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The Journal of Immunology, 2006, 177: 4122-4131.
Copyright © 2006 by The American Association of Immunologists, Inc.

Vascular Endothelial Growth Factor Receptor 2-Based DNA Immunization Delays Development of Herpetic Stromal Keratitis by Antiangiogenic Effects1

Bumseok Kim*, Susmit Suvas*, Pranita P. Sarangi*, Sujin Lee{dagger}, Ralph A. Reisfeld{ddagger} and Barry T. Rouse2,*

* Department of Microbiology and Pathobiology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN 37996; {dagger} Department of Pediatric Infectious Disease, Vanderbilt University, Nashville, TN 37232; and {ddagger} Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stromal keratitis (SK) is an immunoinflammatory eye lesion caused by HSV-1 infection. One essential step in the pathogenesis is neovascularization of the normally avascular cornea, a process that involves the vascular endothelial growth factor (VEGF) family of proteins. In this report, we targeted the proliferating vascular endothelial cells expressing VEGFR-2 in the SK cornea by immunization with recombinant Salmonella typhimurium containing a plasmid encoding murine VEGFR-2. This form of DNA immunization resulted in diminished angiogenesis and delayed development of SK caused by HSV-1 infection and also reduced angiogenesis resulting from corneal implantation with rVEGF. CTL responses against endothelial cells expressing VEGFR-2 were evident in the VEGFR-2-immunized group and in vivo CD8+ T cell depletion resulted in the marked reduction of the antiangiogenic immune response. These results indicate a role for CD8+ T cells in the antiangiogenic effects. Our results may also imply that the anti-VEGFR-2 vaccination approach might prove useful to control pathological ocular angiogenesis and its consequences.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Virus infections often result in chronic immunoinflammatory lesions (1, 2, 3, 4, 5, 6, 7). One example is stromal keratitis (SK),3 a corneal inflammatory lesion that follows ocular infection with HSV-1 (8, 9, 10). In the mouse model, lesions of SK appear to be orchestrated by activated CD4+ T cells (5, 11), but multiple other cell types and humoral molecules are also involved (1, 12, 13, 14, 15). One essential event in the pathogenesis of SK appears to be neovascularization of the normally avascular cornea (16). In support of this, inhibition of neovascularization by antiangiogenic approaches results in diminished SK lesions (17), and several strategies to inhibit unwanted angiogenesis have been proposed for SK lesion therapy (17, 18, 19).

Recently, findings indicate that multiple angiogenic molecules were responsible for the HSV-induced angiogenesis (16, 20, 21, 22). These include vascular endothelial growth factor (VEGF) (16, 23, 24), which in the case of HSV infection may result from induction by the CpG-containing motifs of HSV DNA (25), as well as from infiltrated inflammatory cells (16). Additionally, virus infection stimulates release of various cytokines including IL-1 (22, 26) and IL-6 (21), which indirectly induce VEGF production (21, 22, 26). Accordingly, targeting VEGF represents a logical approach to control HSV-induced angiogenesis (17, 18, 27, 28).

It is known that VEGF causes angiogenesis by signaling through two receptor tyrosine kinases, VEGFR-1 and–2 (29). Although both receptors are involved in angiogenesis, the VEGFR-2-signaling pathway is used more for studying pathological angiogenesis. This VEGFR-2 is overexpressed in activated endothelial cells and its signaling induces proliferation and migration of endothelial cells to the inflammatory area (30). Several antiangiogenic or immunotherapeutic approaches targeting endothelial cells expressing VEGFR-2 have been used to inhibit pathological angiogenesis and tumor growth. These include immunization with VEGFR-2 protein (31, 32), VEGFR-2 mRNA-loaded dendritic cells (33), or paraformaldehyde-fixed endothelial cells (34) as well as disruption of VEGFR genes by various methods (18, 35). These approaches indicate that VEGFR-2 is a critical therapeutic target for inhibition of unwanted angiogenesis. In the present study, we have immunized mice with an attenuated Salmonella typhimurium harboring VEGFR-2 cDNA to induce VEGFR-2-specific immune responses. In this study, we show that the immunization stimulates a potent CD8+ T cell-mediated antiangiogenic response that delays development of SK lesions via inhibitory effects on HSV-induced ocular angiogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Bacterial strains and construction of recombinant S. typhimurium encoding VEGFR-2

Attenuated S. typhimurium Aro/A (strain SL7207) (provided by B. A. D. Stocker, Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA) transformed with pcDNA3.1-VEGFR-2 or -GFP was prepared by R. A. Reisfeld (The Scripps Research Institute, La Jolla, CA). The construction of the expression vector encoding murine VEGFR-2 was described previously (30). Briefly, DNA encoding murine VEGFR-2 (provided by I. Lemischka, Princeton University, Princeton, NJ) was cloned with the primers 3'-CCGGTACCATGGAGAGCAAGGCGCTG-5' and 5'-CCTCTAGACAGC-AGCACCTCTCTC-3' and inserted into the pcDNA3.1 vector (Invitrogen Life Technologies) between restriction sites KpnI and XbaI generating pcDNA3.1-VEGFR-2. Bacteria were electroporated as described (36, 37). Protein expression of VEGFR-2 was demonstrated by immunohistochemical staining of transfected COS-7 cells (Fig. 1).


Figure 1
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  		FIGURE 1. Functionality of VEGFR-2 expression. VEGFR-2 expression was verified by immunohistochemical staining by transfected COS-7 cells. The black arrows indicate the cells expressing VEGFR-2 protein.

 
Mice

Female 5- to 6-wk-old BALB/c or C57BL/6 mice were purchased from Harlan Sprague Dawley. To prevent bacterial infection, all mice received treatment with sulfamethoxazole/trimethoprim (Biocraft) at the rate of 5 ml/200 ml drinking water after virus infection. All investigations followed the guidelines of the Committee on the Care of Laboratory Animals Resources, Commission of Life Sciences, National Research Council. The animal facilities of the University of Tennessee (Knoxville, TN) are fully accredited by the American Association of Laboratory Animal Care.

