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1From the Massachusetts Eye and Ear Infirmary and 4Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts; the 3Department of Ophthalmology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; 5Imperial Cancer Research, London, United Kingdom; and 6Eyetech Research Center, Woburn, Massachusetts.
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Abstract |
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METHODS. Controlled-release pellets containing equimolar amounts of VEGF120 and VEGF164 were implanted in corneas. The mechanisms underlying this differential response of VEGF isoforms were explored. The response of VEGF in cultured endothelial cells was determined by Western blot analysis. The response of VEGF isoforms in leukocytes was also investigated.
RESULTS. VEGF164 was found to be significantly more potent at inducing inflammation. In vivo blockade of VEGF receptor (VEGFR)-1 significantly suppressed VEGF164-induced corneal inflammation. In vitro, VEGF165 more potently stimulated intracellular adhesion molecule (ICAM)-1 expression on endothelial cells, an effect that was mediated by VEGFR2. VEGF164 was also more potent at inducing the chemotaxis of monocytes, an effect that was mediated by VEGFR1. In an immortalized human leukocyte cell line, VEGF165 was found to induce tyrosine phosphorylation of VEGFR1 more efficiently.
CONCLUSIONS. Taken together, these data identify VEGF164(165) as a proinflammatory isoform and identify multiple mechanisms underlying its proinflammatory biology.
The VEGF gene produces alternatively spliced mRNA variants, leading to at least three distinct major VEGF isoforms.1 They encode 120-, 164-, and 188-amino-acid-containing murine proteins (the human proteins are one residue longer, and thus produce isoforms of 121, 165, and 189 amino acids, respectively).11 12 13 The isoforms are highly conserved among species; however, their differential biology is poorly understood. Biochemically, the VEGF isoforms differ in their affinity for heparin, with the larger isoforms binding to heparin more avidly.14 15 16 The isoforms also vary in their affinity for their cognate receptors. For example, VEGF121 binds to VEGFR1 up to 20 times less efficiently than VEGF165 (Kd = 200 pM vs. 10 pM for 121 and 165, respectively) in vitro.17 Finally, VEGF165 is unique among the various VEGF isoforms in its ability to bind to the receptor neuropilin-1,18 a coreceptor for VEGFR2 (flk-1 or KDR). These studies were restricted to isoform-specific in vitro mitogenesis.
Studies in VEGF isoform-specific knockout mice have shed some light on the differential biology of the VEGF isoforms during development. Homozygous mice expressing only the VEGF120 isoform, but normal absolute VEGF levels, die shortly after birth and exhibit an abnormal coronary microvasculature and defective vascular patterning.19 Mice expressing only the VEGF188 isoform display impaired arterial development.20 It is also known that relative and absolute VEGF isoform expression levels vary among normal tissues and that they are altered in disease.13 21 22 23 For example, the selective upregulation of VEGF164 is observed in rheumatoid arthritis and diabetic retinopathy.21 22 23 A similar pattern is observed in injured cornea undergoing inflammation and neovascularization.9 Finally, others have demonstrated the inflammatory potential of VEGF164 in vivo,24 25 but isoform-specific comparisons have never been attempted.
In the present study, we compared and contrasted the biology of two prevalent VEGF isoforms: VEGF120(121) and VEGF164(165). The cornea, owing to its optical transparency and avascular state, was used to study the differential effects of these isoforms on inflammation, and the mechanisms underlying any variances were explored.
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Materials and Methods |
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To assess the effect of VEGFR-1 inhibition in a mouse corneal pocket assay, animals were randomized to receive intraperitoneal injections of 1 mg/kg anti-VEGFR1-neutralizing antibody (AF471; R&D Systems) 2 hours before pellet implantation. Control animals received an equivalent amount of a preimmune control goat IgG (Alpha Diagnostic International, San Antonio, TX).
