reaction was stopped with 50 µL/well 1 mol/L H2SO4 (10276; BDH Chemicals; Poole, Dorset, United Kingdom), and absorbance read immediately in an ELISA plate reader (Labsystems Multiskan Bichromatic; Life Sciences International) at 492 nm.
Rabbit Corneal Angiogenesis Assay
MCF-7 cells were transfected with pcDNA3-VEGF165, pcDNA3-VEGF165b, or pcDNA3 expression vector. Stable cell lines were selected in growth media containing 500 µg/mL G418. VEGF165b lines expressed 17.8 fg/cell/hour, whereas VEGF165 and pcDNA3 lines expressed 4.9 and 0.08 fg/cell/hour, respectively. Corneal assays were done in female New Zealand albino rabbits (Charles River; Calco, Como, Italy). Cells were detached using trypsin and the reaction stopped by the addition of EMEM-10% FCS, followed by pelleting and suspension in EMEM-10% FCS. Cell suspensions containing 2.5 x 105 cells per 5 µL (n = 4 eyes for each group) were implanted into a micropocket (1.5 x 3 mm) incised into the cornea under local anesthesia (30 mg/kg sodium pentothal). Animals were observed every 2 days with a slit-lamp stereomicroscope, without anesthesia. Neovascular responses, presence of hyperemia and edema, and infiltration with inflammatory cells in the cornea were monitored. Positive angiogenic responses were scored according to the product of vessel density and distance from the limbus (in mm), as described previously (29)
. Briefly, a density value of 1 corresponded to the presence of 0 to 25 vessels, a value of 2 to 25–50 vessels, a value of 3 to 50–75 vessels, a value of 4 to 75–100 vessels, and a score of 5 to >100 vessels. The distance from the limbus was measured using an ocular grid.
Construction of Adenovirus
Adenovirus-expressing human VEGF165b was created with the AdEasy Vector system (Qbiogene, Inc., Nottingham, United Kingdom). Briefly, a reverse transcription-PCR fragment encoding a full-length coding region of VEGF165b was cloned into pShuttle-CMV and sequenced in both directions to confirm the correct sequence. pShuttle-CMV-VEGF165b was linearized with EcoRI and cotransformed with pAdEasy-1 into Escherichia coli (BJ5183). Recombinant clones were screened with kanamycin resistance and checked by restriction digest. Purified, correct pAd-VEGF165b clones were linearized and transfected into HEK293Q cells and viral plaques eluted and used to infect 1 x 105 HEK293A cells
Production of VEGF165b was assessed by ELISA. Subsequent passages of virus were generated by infecting HEK293A cells with viral stock of the previous amplification with a multiplicity of infection of 5. Virus was purified by double ultra-centrifugation in saturated CsCl solution. (Sorvall Discovery Ultracentrifuge 90SE; Kendro) at 65,000 rpm.
Rat Mesenteric Angiogenesis Assay
Surgical Procedure.
All surgical procedures were done under sterile conditions. Male Wistar rats (200 to 300 g) were anesthetized by inhalation of 5% halothane and maintained on 3% halothane. Body temperature was maintained at 37°C by a thermostatic heating pad and rectal temperature probe. After ventral laparotomy, part of the intestine was exteriorized and a mesenteric panel with few vessels was exposed under an intravital microscope (Leica DMIL). The panel was imaged with either a Nikon Coolpix 800 (Nikon Instruments) or a Leica DC350F (Leica, Bucks, United Kingdom) digital camera, and 25 µL of virus (107 plaque forming units) were injected into the nearby fat pad with a Hamilton syringe fitted with a 30-gauge needle. Ad-VEGF165b, Ad-VEGF165 (30)
, or adenovirus-expressing enhanced GFP, (a kind gift of James Uney; University of Bristol) was used. In double infection experiments, 107 plaque forming units of each virus were injected into separate, nonoverlapping parts of the fat pad (to prevent co-infection and heterodimer formation). Ten microliters of Monastral blue (0.6%, diluted in saline) were injected into the fat pads on either side of the virus-injected panel. The intestine was replaced, and the animal sutured and recovered. Six days later (day 7), the animal was re-anesthetized with halothane, and a laparotomy was done. The mesentery was exposed, and the virus injected panel located from Monastral blue injection sites and re-imaged.