Oral immunization and corneal HSV infection

Mice were immunized by oral gavage three times at 2-wk intervals with 100 µl of sodium bicarbonate (NaHCO3) buffer containing 108 S. typhimurium transformed with pcDNA3.1-VEGFR-2 or -GFP as a control. Corneas of mice were infected 1 wk after final immunization. HSV-1 strain RE (provided by R. L. Hendricks, University of Pittsburgh, Pittsburgh, PA) was used in all procedures. Virus was grown in Vero cell monolayers (catalog no. CCL81; American Type Culture Collection (ATCC)), titrated, and stored in aliquots at –80°C until used. Corneal infections of all mouse groups were conducted under deep anesthesia induced by Avertin. The mice were scarified lightly on their corneas with a 32-gauge needle and infected with a 2-µl drop containing 5 x 105 PFU of HSV-1 that was applied to the eye and gently massaged with the eyelids.

Clinical observations

The eyes were examined on different days after infection for the development of clinical lesions by slit-lamp biomicroscopy (Kawa Company), and the clinical severity of keratitis of individually scored mice was recorded. The scoring system was as follows: 0, normal cornea; +1, mild corneal haze; +2, moderate corneal opacity or scarring; +3, severe corneal opacity but iris visible; +4, opaque cornea and corneal ulcer; +5, corneal rupture and necrotizing stromal keratitis. The severity of angiogenesis was recorded as described previously (17). In reference to the angiogenic scoring system, the method relied on quantifying the degree of neovessel formation based on three primary parameters: 1) the circumferential extent of neovessels (as the angiogenic response is not uniformly circumferential in all cases); 2) the centripetal growth of the longest vessels in each quadrant of the circle; and 3) the longest neovessel in each quadrant was identified and graded between 0 (no neovessel) and 4 (neovessel in the corneal center) in increments of ~0.4 mm (radius of the cornea is ~1.5 mm). According to this system, a grade of 4 for a given quadrant of the circle represents a centripetal growth of 1.5 mm toward the corneal center. The score of the four quadrants of the eye were then summed to derive the neovessel index (range, 0–16) for each eye at a given time point.

Corneal micropocket assay

In vivo angiogenic activity was assayed in the avascular cornea of mouse eyes as previously described (17). Briefly, corneas of mice immunized with S. typhimurium transformed with pcDNA3.1-VEGFR-2 or -GFP as a control were implanted pellets containing 200 ng of rVEGF protein (R&D Systems). Pellets for insertion into the cornea were made by combining known amounts of VEGF protein, sucralfate (10 mg; Bulch Meditec) and hydron polymer in ethanol (120 mg/ml ethanol; IFN Sciences), and applying the mixture to a 15 x 15-mm2 piece of synthetic mesh (Tekto). The mixture was allowed to air dry and fibers of the mesh were pulled apart, yielding pellets containing 200 ng of VEGF. Pellets containing VEGF were implanted into an intracorneal pocket (1 mm from the limbus). The eyes were then evaluated for corneal neovascularization. The extent of the neovessel ingrowth was recorded by direct measurement using calipers (Symbol of Quality; Biomedical Research Instruments) under stereomicroscopy (Leica Microsystems). The length of the neovessels originating from the limbal vessel ring toward the center of the cornea and the width of the neovessels presented in clock hours were measured. Each clock hour is equal to 30° at the circumference. The angiogenic area was calculated according to the formula for an ellipse: A = ((clock hours) x 0.4 x (vessel length in mm) x {pi})/2.

ELISA

The lysates from HSV-1-infected cornea were used for the measurement of VEGFR-2 or the supernatants from cocultured splenocytes with endothelial cells expressing VEGFR-2 were used for the measurement of IFN-{gamma} by a standard ELISA protocol. For preparation of corneal lysates, two corneas/time point were collected and minced with liquid nitrogen. Minced pieces were collected in 1 ml of DMEM without FCS and homogenized using an ultra sonicater (Heat Systems-Ultrasonics). The lysates were then clarified by centrifugation at 12,000 rpm for 5 min at 4°C. The supernatant was collected and stored at –80°C until further use. The corneal lysates or coculture supernatants were added to the plate (100 µl/well) and incubated at 4°C overnight. The plate was washed with 0.05% Tween 20/PBS and blocked with 3% BSA for 2 h at 37°C. After washing, the plate was incubated with anti-VEGFR-2 or anti-IFN-{gamma} biotinylated detection Ab (100 ng/ml; R&D Systems) for 2 h. Finally, peroxidase-conjugated streptavidin (Jackson ImmunoResearch Laboratories) was added. The color reaction was developed using ABTS (Sigma-Aldrich) and measured with an ELISA reader (Spectramax 340; Molecular Devices) at 405 nm. Quantification was performed with Spectramax ELISA reader software version 1.2.

Activation of CD8+ T cells

One week after final immunization, splenocytes were collected from mice immunized against VEGFR-2 or GFP, and cocultured 24 h with high endothelial venules (HEV) (murine endothelial cell lines expressing VEGFR-2). Flow cytometric analyses were performed using PE-conjugated Ab to CD8 in combination with FITC-conjugated Abs to CD69 or CD25 (BD Pharmingen). Supernatant of cell cultures were used to measure IFN-{gamma} levels.

CTL assay

CTL activity was assessed by a standard 4-h 51Cr-release assay against labeled target cells as previously described (30). One week after final immunization, splenocytes were collected from mice immunized with VEGFR-2 or GFP DNA. Splenocytes were cocultured with irradiated (3000 rad) and mitomycin C-incubated HEV, CT-26 (murine colon carcinoma cells), YAC cell lines. Five days later, the activated T cells were harvested and tested for cytotoxicity.

Depletion of CD8+ T cells

CD8+ T cells were depleted from animals vaccinated with pcDNA3.1-VEGFR-2 or GFP by i.p. injection of 1 mg of rat anti-mouse mAb to CD8 (CRL-1971; ATCC) 1 day before VEGF pellet implantation.