VEGF ELISA
On days 2 and 7 after corneal pellet implantation, the eyes were enucleated and the pellets removed. The corneal samples were placed into 150 mL of lysis buffer (20 mM imidazole HCl, 10 mM KCl, 1 mM MgCl2, 10 mM EGTA, 1% Triton, 10 mM NaF, 1 mM sodium molybdate, and 1 mM EDTA [pH 6.8]) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and sonicated. The lysate was centrifuged at 14,000 rpm for 15 minutes at 4°C, and the VEGF levels in the supernatant determined with a mouse VEGF kit (Quantikine; R&D Systems), according to the manufacturer’s protocol. The assay recognized all VEGF isoforms. Total protein was determined with a bicinchoninic acid (BCA) assay kit (Bio-Rad, Hercules, CA) and was used for the normalization of corneal VEGF protein levels.
Leukocyte Counts
Eyes were enucleated 2 days after pellet implantation, embedded in optimal cutting temperature (OCT) compound, snap frozen in liquid nitrogen, and cut into 7-µm-thick sections. After fixation with ice-cold acetone and blocking with normal goat serum, the sections were stained with a monoclonal rat anti-mouse CD45 (leukocyte common antigen, clone 30-F11; BD Pharmingen, San Diego, CA) antibody to detect infiltrating leukocytes, followed by staining with FITC-conjugated anti-rat IgG (Santa Cruz Biotechnology, Santa Cruz, CA). The cells were visualized by fluorescence microscopy and counted in a masked fashion. Eight consecutive serial sections extending through the pellet and limbus were studied per cornea. The number of CD45 cells between the limbus and pellet was determined at 50x magnification.
Cell Culture
Single-donor human umbilical venous endothelial cells (HUVECs) were purchased from Cascade Biologics Inc. (Portland, OR) and cultured in Medium 200 with low serum growth supplement (Cascade Biologics Inc). The cells were used within five passages. The human leukocyte cell line (Jurkat cells) was kindly provided by Nicholas Mitsiades (Dana Farber Cancer Institute, Boston, MA) and cultured in RPMI 1640 with 10% fetal bovine serum (FBS).
Western Blot Analysis
Intracellular adhesion molecule (ICAM)-1 expression in the HUVECs was studied by Western blot analysis. After exposure to medium 200 with 1% FBS for 24 hours, recombinant human VEGF121 or VEGF165 (R&D Systems) was added to the HUVEC cultures. Some HUVECs were preincubated with 30 ng/mL of anti-VEGFR2-neutralizing antibody (clone CUE04; R&D Systems) 1 hour before administration of VEGF. After incubation for 6 hours with recombinant VEGF, the cells were washed with PBS and suspended in sample buffer. The samples were boiled for 5 minutes, separated by SDS-polyacrylamide gel electrophoresis under denaturing conditions, and electroblotted to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membranes were incubated in blocking buffer, followed by the monoclonal anti-ICAM-1 antibody (Santa Cruz Biotechnology), washed, and incubated with a horseradish-peroxidase-conjugated secondary antibody. The blot was visualized with a chemiluminescence kit (ECL Plus; Amersham Pharmacia, Piscataway, NJ) according to the manufacturer’s instructions.
Cell Preparation and Sorting
Splenocytes from C57/B6 mice were prepared by teasing the spleen through a nylon screen. Erythrocytes were lysed by osmotic shock, and single-cell suspensions were prepared. The cells were first incubated with 50 µg normal rat immunoglobulin and anti-mouse immunoglobulin FcR
antibody (clone 2.4G2; BD Pharmingen) before the addition of the appropriate fluorescein-conjugated antibodies against cell surface markers for leukocyte subpopulations. The following antibodies were used for flow cytometry: anti-CD3 for T cells (BD Pharmingen), anti-F4/80 for monocytes and macrophages (Serotec, Raleigh, NC), and 7/4 for neutrophils (Serotec). The fluoresceinated reagents used for the second-stage were FITC-avidin and phycoerythrin (PE)-avidin. Flow cytometry was then performed (model XLEPICS XL flow cytometer; Beckman Coulter, Miami, FL). Dead cells were excluded based on low forward light scattering.