Immunofluorescence on Whole Mount Mesentery.
After imaging, the mesenteric panel was fixed in vivo with 4% paraformaldehyde for 5 minutes, and the rat killed by cervical dislocation. The same mesenteric panel was excised and additionally fixed for 30 minutes. Immediately after fixation, the mesentery was washed with 0.5% Triton X-100 in PBS (0.5% PBX) twice for 1 hour, then blocked with 1% BSA in 0.5% PBX for 1 hour at room temperature. The mesentery was incubated with biotinylated Griffonia simplicifolia lectin IB4 (GS-IB4; Molecular Probes, Cambridge, United Kingdom) 1:250, and mouse monoclonal antibodies to either Ki-67 (NCL-L-Ki-67- MM1, 1:100; Novocastra Lab, Newcastle upon Tyne, United Kingdom) or VEGF165b, (8 µg·mL-1) diluted in the blocking solution overnight at 4°C on a rocker. The mesentery was then washed with 0.5% PBX five times for 1 hour and then incubated with tetramethylrhodamine isothiocyanate-labeled streptavidin (Molecular Probes, Cambridge, United Kingdom) 1:1000, Alexa Fluor 488-labeled goat-antimouse IgG (Molecular Probes) 1:500, and Alexa Fluor 633-labeled phalloidin (Molecular Probes) 1:1000, diluted in blocking solution for 2 hours at room temperature on a rocker. The mesentery was then washed in PBX five times for 1 hour and incubated with Hoechst 33324 (1 µmol/L) for 15 minutes at room temperature on a rocker. The mesentery was mounted as flat as possible with VectaShield (Vector Lab) on a slide and coverslipped. The whole mount mesentery was imaged with a Leica Confocal Microscope (Leica confocal SP2 system; Leica), or Nikon E400 Eclipse epifluorescence microscope (Nikon). Proliferating endothelial cells, sprouts, and microvessels were imaged. VEGF165b secretion from the fat pad into the mesentery was checked by staining mesenteries with the anti-VEGF165b antibody (Fig. 3)
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Microvessel Measurement.
After immunofluorescence staining, the whole mount mesentery was used for microvessel measurement. For each mesentery, 8 to 12 views were selected randomly with a 40x objective on a Nikon E400 Eclipse epifluorescence microscope and images were taken with the Nikon Coolpix 800 Digital Camera (Nikon, Surrey, United Kingdom). Openlab software (Improvision, Coventry, United Kingdom) was used to measure the area analyzed and vessel parameters between two adjacent branch points. The total vessels were counted and labeled, and the branch points, proliferating endothelial cells, and sprouts in each image were counted. The diameter and length of each vessel were measured. Branch point density, sprout density, and proliferating endothelial cell density were calculated as the number per unit area within five randomly selected fields of view (x40 objective) containing vessels as described previously (31)
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Tumor Model
A total of 1 x 106A375 human melanoma cells stably transfected with 2.4 µg of pcDNA3 (control) vector or pcDNA3-VEGF165 or pcDNA3-VEGF165b (both expressing 4 fg.cell-1·day-1 VEGF) was s.c. injected into each of six nude mice. To determine the effect of a mixture of populations, 5 x 105 of VEGF165b and VEGF165-expressing cells were combined in PBS and injected as above. Tumor width and length was measured using Vernier’s calipers, and tumor volume was calculated as the product of length width and the average of the two measurements. The mice were humanely killed once the tumors reached 16 mm in diameter or 28 days, whichever occurred soonest.
Statistical Analysis
One-way ANOVA was used to analyze the difference of parameters between the groups and Student Newmann Keuls post hoc test used to compare the individual groups. P < 0.05 was considered significant.