Confocal microscopy

For immunofluorescence staining, eyes were frozen in optimum cutting temperature (OCT) compound (Miles). Seven-micrometer-thick sections were cut, air-dried, and fixed in cold acetone for 10 min at –20°C. Nonspecific binding was blocked with 3% BSA-PBS-0.05% Tween 20 for 2 h at 37°C. For detection of endothelial cells expressing VEGFR-2, the sections were incubated with a mixture of FITC-coupled monoclonal anti-mouse CD31 (0.5 µg/ml; BD Pharmingen) and biotin-labeled anti-VEGFR-2 Ab (1 µg/ml; R&D systems) in 1% BSA-PBS-0.05% Tween 20. For detection of both endothelial and CD8+ T cells in the stroma, the sections were incubated with a mixture of FITC-coupled monoclonal anti-mouse CD8 and biotin-labeled anti-CD31 Ab (0.5 µg/ml; BD Pharmingen) in 1% BSA-PBS-0.05% Tween 20. For detection of both CD8+ and CD4+ T cells, the sections were incubated with a mixture of FITC-coupled monoclonal anti-mouse CD8 and biotin-labeled anti-CD4 Ab (0.5 µg/ml; BD Pharmingen) in 1% BSA-PBS-0.05% Tween 20. After incubating overnight at 4°C, slides were washed thoroughly in PBS, and streptavidin-conjugated Alexa Fluor 546 (1 µg/ml; Molecular Probes) in 1% BSA-PBS was added for 1 h at room temperature. Slides were mounted with Vectashield without propidium iodide (Vector Laboratories). Images were captured with a Leica SP2 laser scanning confocal microscope (Leica).

Adoptive transfer of Thy1.1 CD8+ T cells

CD8+ T cells were isolated from VEGFR-2-based DNA immunized Thy 1.1+ C57BL/6.PL (H-2b) mice. Single-cell suspensions of pooled spleens were prepared from immunized mice at day 7 after final immunization, and CD8+ T cells were purified using a CD8+ T cell purification column (Miltenyi Biotec) according to the suggested protocol. The purity of cells ranged from 90 to 95%. The purified Thy 1.1 CD8+ T cells (5 x 106/mouse) were adoptively transferred into Thy 1.2 mice day 1 before and after virus ocular infection or VEGF pellet implantation.

Isolation and flow cytometric analysis of cornea

For preparation of single-cell suspension of cornea, two corneas per time point were collected and digested with Liberase (60 U/ml; Roche Diagnostics) at 37°C for 1 h in complete RPMI 1640 (catalog no. R8758; Sigma-Aldrich) in a humidified atmosphere of 5% CO2. After incubation, the corneas were disrupted by grinding with a syringe plunger on a strainer (70 µm) and a single-cell suspension was made in complete RPMI 1640 medium. Next, the single-cell suspension obtained from corneal samples was stained for FACS. Briefly, single cells were first blocked with an unconjugated anti-CD32/CD16 mAb (0.5 µg/ml; BD Pharmingen) for 30 min in FACS buffer. To measure endothelial cells in the cornea, total cells were used for cell surface staining for CD31 by using biotin-labeled rat anti-mouse (m) PECAM-1 Ab (0.2 µg/well; BD Biosciences) followed by streptavidin-FITC (0.2 µg/well; BD Pharmingen) and for VEGFR-2 by using PE-labeled rat-anti-mVEGFR-2 Ab (0.2 µg/well; BD Biosciences). To detect adoptively transferred cells, total cornea cells were stained by anti-Thy 1.1-PerCP Ab (BD Pharmingen) and anti-CD8-FITC Ab for 30 min. Finally, the cells were washed three times and positive cells were measured by flow cytometry using a FACSCalibur (BD Biosciences). The data were analyzed using CellQuest 3.1 software (BD Biosciences).

Statistical analysis

Significant differences between groups were evaluated by using the Student t test. A value of p < 0.05 was regarded as a significant difference between the two groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
VEGFR-2 is overexpressed in the neovessels of SK cornea

Previous studies on HSV-1-induced angiogenesis identified VEGF as a protein that is highly expressed in the infected cornea and involved in HSV-specific angiogenesis (16). In addition VEGFR-2, one of the VEGF receptors, is overexpressed in proliferating endothelial cells under pathological conditions (30, 38). To test whether this receptor is similarly overexpressed in the HSV-1-induced abnormal blood vessels, VEGFR-2 expression was measured at various times after infection. Such infection caused vascular sprouting from the limbal vessels, starting at day 2 after virus infection. In the first approach, corneal lysates from mice infected with virus were measured for VEGFR-2 expression by ELISA. As shown in Fig. 2A, the levels of VEGFR-2 in SK cornea increased as lesions progressed compared with the naive cornea.


Figure 2
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  		FIGURE 2. Expression of VEGFR-2 on the proliferating endothelial cells of neovessels of SK cornea. The mice were scratched lightly on their corneas with a 32-gauge needle and infected with a 2-µl drop containing 5 x 105 PFU of HSV-1 RE that was applied to the eye. At various time points, corneas were collected and processed to measure the VEGFR-2 protein. Levels of VEGFR-2 protein were estimated from supernatants of corneal lysates of mice by ELISA as outlined in Materials and Methods (A). At day 21 p.i., eyes were frozen and 7-µm-thick sections were cut, air-dried, and processed for immunofluorescences staining as described in Materials and Methods (B). Briefly, for detection of endothelial cells expressing VEGFR-2, the sections were incubated with a mixture of FITC-coupled monoclonal anti-mouse CD31 (0.5 µg/ml) and a biotin-labeled anti-VEGFR-2 Ab (1 µg/ml). On the next day, slides were incubated with streptavidin-conjugated Alexa Fluor 546 (1 µg/ml). Slides were mounted with Vectashield without propidium iodide. Images were captured with a Leica SP2 laser scanning confocal microscope. The white arrows indicate the cross-section of endothelial cells expressing CD31 as well as VEGFR-2, lining the lumen of a blood vessel in the corneal stroma. Increases in the number of both CD31 and VEGFR-2-positive cells are associated with heightened angiogenic response following HSV-1 corneal infection (C). Number and percentage of CD31-positive endothelial cells expressing VEGFR-2 in the SK cornea was measured by flow cytometry at various time points.