Reverse Transcription-Polymerase Chain Reaction
The expression of VEGF receptors on mouse leukocytes at the mRNA level was investigated by reverse transcription-polymerase chain reaction (RT-PCR), which was performed on subpopulations of leukocytes after cell sorting. Total RNA was isolated from each leukocyte subpopulation (1 x 106 cells; TRIzol; Invitrogen-Gibco, Grand Island, NY), and cDNA was produced with reverse transcriptase (SuperScript II; Invitrogen, San Diego, CA). The PCR conditions were as follows: 30 cycles of 45 seconds at 94°C, 45 seconds at 55°C, and 45 seconds at 72°C, with an initial 5-minute denaturation step and a final 7-minute elongation step. The oligonucleotide primers for VEGFR1 and VEGFR2 were purchased from R&D Systems (human/mouse VEGF R1 and VEGF R2 Primer Pair). The primers for NP-1 were based on the sequences of mouse NP-1. The primers were 5'-TCA GGA CCA TAC AGG AGA TGG-3' (sense), corresponding to nucleotides 2396-2416 in the mouse NP1 mRNA, and 5'-TGA CAT CCC ATT GTG CCA AC-3' (antisense), corresponding to nucleotides 2995-3014 in the mouse NP1 mRNA. The fragments amplified by RT-PCR were subcloned into the TA vector (Invitrogen).
Cell Migration Assay
VEGF isoform-induced chemotaxis was evaluated with a 48-well microchemotaxis chamber (Neuro Probe, Bethesda, MD), as previously described.27 Briefly, serum-free RPMI 1640 medium was added, with or without mouse VEGF120 and VEGF164 (R&D Systems) at a concentration of 10 ng/mL. A 5-µm pore size PVP(-) polycarbonate filter, (Neuro Probe) was overlaid, and 50 µL of cell suspension per well (1 x 106 cells/mL) was seeded in the upper compartment and incubated at 37°C for 2 (monocytes) or 4 (T lymphocytes) hours in a 5% CO2 incubator. Some cells were preincubated with an anti-VEGFR1-neutralizing antibody (10 ng/mL) at 37°C for 1 hour. Migrated cells were counted in a masked fashion.
VEGFR1 Activation
Jurkat cells (1 x 107) were incubated in 1% FBS in RPMI 1640 with human recombinant VEGF121 or VEGF165 (R&D Systems) for 2 minutes at 37°C after incubation in 1% FBS in RPMI 1640 for 12 hours. The cells were then centrifuged, lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 1% NP40, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM Na3VO4) supplemented with a protease inhibitor cocktail (Roche Diagnostics) and sonicated. The lysates were cleared by centrifugation at 14,000 rpm for 15 minutes at 4°C. The supernatants were then precleared by incubation for 1 hour with rabbit anti-mouse IgG-agarose and then incubated for 1 hour with an anti-phosphotyrosine monoclonal antibody 4G10 (10 µg/mL; UBI, Salt Lake City, UT). The immune complexes were recovered on rabbit anti-mouse IgG-agarose. Immunoprecipitates were washed four times with lysis buffer, twice with the same buffer without detergent, and once with Tris-buffered saline. The proteins were separated by 7.5% SDS-PAGE, blotted onto PVDF membrane, and probed with polyclonal anti-VEGFR1 antibody (C-17; Santa Cruz Biotechnology). Immunoreactivity was determined by the enhanced chemiluminescence reaction (Amersham Pharmacia Biotech).
Lectin Angiography and Neovascularization Quantitation
Corneal neovascularization was imaged through lectin angiography. Mice received intravenous isolectin B4 conjugated with FITC (500 µg; Vector Laboratories, Burlingame, CA) and were killed 30 minutes later. The eyes were enucleated and fixed with 1% paraformaldehyde for 15 minutes. After fixation, the corneas were placed on glass slides and studied through fluorescence microscopy (Leica, Deerfield, IL). Images were captured using a CD-330 charged-coupled device camera (Dage-MTI Inc., Michigan City, IN; controlled by Openlab software; Improvision, Lexington, MA), as described previously.28 Briefly, NIH Image 1.62 (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD) was used for image analysis. The neovascularization was quantified by setting a threshold level of fluorescence, above which only vessels were captured. The entire flatmounted cornea was analyzed to minimize sampling bias. The neovascularization quantitation was performed in a masked manner. The vascularized area was outlined using the innermost vessel of the limbal arcade as the border.