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RESULTS |
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shows that this antibody specifically detects VEGF165b and does not detect VEGF165. This was true even in the presence of 50-fold greater amounts of VEGF165. To determine whether VEGF165b protein could be detected in normal human cells, this antibody was used on a Western blot analysis containing protein extracted from terminally differentiated conditionally immortalized human glomerular visceral epithelial cells (podocytes, a kind gift of Moin A. Saleem), which express VEGF165b mRNA (35)
. The protein was then extracted from 106 cells and subjected to SDS-PAGE and Western blotting. The membrane was first probed with the VEGF165b antibody and then a commercial pan-VEGF antibody to ensure specificity. Fig. 4A
shows that the antibody detected a number of different proteins, which appear to correlate with expected molecular masses for most isoforms of VEGF, including VEGF121b (monomer-band a, dimer-band b), VEGF145b (band c), VEGF165b (band d), VEGF189b (band e), and large VEGFbs (bands at f). The pan-VEGF antibody confirmed that these bands were VEGF isoforms (Fig. 4A)
. These isoforms are consistent with VEGF165b, VEGF189b, VEGF145b, and VEGF121b. A larger molecular mass isoform, which corresponds to large-VEGF formed by alternate start codon usage (33)
, was present in both blots. Interestingly, however, the relative intensities of the bands were different for the pan-VEGF antibody compared with the VEGFxxxb antibody. This suggests that the relative amount of the different splice forms is not the same for VEGFxxxb isoforms as for the conventional isoforms. Moreover, Western blotting of prostate tissue also demonstrated VEGF165b and other isoforms, showing that VEGF165b is expressed in human tissue (Fig. 4B)
. We therefore speculate that podocytes and other human cells produce a sister family of inhibitory isoforms, including that to large-VEGF. We have described these as the VEGFxxxb family of isoforms. To determine whether the expression of VEGFxxxb also occurred in podocytes in human glomeruli, VEGFxxxb protein expression was detected in sections of nephrectomy specimens by immunohistochemistry. Fig. 4C
shows clear expression of VEGFxxxb isoforms in the podocytes on the periphery of the glomerulus (arrows)—shown to be the location of VEGF expression by numerous studies (24)
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. To determine whether VEGF splicing was altered in prostate tissue, mRNA was extracted from transurethral resection of the prostate chips taken from 17 patients with benign prostatic hyperplasia and 9 with malignant prostate cancer. VEGF165b mRNA was found in 82% of the benign (14 of 17) but only 22% (2 of 9) of the malignant samples (P < 0.05, Fisher’s exact test, three of each group shown in Fig. 4E
). VEGF165b is therefore an endogenously produced protein and is down-regulated at the mRNA level in prostate tumors.
VEGF165b Inhibits VEGF165-mediated Signaling by Acting as a Competitive Antagonist of VEGFR-2.
To determine whether VEGF165b could inhibit VEGF165-mediated signaling, we measured the ability of VEGF165b to compete for binding of its receptors. Human umbilical vein endothelial cells were incubated with a mixture of 20 ng·ml-1 125I-VEGF165 and increasing concentrations of either unlabeled recombinant VEGF165 (Peprotech, Rocky Hill, NJ) or purified recombinant VEGF165b (R&D Systems). VEGF165b competed with 125I-VEGF for binding to the cells with the same affinity as unlabeled VEGF165 (Fig. 5A)
. To determine whether this was due to binding to the active signaling receptor VEGFR-2, we measured the ability of VEGF165b to compete with 20 ng·ml-1 125I-VEGF165 binding to the receptor immobilized on an ELISA plate. VEGF165b competed with the same affinity (IC50 = 19.8 ± 1.3 ng/mL) as unlabeled VEGF165 (IC50 = 17.3 ± 1.3 ng/mL; Fig. 5B
). These results show that VEGF165b can bind to VEGFR-2 with the same affinity as VEGF165, i.e., the altered COOH terminus of VEGF165b does not affect binding to the receptor.