 
To determine whether VEGFR-2 is mainly expressed in the corneal neovascular cells, the endothelial cells in the SK corneas were identified using anti-CD31 labeled with FITC and additionally stained with biotinylated anti-VEGFR-2 with streptavidin-Alexa Fluor 546 to identify VEGFR-2. Confocal microscopic observation revealed that the endothelial cells of the newly grown blood vessels expressed CD31 and such cells expressed the VEGFR-2 (Fig. 2B). Furthermore, to evaluate whether VEGFR-2 is overexpressed in corneal neovascular endothelial cells after HSV infection, the extracellular expression of VEGFR-2 was measured on CD31-positive endothelial cells isolated from the SK cornea at different time points. As is evident in Fig. 2C, the number of endothelial cells expressing both VEGFR-2 and CD31 was increased after HSV infection and ~53.7% of CD31-positive endothelial cells expressed the VEGFR-2 at day 21 postinfection (p.i.). Such data support the idea that VEGFR-2 is overexpressed in the proliferating endothelial cells of the HSV-infected cornea.

A VEGFR-2-encoded DNA immunization diminishes the severity of SK and HSV-1-induced angiogenesis

Because VEGFR-2 is overexpressed in the SK cornea, inhibition of VEGFR-2 signaling may result in diminished angiogenesis and SK severity. To evaluate this idea, a DNA oral vaccine encoding mVEGFR-2 carried by attenuated S. typhimurium was developed as described elsewhere (30). To determine its efficacy and to establish whether orally administered Salmonella efficiently infected and transferred the target gene into Peyer’s patches, mice were orally administered with 108 CFU of S. typhimurium encoding GFP DNA, and Peyer’s patches were sampled and cryosectioned. As shown in Fig. 3, GFP gene transfer from attenuated S. typhimurium into Peyer’s patches by GFP expression at 16 h after oral administration was demonstrated by confocal microscopic observation.


Figure 3
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  		FIGURE 3. Gene transfer from attenuated S. typhimurium to Peyer’s patches. Gene transfer from attenuated S. typhimurium into Peyer’s patches by GFP expression at 16 h after oral administration was demonstrated by confocal microscopic observation. The white arrows indicate the cells expressing GFP transferred from attenuated S. typhimurium-based GFP DNA.

 
To evaluate whether immunization with recombinant S. typhimurium encoding VEGFR-2 inhibits the development of SK, mice were given orally 108 CFU of S. typhimurium encoding VEGFR-2 or GFP as a control three times over a 4-wk period, at 2-wk intervals. One week after the final immunization, the corneas of mice were scratched and infected with 5 x 105 HSV-1 RE. The levels of SK severity and neovascularization were measured in the corneas by biomicroscopy in groups of mice either prevaccinated with pcDNA3.1-VEGFR-2 or control GFP plasmid before HSV infection. As shown in Fig. 4, significantly decreased average SK severity and percentage of animals with clinical lesions (66% of VEGFR-2 group compared with 91% of control group) was observed in mice following immunization with the VEGFR-2-encoded DNA. A comparison of average severity scores after day 4 p.i. were all significantly less in the VEGFR-2 DNA immunized than the control group (p < 0.05). Furthermore, the results shown in Fig. 4 indicate that levels of angiogenesis were significantly less at most time points tested in the S. typhimurium encoding VEGFR-2 DNA compared with control plasmid-immunized animals (p < 0.01).


Figure 4
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  		FIGURE 4. Reduction of SK lesion severity, incidence, and HSV-induced angiogenesis by VEGFR-2 DNA immunization. Groups of animals (n = 4) were orally administered with 108 CFU of attenuated S. typhimurium three times over a 4-wk period, at 2-wk intervals. One week after final immunization, the corneas were scratched and infected with 5 x 105 HSV-1 RE. The levels of SK severity and neovascularization were measured in the corneas by biomicroscopy in groups of mice either prevaccinated with pcDNA3.1-VEGFR-2 ({circ}) or control GFP (•) plasmid before HSV infection. The data are compiled from three independent experiments. Statistically significant differences in SK and angiogenic score were observed between the groups (*, p < 0.05; **, p < 0.01). Images were taken by stereomicroscopic imaging system at day 21 after virus infection (original magnification, x40).

 
CD8+ T cells are responsible for the antiangiogenic effects in SK cornea

It has been reported that immunization with recombinant Salmonella encoding Ags induces cytotoxic CD8+ T cell response against the recombinant protein (30, 36, 39, 40). It was suspected that the induction of CD8+ T cell immunity against the VEGFR-2 protein was the mechanism by which the immunization with recombinant Salmonella encoding VEGFR-2 controlled HSV-induced angiogenesis and SK lesion severity. Initially, we collected corneas from recombinant S. typhimurium encoding VEGFR-2- and GFP-immunized mice that were subsequently HSV infected to look for the presence of CD8+ T cells by confocal microscopy. In such studies, endothelial cells were identified using CD31 as a marker. In sample analyzed 10 and 21 days after infection, CD8+ T cells could only be observed in the corneas of recombinant S. typhimurium encoding VEGFR-2-immunized animals (Fig. 5). However, in both S. typhimurium encoding VEGFR-2 and GFP-immunized mice CD4+ T cells were present in SK corneas (SK day 10). Interestingly, the CD8+ T cells were mainly located with neovessels in the corneal stroma of mice immunized against VEGFR-2 and infected with HSV and almost no CD8+ T cells were observed in the corneas of control mice. These results indicate that immunization against VEGFR-2-induced infiltration of CD8+ T cells into angiogenic area and that these CD8+ T cells were likely responsible for the antiangiogenic effects on HSV-1-induced angiogenesis.


Figure 5
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  		FIGURE 5. Involvement of CD8+ T cells for the antiangiogenic effects in SK cornea. Upper panels, The localization of CD8+ T cells (FITC, green) and endothelial cells (biotin + Alexa, red) in the corneal stroma (SK day 21) of mice immunized and infected with HSV-1. Lower panels, The infiltration of CD8+ (FITC, green) and CD4+ (biotin + Alexa, red) T cells in the SK (day 10) cornea of mice immunized and infected with HSV-1. The white arrows indicate the infiltrated CD8+ T cells in the corneal stroma.