Concanavalin A (ConA) lectin (Vector Laboratories) was used for imaging adherent leukocytes and the vasculature, as described previously.29 Briefly, after anesthesia with xylazine hydrochloride (5 mg/kg) and ketamine hydrochloride (35 mg/kg), the mouse’s chest cavity was carefully opened and a 24-gauge cannula was introduced into the left ventricle. The animals were perfused with 10 mL PBS to remove erythrocytes and nonadherent leukocytes, after which, 10 mL FITC-conjugated ConA lectin (20 µg/mL) was perfused. Residual unbound lectin was removed with 10 mL PBS solution. After the eyes were enucleated, the corneas were excised and fixed with 1% paraformaldehyde for 15 minutes. The corneas were flatmounted on glass slides and observed by fluorescence microscopy (Leica).
Statistical Analysis
All results are expressed as the mean ± SD. The data were analyzed with the Mann-Whitney test, and post hoc comparisons were tested with the Fisher protected least significance procedure. Differences were considered statistically significant at P < 0.05.
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VEGF-Mediated Leukocyte Adhesion and Inflammation
Leukocyte adhesion to the limbal vascular endothelium was examined after VEGF pellet implantation. ConA was used to image the limbal vasculature and adherent leukocytes. At day 2, the number of adherent leukocytes in the limbal vasculature of the VEGF164-implanted corneas was significantly greater than in the VEGF120-implanted corneas (Fig. 1 , 21.4 ± 3.1 VEGF164 vs. 8.4 ± 2.1 VEGF120 leukocytes/cornea, n = 6 to 8/each condition, P < 0.05). However, leukocyte adhesion to the limbal vasculature was not inhibited by the systemic administration of a neutralizing antibody against VEGFR1 (Fig. 1) .
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Discussion |
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VEGF120(121) is a non-heparin-binding acidic protein that is freely diffusible. In contrast, VEGF164(165) binds to heparin-containing proteoglycan, suggesting that extracellular matrix may represent a major repository of VEGF.31 Although it is has been speculated that the differential affinity for heparin among the VEGF isoforms may provide for spatial targeting, this has not been demonstrated in vivo. The current data demonstrate that VEGF164 resides in the heparin proteoglycan-rich cornea for a longer period than VEGF120. Therefore, one potential explanation for the superior potency of VEGF164 is its prolonged corneal clearance and hence bioavailability.
VEGF can upregulate ICAM-1 expression on cultured endothelial cells32 as well as retinal endothelial cells in vivo.33 The current data demonstrate that VEGF165 is more potent at inducing ICAM-1 expression than VEGF121 (Fig. 3) . This effect was largely abrogated with an anti-VEGFR2 antibody in vitro. In vivo, the antibody blockade of VEGFR1 did not inhibit VEGF-induced leukocyte adhesion in the limbal vasculature. A VEGFR1 antibody was used because a suitable anti-mouse VEGFR2 antibody was not available for in vivo use. However, the in vivo result is consistent with the VEGFR2-mediated ICAM-1 increases observed in vitro. The more pronounced VEGF165-induced ICAM-1 increases are also consistent with the differential VEGFR2 tyrosine phosphorylation activities observed. Previous work has shown that the EC50 for VEGFR2 phosphorylation is lower with VEGF165 than with VEGF121 (34.9 pM and 2.09 nM for VEGF165 and VEGF121, respectively).34
Unlike VEGFR1 on endothelial cells, VEGFR1 on leukocytes is active and serves to trigger chemotactic migration.27 35 36 37 Early studies using radiolabeled VEGF revealed that VEGF bound almost exclusively to the vasculature.38 However, subsequent studies have confirmed the presence of high-affinity VEGF receptors on monocyte-macrophage subpopulations27 35 36 37 and neutrophils.27 The identification of these receptors further highlights the intertwined nature of the angiogenic and inflammatory pathways. It is also notable that the T lymphocytes expressed neuropilin-1, which was also recently suggested by Tordjman et al.39 We speculate that T-lymphocyte-expressed neuropilin-1 is not involved in mediating lymphocyte migration, because administration of VEGF did not show chemotaxis in vitro (Fig. 5) . Instead, neuropilin-1 may be involved in the cellular immune response, and VEGF164(165) may be involved in this process. More work is needed to delineate more clearly the effect of VEGF stimulation on T lymphocytes.