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shows that VEGFR-2 was less phosphorylated by VEGF165b than VEGF165 and was no more phosphorylated than untreated cells. Moreover, addition of both isoforms resulted in less phosphorylation than VEGF165 alone or saline (P < 0.05, ANOVA, n = 3). VEGF stimulates angiogenesis and vascular permeability by activating VEGFR-2. Human microvascular endothelial cells express both VEGFR-2 and VEGFR-1, which have been shown to interact. To determine whether VEGF165b inhibited VEGF165 by inhibiting phosphorylation of VEGFR-2 directly, we measured the effect of VEGF165b on VEGFR-2 phosphorylation by incubating CHO cells transfected with VEGFR-2 with saline, VEGF165, VEGF165b, or both isoforms as above. Cells were then immunoprecipitated with anti-phosphotyrosine antibody and both supernatant and pellet subjected to Western blotting with an antibody to VEGFR-2. Fig. 6Bi
shows that transfected cells have an endogenous level of phosphorylation of VEGFR-2 that is increased by VEGF165 and decreased by VEGF165b. Moreover, VEGF165b incubation inhibited phosphorylation by VEGF165.
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. The blot was then stripped and reprobed with a p42/p44 MAPK antibody. Although treatment with VEGF165 resulted in a significant increase in phospho-MAPK blot density, this was not the case for incubation with VEGF165b (Fig. 6Bii)
. Moreover, treatment with both VEGF165 and VEGF165b did not result in a significant increase in phosphorylation. Fig. 6Bii
shows the mean density of the phospho-p42/p44 MAPK band relative to total MAPK. Furthermore Fig. 6Biii
shows that, unlike VEGF165, VEGF165b incubation did not result in phosphorylation of AKT, and incubation with both isoforms inhibited AKT phosphorylation compared with VEGF165 treatment alone (P < 0.01, ANOVA; Fig. 6Biii
). This shows that VEGF165b can inhibit VEGF165-mediated signaling of at least two different pathways stimulated by activation of VEGFR-2.
VEGF165 has previously been shown to stimulate both Akt and p44/p42 MAPK phosphorylation in endothelial cells through VEGFR-2 activation (39, 40, 41)
. To determine whether VEGF165b could inhibit VEGF-mediated signaling in endothelial cells, human microvascular endothelial cells were treated with either saline, 1 nmol/L VEGF165, 1 nmol/L VEGF165b, or 1 nmol/L of each isoform for 20 minutes. Protein extracted from these cells was then subjected to SDS-PAGE and Western blotting and probed with antibodies to phospho-Akt and total Akt or phospho-MAPK and total MAPK (Fig. 7)
. These studies showed that treatment with VEGF165 resulted in greater phosphorylation than no treatment but treatment with VEGF165b did not. However, surprisingly VEGF165b did not appear to inhibit VEGF165-mediated phosphorylation of Akt (Fig. 7A)
. Furthermore, VEGF165b treatment resulted in a significant increase in phosphorylation of p42/p44 MAPK, very similar to that elicited by VEGF165. Moreover, treatment with both isoforms also increased phosphorylation of MAPK to at least as great a level as treatment with either 1 nmol/L VEGF165 or VEGF165b (Fig. 7B)
.
|
shows examples of the vasculature of the cornea 10 days after implantation of the cells. pcDNA3-transfected cells showed no angiogenesis, whereas VEGF165-transfected cells expressing 2 fg·cell-1·day-1 VEGF165 resulted in significant angiogenesis after 5 days that was marked by 10 days.
|
). Moreover, when cells transfected with VEGF165b were mixed with cells transfected with VEGF165 and implanted into the cornea, no angiogenesis was seen (Fig. 8B)
, despite significant angiogenesis when VEGF165-transfected cells were mixed with pcDNA3-transfected cells and implanted (P < 0.0001, two-way ANOVA; Fig. 8, B and D
).
VEGF165b Inhibits VEGF165-mediated Angiogenesis in the Viscera.