 
Activation of CD8+ T cells after coincubation with endothelial cells expressing VEGFR-2

To assess whether CD8+ T cells of mice immunized against VEGFR-2 were activated by VEGFR-2 stimulation, the expression of T cell activation markers was compared between the groups. As shown in Fig. 6A, expression of CD69 and CD25 on CD8+ T cells of splenocytes from mice immunized against VEGFR-2 was increased after a 24-h coincubation with endothelial cell lines (HEV), which constitutively express VEGFR-2. Coincubation with HEV cells for 3 days resulted in increased population of CD8+ T cells among total splenocytes from mice immunized against VEGFR-2 (Fig. 6B). In addition, increased IFN-{gamma} secretion was noted in the coculture supernatants from the VEGFR-2-immunized group (Fig. 6C). These results indicate that VEGFR-2 restimulation activated CD8+ T cells of splenocytes from mice immunized against VEGFR-2, but not from control mice.


Figure 6
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  		FIGURE 6. Activation of CD8+ T cells by restimulation with VEGFR-2. Splenocytes from VEGFR-2 or GFP DNA-immunized mice were collected and coincubated with cells expressing VEGFR-2. Shown is the percentage of CD8+CD69+ and CD8+CD25+ T cells (A) or population of CD8+ T cells (B) isolated from splenocytes of mice immunized with VEGFR-2 or GFP after coincubation with cells expressing VEGFR-2. Levels of IFN-{gamma} in the culture supernatants from both groups were measured by ELISA (C). Statistically significant differences were observed between the groups (*, p < 0.05).

 
Induction of T cell-mediated lysis by immunization against VEGFR-2

To test for VEGFR-2-specific cytotoxicity, endothelial cell lines (HEV), which constitutively express VEGFR-2, were labeled with 51Cr- and 4-h release assay performed using splenocytes from mice immunized against VEGFR-2 or mice immunized and infected with HSV. As shown in Fig. 7A, immunization with the vector encoding VEGFR-2 resulted in a higher level (29.5%, 100:1 E:T) of CTL responses against the endothelial cells expressing VEGFR-2, compared with control immunization (9.7%, 100:1 E:T). However, neither immunization was effective in evoking cytotoxicity against CT-26 cells not expressing VEGFR-2, indicating VEGFR-2-specific lysis by VEGFR-2 immunization (Fig. 7B). In addition, neither vaccination induced cytotoxicity against YAC cells, which are specific cell lines for NK cell cytotoxicity, excluding direct lysis by NK cells (Fig. 7C).


Figure 7
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  		FIGURE 7. Specific lysis of endothelial cells by CD8+ T cells. Splenocytes from VEGFR-2 or GFP DNA-immunized mice were collected and coincubated with irradiated HEV (A), CT-26 (B), or YAC (C) cells. After 5 days coincubation, the activated T cells were harvested and tested for cytotoxicity.

 
Reduction of VEGF-induced corneal neovascularization by VEGFR-2 immunization

To test the antiangiogenic effects of immunization against VEGFR-2 in the eye, the corneas of immunized mice were implanted with 200 ng of rVEGF pellets and the angiogenic area was measured at different time points. Immunization with VEGFR-2 DNA markedly delayed (~50% reduction) the extent of VEGF-induced angiogenesis compared with control DNA-immunized animals at days 4 and 7 after pellet implantation (Fig. 8).


Figure 8
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  		FIGURE 8. Reduction of VEGF-induced corneal angiogenesis by VEGFR-2 immunization. The cornea of mice (n = 4) immunized with either attenuated S. typhimurium harboring pcDNA3.1-VEGFR-2 or -GFP was given 200 ng of rVEGF pellets (*) using a corneal micropocket assay method. Furthermore, to demonstrate the involvement of VEGFR-2-specific CD8+ T cells with antiangiogenic response in the eye, CD8+ T cells of immunized mice were depleted before VEGF pellet implantation. The angiogenesis area (A) was measured on days 4 and 7 after the VEGF pellet implantation. Images were taken by stereomicroscopic imaging system at day 4 after VEGF pellet implantation (original magnification, x40) (B).

 
To demonstrate the involvement of VEGFR-2-specific CD8+ T cells in the antiangiogenic response in the eye, CD8+ T cells were depleted before VEGF pellet implantation. As shown in Fig. 8, in vivo depletion of CD8+ T cells resulted in a significant reduction (p < 0.05) of the antiangiogenic response induced by VEGFR-2 immunization. This result demonstrates the antiangiogenic effects of CD8+ T cells targeting endothelial cells overexpressing VEGFR-2 by immunization against VEGFR-2.

Adoptive transfer of VEGFR-2-specific CD8+ T cells efficiently delays angiogenesis

To further establish the role of VEGFR-2-specific CD8+ T cells in suppressing corneal angiogenesis, an adoptive transfer system was used. Mice were infected ocularly with HSV-1 or implanted with VEGF pellets into the cornea to induce angiogenesis. At day 1 before and after infection or implantation, CD8+ T cells were isolated from VEGFR-2-based DNA immunized Thy 1.1+ C57BL/6.PL (H-2b) mice and transferred into Thy 1.2 recipients. The results expressed in Fig. 9A indicate that the severity of lesions and extent of angiogenesis induced by HSV infection or VEGF pellets implantation were low if animals received CD8+ T cells of VEGFR-2-based DNA-immunized mice. To demonstrate the infiltration of adoptive-transferred Thy 1.1+CD8+ T cells in the recipient SK cornea, liberase-digested SK cornea cells were stained against Thy 1.1 and CD8+ T cells at day 14 p.i. Flow cytometry analysis revealed an increase in the number of Thy 1.1+CD8+ T cells per cornea of mice adoptively transferred with Thy 1.1+CD8+ T cells of VEGFR-2-based DNA-immunized mice than the control eye (Fig. 9B).