The migration of monocytes appeared to be mediated in large part by VEGFR1. The administration of an anti-VEGFR1-neutralizing antibody blocked both inflammation in vivo and leukocyte chemotaxis in vitro (Figs. 2 5) . These results confirm and extend previous work in this area. Hiratsuka et al.36 showed that VEGFR1 tyrosine-kinase-deficient mice produced macrophages deficient in VEGF-dependent migration. The mechanisms underlying the selective migration of leukocytes to VEGF164(165) may lie in the greater affinity of VEGF164(165) for VEGFR1. Keyt et al.17 demonstrated that VEGF164(165) binds VEGFR1 with greater affinity. Other mechanisms may apply; however, the fact that leukocyte VEGFR1 undergoes greater phosphorylation on binding VEGF164(165) suggests that the differential affinity for VEGFR1 may underlie the differential activation observed in this study.
Because inflammatory cells enhance the angiogenic process,28 it is not surprising that VEGF164(165) was also more potent at inducing angiogenesis. The mechanisms underlying the enhanced angiogenic response are probably complex; however, leukocytes make and release VEGF into the extracellular milieu.40 41 42 43 The recruitment of leukocytes to sites of angiogenesis may produce an amplification of local VEGF production and release. In addition, some leukocytes recruited to sites of angiogenesis may in fact be endothelial progenitor cells, exhibiting features of both leukocytes and endothelial cells.44 Some of these endothelial progenitor cells can be directly incorporated into the growing corneal neovasculature (Usui et al., manuscript submitted). VEGF164(165) may serve to recruit these cells to sites of angiogenesis differentially, thereby contributing to the VEGF164(165) induced "inflammation." Last, others have shown that VEGF164(165) is a more potent endothelial cell mitogen, an effect that may also be operative, in part, in the differential responses observed in the present study.45
Finally, the expression pattern of the VEGF isoforms appears to be altered in ocular disease. RT-PCR analysis has been used in severe cases of human proliferative diabetic retinopathy to show preferential expression of VEGF165.22 In an animal model of diabetes, the expression VEGF164 greatly predominated over VEGF120.23 Diabetic blood-retinal barrier breakdown was also more potently induced by VEGF164 than VEGF120.46 VEGF164 was also selectively upregulated in the ischemic retina in a rat model of rat retinopathy of prematurity.46 These data suggest that targeting VEGF164(165), which predominates in ocular vascular disease and is more potent at inducing disease, may represent a more targeted therapeutic strategy in ocular neovascular disease.
In summary, the current data further define the differential bioactivities of two major VEGF isoforms and demonstrate that they have differential effects on inflammation and angiogenesis. VEGF164(165) was more potent at inducing endothelial ICAM-1 expression by VEGFR-2, leukocyte migration by VEGFR-1, and inflammation and angiogenesis in the adult cornea. These data provide additional insights into the unique biology of the VEGF isoforms.
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Footnotes |
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Supported by the Roberta Siegel Fund (APA), Juvenile Diabetes Research Foundation International (APA), the Falk Foundation (APA), the Iacocca Foundation (APA), National Eye Institute Grant EY12611 (APA), and Eyetech Pharmaceuticals.
Submitted for publication February 1, 2003; revised May 29, 2003; accepted May 31, 2003.
Disclosure: T. Usui, None; S. Ishida, None; K. Yamashiro, None; Y. Kaji, None; V. Poulaki, None; J. Moore, None; T. Moore, None; S. Amano, None; Y. Horikawa, None; D. Dartt, None; M. Golding, None; D.T. Shima, Eyetech Pharmaceuticals, Inc. (F, E); A.P. Adamis, Eyetech Pharmaceuticals, Inc. (F, E)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Anthony P. Adamis, Eyetech Research Center, 42 Cummings Park, Woburn, MA 01801; tony.adamis{at}eyetk.com.
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