To determine whether VEGF165b could inhibit VEGF165-mediated angiogenesis in a separate assay in rats, we used a modification of a mesenteric angiogenesis assay (31)
. Adenoviruses expressing EGFP, VEGF165b, or VEGF165 were injected into the mesenteric fat pad of an anesthetized rat. In rats that received injections of adenovirus-expressing GFP, a small increase in visualized vascular area was seen (Fig. 9A, i to ii)
. When receiving injections of Ad- VEGF165, however, extensive and florid vasculature was induced in the adjacent mesenteric connective tissue panels (Fig. 9A, iii to iv)
as shown previously (30)
. In rats that received injections of Ad-VEGF165b, the increase in vasculature seen in the fat pad was no greater (Fig. 9A, v to vi)
than that seen with control virus. Injection of both Ad-VEGF165b and Ad-VEGF165 did not result in an increase in vascular area (Fig. 9A, vii to viii)
. The perfused blood vessel area was significantly increased by Ad-VEGF165 relative to adenovirus-expressing enhanced GFP but not by Ad-VEGF165b or both Ad-VEGF165b and Ad-VEGF165 (P < 0.0001, ANOVA; Fig. 9B
). Quadruple fluorescent staining (e.g., Fig. 10A
) of the mesenteries with Griffonia simplicifolia isolectin B4 (stains endothelial cells), Ki-67 (proliferating cells), Hoescht (nuclei), and phalloidin (stains actin filaments in sprouts of endothelial cells undergoing migration) enabled quantification of parameters that require angiogenesis to occur. The density of branch points (Fig. 10B)
, sprouts (Fig. 10C)
, proliferating endothelial cells (Fig. 10D)
, and blood vessels (Fig. 10E)
and the mean vessel length (Fig. 10F)
were measured. VEGF165 increased all of these measurements, except mean vessel length, which was decreased as expected if sprouting angiogenesis occurred. Injection of Ad-VEGF165b, however, did not result in an increase in perfused vessel area, vascular density, sprouting, branching, or proliferation and did not decrease mean vessel length. Moreover, when both viruses were injected, the effect of VEGF165 on all these parameters was significantly inhibited (see Fig. 10B–F
).
|
|
than cells expressing VEGF165 (Fig. 11A)
. Moreover, injection of a mixture of the two isoform-expressing cells resulted in delayed tumor growth (Fig. 11C)
, and at 20 days, the tumors were significantly smaller than in mice that received injections of VEGF165-expressing cells. (Fig. 11D)
. Moreover, mixing the tumor cell populations resulted in growth at a slower rate, with an increased doubling time, suggesting that growth was inhibited by VEGF165b expression, not simply the number of cells developing into a tumor was different (Fig. 11E)
.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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VEGF has been the most widely implicated protein in stimulating angiogenesis in cancer and other major health concerns of the developed world (3) . That VEGF can be differentially spliced to form an inhibitory isoform, which is endogenously expressed, and is antiangiogenic requires a major reevaluation of our current understanding of how angiogenesis is regulated. Because of the nature of this splice variant—distally splicing into the 3'-untranslated region of the VEGF mRNA, most previously investigated expression studies will not have distinguished VEGFxxxb from other isoforms. This may explain some of the data that does not show clear relationships between VEGF expression and angiogenesis, such as in melanoma and other angiogenic conditions (49) . Moreover, recent studies showing that VEGF-neutralizing antibodies are an effective therapy for some types of cancer, retinopathy, and other angiogenic conditions (45 , 46 , 50 , 51) could be made more effective by targeting the proangiogenic splice variants rather than a pan-VEGF strategy. There appears to be a splicing switch in at least two types of cancer—renal and prostate—but the mechanisms that regulate splicing of VEGF are almost completely unknown. Regulation of splicing from anti- to proangiogenic growth factor isoforms may be a component of the control of development of cancers, progression of heart disease, and other pathological states. It has not escaped our notice that other proteins involved in angiogenesis, such as VEGFR-1, fibroblast growth factor, fibronectin, and collagen (52, 53, 54, 55, 56) , also have differentially spliced inhibitory isoforms. A common mechanism may therefore exist for the regulation of these antiangiogenic splicing events.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: David Bates, Microvascular Research Laboratories, Department of Physiology, Preclinical Veterinary School, Southwell Street, Bristol, BS2 8EJ, United Kingdom. Fax: 0117-928-8151; E-mail: Dave.Bates{at}bris.ac.uk
Received 3/16/04. Revised 7/ 9/04. Accepted 9/ 1/04.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or pan-VEGF antibody (
). Both ELISAs detected respective VEGF isoforms at concentrations < 100 pg·ml-1.