Figure 9
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  		FIGURE 9. Delay of angiogenesis by adoptive transfer of VEGFR-2-specific CD8+ T cells. Mice (n = 6) were infected ocularly with HSV-1 RE or implanted with VEGF (200 ng) pellets into the cornea to induce angiogenesis. CD8+ T cells were isolated from VEGFR-2-based DNA-immunized Thy 1.1+ C57BL/6.PL (H-2b) mice (n = 4) and transferred into Thy 1.2 recipients at day 1 before and after infection or implantation. The levels of SK severity and angiogenic area were measured in the corneas by biomicroscopy in groups of mice that received CD8+ T cells of VEGFR-2- or GFP-based DNA-immunized mice (A). Statistically significant differences in SK and angiogenic scores were observed between the groups (*, p < 0.05; **, p < 0.01). Four corneas of mice of each group were pooled together and the number and percentage of Thy 1.1+CD8+ T cells was enumerated by FACS analysis as described in Materials and Methods (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Our study shows that oral immunization with recombinant S. typhimurium that contains a plasmid encoding the gene for the VEGFR-2 was inhibitory to ocular neovascularization caused by HSV infection or implantation of VEGF into the cornea. It also delayed the development of HSV-induced chronic SK lesions. Our results confirm observations made previously in a tumor angiogenesis system (30). The antiangiogenic effect induced appeared to be mediated by a CD8+ T cell response against vascular endothelial cells. Thus, such cells could be demonstrated to be present only in the corneas of recombinant S. typhimurium encoding VEGFR-2-immunized mice after HSV infection and not in infected eyes of mice immunized with S. typhimurium encoding GFP. Moreover, cells with VEGFR-2-specific cytotoxic activity could be demonstrated in the lymphoid tissue of VEGFR-2 but not in GFP-immunized mice. Finally, CD8+ T cell depletion after immunization significantly diminished the antiangiogenic effect. We interpret these observations to indicate that immunization against VEGFR-2 efficiently induces immune responses against activated endothelial cells that overexpress VEGFR-2 and negatively regulates HSV-induced angiogenesis resulting in delayed SK lesion development.

Previous studies showed that the VEGF family of proteins are major factors involved in HSV-1-induced angiogenesis, but not the only ones involved in neovascularization (16, 20, 21). Thus, merely inhibiting VEGF, as has been done previously with neutralizing Ab or siRNA-based technology, is not expected to fully curtail angiogenesis (16, 18). In the present study, where we immunized against the main receptors for VEGF involved in pathological angiogenesis, we did achieve significant, but not complete, antiangiogenesis. Other approaches to targeting vascular endothelial cells to control SK have also been explored, but the effect was less effective than the present procedure and was only transitory (17). The present approach, which is novel for ocular angiogenesis, should be effective for long periods, a status not achieved by any gene-silencing type effect or with the use of passive Abs against angiogenic factors.

The current antiangiogenic approach shows effective control of newly proliferating vascular endothelial cells, but this strategy may be less effective for the therapeutic control of already established pathological neovessels, because overexpression of VEGFR-2 is mainly found on newly proliferating endothelial cells. However, some efficacy of targeting VEGFR-2 has been shown in some tumor models to reverse neovascularization in established metastases (30). However, therapeutic antiangiogenesis has not proven effective when tested previously in the animals with established SK (17) and in the present study was not formally tested. In fact it is probable that multiple targeting VEGFR-2 combined with other anti-inflammatory or antiangiogenic therapies may be necessary to control established SK and the angiogenic events as we are currently evaluating.

Various immunotherapeutic strategies targeting VEGFR-2 of uncontrolled endothelial cells have been suggested by investigators mainly for the purpose of inhibition of tumor progression and angiogenesis (30, 31). Surprisingly, although VEGFR-2 is an autologous protein, our vaccination strategy, as well as those that have been previously introduced, could efficiently break peripheral T cell tolerance against self-Ag, VEGFR-2 (30, 33). The precise mechanism by which oral DNA vaccination targeting self-Ag to induce a potent and sustained immune response remains unresolved and this issue is under further investigation. Recently, several research groups identified the mouse H-2Db-restricted epitope peptides (41, 42) as well as the human epitope peptides from VEGFR-2 (43), specifically targeted by CTLs. Additionally, one of those groups observed prolonged antiangiogenic effects of immunization for up to 10 mo after tumor challenge. Similarly, it is likely that the memory VEGFR-2-specific CD8+ T cells induced by this immunization strategy can be used in controlling angiogenesis of recurrent human herpetic SK.

Induction of CTL responses against VEGFR-2 by oral immunization demonstrates that VEGFR-2-specific CD8+ T cells might directly kill or control the proliferating endothelial cells, leading to reduced angiogenesis and, subsequently, delayed development of SK lesions. We further showed that angiogenesis induced by VEGF pellet implantation was reduced in the immunized mice. To demonstrate the involvement of CD8+ T cells in antiangiogenic immune response, neutralizing Ab to deplete CD8+ T cells was used. As was expected, depletion of CD8+ T cells abrogated the antiangiogenic activity in the immunized and VEGF pellet-implanted mice. Our data again support the idea that VEGFR-2-specific CD8+ T cells demonstrate an antiangiogenic function and indirectly show this function in the SK model.

In summary, we demonstrated that oral immunization with recombinant S. typhimurium harboring mVEGFR-2 could break peripheral T cell tolerance and induce immune response against self-Ag, VEGFR-2. This VEGFR-2-specific immune response resulted in reduction of herpetic SK lesion severity by its antiangiogenic effects. This novel approach might also prove useful to help control the severity of recurrent SK as is typically the pattern of events in the natural human disease.


   Acknowledgments
 
We thank Dr. John R. Dunlap for his expertise on confocal microscopy and Carrie Dolman for technical assistance. We also thank Dr. Udayasankar Kumaraguru and Christopher D. Pack for advice and Dr. Shilpa Deshpande Kaistha for editorial assistance with manuscript preparation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grant RO1 EY05093 (to B.T.R.) and CA 83856 (to R.A.R.). Back

2 Address correspondence and reprint requests to Dr. Barry T. Rouse, University of Tennessee, M409 Walters Life Sciences Building, Knoxville, TN 37996-0845. E-mail address: btr{at}utk.edu Back

3 Abbreviations used in this paper: SK, stromal keratitis; VEGF, vascular endothelial cell growth factor; HEV, high endothelial venule; m, mouse; p.i., postinfection. Back

Received for publication November 22, 2005. Accepted for publication June 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Biswas, P. S., B. T. Rouse. 2005. Early events in HSV keratitis—setting the stage for a blinding disease. Microbes Infect. 7: 799-810. [Medline]
  2. Decrion, A. Z., I. Dichamp, A. Varin, G. Herbein. 2005. HIV and inflammation. Curr. HIV Res. 3: 243-259. [Medline]
  3. Gangappa, S., J. S. Babu, J. Thomas, M. Daheshia, B. T. Rouse. 1998. Virus-induced immunoinflammatory lesions in the absence of viral antigen recognition. J. Immunol. 161: 4289-4300. [Abstract/Free Full Text]
  4. Deshpande, S. P., M. Zheng, S. Lee, B. T. Rouse. 2002. Mechanisms of pathogenesis in herpetic immunoinflammatory ocular lesions. Vet. Microbiol. 86: 17-26. [Medline]
  5. Gangappa, S., S. P. Deshpande, B. T. Rouse. 1999. Bystander activation of CD4+ T cells can represent an exclusive means of immunopathology in a virus infection. Eur. J. Immunol. 29: 3674-3682. [Medline]
  6. Piedimonte, G.. 2003. Contribution of neuroimmune mechanisms to airway inflammation and remodeling during and after respiratory syncytial virus infection. Pediatr. Infect. Dis. J. 22: S66-S74. [Medline]
  7. Auais, A., B. Adkins, G. Napchan, G. Piedimonte. 2003. Immunomodulatory effects of sensory nerves during respiratory syncytial virus infection in rats. Am. J. Physiol. 285: L105-L113.
  8. Garweg, J. G., M. Halberstadt. 2002. [The pathogenesis of herpetic keratitis]. Klin. Monatsbl. Augenheilkd. 219: 477-486. [Medline]
  9. Deshpande, S. P., S. Lee, M. Zheng, B. Song, D. Knipe, J. A. Kapp, B. T. Rouse. 2001. Herpes simplex virus-induced keratitis: evaluation of the role of molecular mimicry in lesion pathogenesis. J. Virol. 75: 3077-3088. [Abstract/Free Full Text]
  10. Thomas, J., B. T. Rouse. 1997. Immunopathogenesis of herpetic ocular disease. Immunol. Res. 16: 375-386. [Medline]
  11. Gangappa, S., S. P. Deshpande, B. T. Rouse. 2000. Bystander activation of CD4+ T cells accounts for herpetic ocular lesions. Invest. Ophthalmol. Vis. Sci. 41: 453-459. [Abstract/Free Full Text]
  12. Thomas, J., S. Gangappa, S. Kanangat, B. T. Rouse. 1997. On the essential involvement of neutrophils in the immunopathologic disease: herpetic stromal keratitis. J. Immunol. 158: 1383-1391. [Abstract]
  13. Bauer, D., A. Schmitz, N. Van Rooijen, K. P. Steuhl, A. Heiligenhaus. 2002. Conjunctival macrophage-mediated influence of the local and systemic immune response after corneal herpes simplex virus-1 infection. Immunology 107: 118-128. [Medline]
  14. Bauer, D., S. Mrzyk, N. Van Rooijen, K. P. Steuhl, A. Heiligenhaus. 2000. Macrophage-depletion influences the course of murine HSV-1 keratitis. Curr. Eye Res. 20: 45-53. [Medline]
  15. Bauer, D., S. Mrzyk, N. Van Rooijen, K. P. Steuhl, A. Heiligenhaus. 2001. Incidence and severity of herpetic stromal keratitis: impaired by the depletion of lymph node macrophages. Exp. Eye Res. 72: 261-269. [Medline]
  16. Zheng, M., S. Deshpande, S. Lee, N. Ferrara, B. T. Rouse. 2001. Contribution of vascular endothelial growth factor in the neovascularization process during the pathogenesis of herpetic stromal keratitis. J. Virol. 75: 9828-9835. [Abstract/Free Full Text]
  17. Zheng, M., M. A. Schwarz, S. Lee, U. Kumaraguru, B. T. Rouse. 2001. Control of stromal keratitis by inhibition of neovascularization. Am. J. Pathol. 159: 1021-1029. [Abstract/Free Full Text]
  18. Kim, B., Q. Tang, P. S. Biswas, J. Xu, R. M. Schiffelers, F. Y. Xie, A. M. Ansari, P. V. Scaria, M. C. Woodle, P. Lu, B. T. Rouse. 2004. Inhibition of ocular angiogenesis by siRNA targeting vascular endothelial growth factor pathway genes: therapeutic strategy for herpetic stromal keratitis. Am. J. Pathol. 165: 2177-2185. [Abstract/Free Full Text]
  19. Deshpande, S., K. Banerjee, P. S. Biswas, B. T. Rouse. 2004. Herpetic eye disease: immunopathogenesis and therapeutic measures. Expert Rev. Mol. Med. 2004: 1-14. [Medline]
  20. Lee, S., M. Zheng, B. Kim, B. T. Rouse. 2002. Role of matrix metalloproteinase-9 in angiogenesis caused by ocular infection with herpes simplex virus. J. Clin. Invest. 110: 1105-1111. [Medline]
  21. Banerjee, K., P. S. Biswas, B. Kim, S. Lee, B. T. Rouse. 2004. CXCR2–/– mice show enhanced susceptibility to herpetic stromal keratitis: a role for IL-6-induced neovascularization. J. Immunol. 172: 1237-1245. [Abstract/Free Full Text]
  22. Biswas, P. S., K. Banerjee, B. Kim, B. T. Rouse. 2004. Mice transgenic for IL-1 receptor antagonist protein are resistant to herpetic stromal keratitis: possible role for IL-1 in herpetic stromal keratitis pathogenesis. J. Immunol. 172: 3736-3744. [Abstract/Free Full Text]
  23. Ferrara, N.. 2004. Vascular endothelial growth factor as a target for anticancer therapy. Oncologist 9: (Suppl. 1):2-10. [Abstract/Free Full Text]
  24. Stewart, R. J., D. Panigrahy, E. Flynn, J. Folkman. 2001. Vascular endothelial growth factor expression and tumor angiogenesis are regulated by androgens in hormone responsive human prostate carcinoma: evidence for androgen dependent destabilization of vascular endothelial growth factor transcripts. J. Urol. 165: 688-693. [Medline]
  25. Zheng, M., D. M. Klinman, M. Gierynska, B. T. Rouse. 2002. DNA containing CpG motifs induces angiogenesis. Proc. Natl. Acad. Sci. USA 99: 8944-8949. [Abstract/Free Full Text]
  26. Biswas, P. S., K. Banerjee, M. Zheng, B. T. Rouse. 2004. Counteracting corneal immunoinflammatory lesion with interleukin-1 receptor antagonist protein. J. Leukocyte Biol. 76: 868-875. [Abstract/Free Full Text]
  27. Kim, B., S. Lee, S. Suvas, B. T. Rouse. 2005. Application of plasmid DNA encoding IL-18 diminishes development of herpetic stromal keratitis by antiangiogenic effects. J. Immunol. 175: 509-516. [Abstract/Free Full Text]
  28. Lee, S., M. Zheng, S. Deshpande, S. K. Eo, T. A. Hamilton, B. T. Rouse. 2002. IL-12 suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis. J. Leukocyte Biol. 71: 469-476. [Abstract/Free Full Text]
  29. Ferrara, N., H. P. Gerber, J. LeCouter. 2003. The biology of VEGF and its receptors. Nat. Med. 9: 669-676. [Medline]
  30. Niethammer, A. G., R. Xiang, J. C. Becker, H. Wodrich, U. Pertl, G. Karsten, B. P. Eliceiri, R. A. Reisfeld. 2002. A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat. Med. 8: 1369-1375. [Medline]
  31. Li, Y., M. N. Wang, H. Li, K. D. King, R. Bassi, H. Sun, A. Santiago, A. T. Hooper, P. Bohlen, D. J. Hicklin. 2002. Active immunization against the vascular endothelial growth factor receptor flk1 inhibits tumor angiogenesis and metastasis. J. Exp. Med. 195: 1575-1584. [Abstract/Free Full Text]
  32. Liu, J. Y., Y. Q. Wei, L. Yang, X. Zhao, L. Tian, J. M. Hou, T. Niu, F. Liu, Y. Jiang, B. Hu, et al 2003. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood 102: 1815-1823. [Abstract/Free Full Text]
  33. Nair, S., D. Boczkowski, B. Moeller, M. Dewhirst, J. Vieweg, E. Gilboa. 2003. Synergy between tumor immunotherapy and antiangiogenic therapy. Blood 102: 964-971. [Abstract/Free Full Text]
  34. Wei, Y. Q., Q. R. Wang, X. Zhao, L. Yang, L. Tian, Y. Lu, B. Kang, C. J. Lu, M. J. Huang, Y. Y. Lou, et al 2000. Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Nat. Med. 6: 1160-1166. [Medline]
  35. Song, W., Q. Sun, Z. Dong, D. M. Spencer, G. Nunez, J. E. Nor. 2005. Antiangiogenic gene therapy: disruption of neovascular networks mediated by inducible caspase-9 delivered with a transcriptionally targeted adenoviral vector. Gene Ther. 12: 320-329. [Medline]
  36. Niethammer, A. G., F. J. Primus, R. Xiang, C. S. Dolman, J. M. Ruehlmann, Y. Ba, S. D. Gillies, R. A. Reisfeld. 2001. An oral DNA vaccine against human carcinoembryonic antigen (CEA) prevents growth and dissemination of Lewis lung carcinoma in CEA transgenic mice. Vaccine 20: 421-429. [Medline]
  37. Niethammer, A. G., R. Xiang, J. M. Ruehlmann, H. N. Lode, C. S. Dolman, S. D. Gillies, R. A. Reisfeld. 2001. Targeted interleukin 2 therapy enhances protective immunity induced by an autologous oral DNA vaccine against murine melanoma. Cancer Res. 61: 6178-6184. [Abstract/Free Full Text]
  38. O’Reilly, M. S.. 2002. Vessel maneuvers: vaccine targets tumor vasculature. Nat. Med. 8: 1352-1353. [Medline]
  39. Feng, K. K., H. Y. Zhao, H. Qiu, J. X. Liu, J. Chen. 2005. Combined therapy with flk1-based DNA vaccine and interleukin-12 results in enhanced antiangiogenic and antitumor effects. Cancer Lett. 221: 41-47. [Medline]
  40. Feng, K., H. Zhao, J. Chen, D. Yao, X. Jiang, W. Zhou. 2004. Anti-angiogenesis effect on glioma of attenuated Salmonella typhimurium vaccine strain with flk-1 gene. J. Huazhong Univ. Sci. Technolog. Med. Sci. 24: 389-391. [Medline]
  41. Zhou, H., Y. Luo, M. Mizutani, N. Mizutani, R. A. Reisfeld, R. Xiang. 2005. T cell-mediated suppression of angiogenesis results in tumor protective immunity. Blood 106: 2026-2032. [Abstract/Free Full Text]
  42. Dong, Y., J. Qian, R. Ibrahim, J. A. Berzofsky, S. N. Khleif. 2006. Identification of H-2Db-specific CD8+ T-cell epitopes from mouse VEGFR2 that can inhibit angiogenesis and tumor growth. J. Immunother. 29: 32-40.
  43. Wada, S., T. Tsunoda, T. Baba, F. J. Primus, H. Kuwano, M. Shibuya, H. Tahara. 2005. Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2. Cancer Res. 65: 4939-4946. [Abstract/Free Full Text]



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