Vaccination Against VEGFR2 Attenuates Initiation and Progression of Atherosclerosis
Objective— Vascular endothelial growth factor receptor 2 (VEGFR2)–overexpressing cells may form an interesting target for the treatment of atherosclerosis because of their involvement in processes that contribute to this disease, such as angiogenesis.
Methods and Results— We vaccinated mice against VEGFR2 by an orally administered DNA vaccine, comprising a plasmid, encoding murine VEGFR2, carried by live attenuated Salmonella typhimurium. This vaccine induces cellular immunity against cells that overexpress VEGFR2. Vaccination of hypercholesterolemic mice against VEGFR2 resulted in a marked induction of CD8+ cytotoxic T cells specific for VEGFR2 and led to an inhibition of angiogenesis in a hindlimb ischemia model. Interestingly, VEGFR2 vaccination attenuated the progression of preexisting advanced atherosclerotic lesions in the brachiocephalic artery of apoE−/− mice. Furthermore, VEGFR2 vaccination strongly reduced the initiation of collar-induced atherosclerosis in the carotid arteries of LDLr−/− mice. In addition, denudation of the carotid artery, as a model for postinterventional lesion formation, resulted in delayed endothelial replacement and significantly increased neointima formation on VEGFR2 vaccination.
Conclusions— These data indicate the prominent role of VEGFR2+ cells in cardiovascular diseases and show that induction of cellular immunity against atherosclerosis-associated cells by means of DNA vaccination may contribute to the development of novel therapies against atherosclerosis.
Atherosclerosis is a multifactorial disease, which is regulated by a variety of inflammatory mechanisms.1 Many cell types contribute to the initiation and progression of the atherosclerotic plaque. The process of atherosclerosis is initiated by the activation of endothelial cells on exposure to various stimuli, such as high levels of atherogenic lipoproteins or shear stress, leading to enhanced migration of leukocytes from the circulation into the intima. The accumulation and activation of macrophages with subsequent foam cell formation after uptake of atherogenic lipoproteins within the intima are key processes that induce the formation of an atherosclerotic plaque. Changes in proliferation, migration, and activation of smooth muscle cells (SMCs) also regulate the process of atherosclerosis. More recently identified inflammatory regulators in atherosclerosis are T cells, B cells, dendritic cells, and NKT cells.1
In the present study, we focused on the role of a specific cell population in atherosclerosis, that highly expresses vascular endothelial growth factor receptor 2 (VEGFR2), and we aimed to diminish the amount of these cells and study the effect thereof on atherosclerosis. Highly VEGFR2 expressing cells comprise proliferating endothelial cells and (endothelial) progenitor cells,2 which are involved in angiogenesis,3–5 a process that potentially contributes to the initiation and progression of atherosclerotic plaques.6,7 Furthermore, VEGFR2 is highly expressed on the activated endothelial cell layer that covers the atherosclerotic plaque, whereas the healthy vascular wall shows much lower expression levels of VEGFR2 on the luminal endothelial cells.8 In addition, some studies indicate that VEGFR2+ progenitor cells or endothelial cells can differentiate into nonendothelial cells, such as SMCs that may contribute to atherosclerosis.9,10 Moreover, VEGF has been demonstrated to promote the progression of atherosclerosis and lead to increased macrophage content in the atherosclerotic plaque, indicating that interference in the interaction of VEGF with its main receptor, VEGFR2, may result in reduced atherogenesis.11 These proatherogenic effects are substantiated by the fact that binding of VEGF to VEGFR2 induces inflammatory responses in endothelial cells by NF-κB activation, leading to elevated expression levels of vascular endothelial cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and endothelial selectin (E-selectin) in endothelial cells resulting in increased adherence of monocytes.12
In the present study, we targeted cells that overexpress VEGFR2 with a novel DNA vaccination technique, which has recently been used to block tumor angiogenesis leading to dramatically reduced tumor growth and prolonged survival of mice that were challenged by several tumor cell lines.13 By oral administration of the live attenuated bacterium, Salmonella typhimurium, containing a VEGFR2-encoding plasmid, we induced a cytotoxic CD8+ T cell response against VEGFR2 overexpressing cells in atherosclerosis prone mice. We show that vaccination against VEGFR2 attenuates both the initiation and progression of atherosclerosis, but leads to enhanced postinterventional neointima formation, which illustrates the important role of VEGFR2 overexpressing cells in stenotic vascular processes. Furthermore, we show for the first time that targeting of specific subpopulations of cells by vaccination may be a valuable strategy in the development of novel therapies against atherosclerosis.
For detailed Methods please see the supplemental materials (available online at http://atvb.ahajournals.org).
Plasmid pcDNA3.1 (Invitrogen) encoding murine VEGFR2 was constructed and electroporated in attenuated S typhimurium Aro/A (strain SL7207) bacteria, as previously described.13
Induction of VEGFR2-Specific Cytotoxic CD8+ T Lymphocytes
The potential of splenocytes, isolated from vaccinated or control apoE−/− mice 3 weeks after the last administration of the vaccine, to lyse VEGFR2-expressing cells was determined in a standard 51Cr release assay.
IFN-γ and VEGF ELISA
Serum IFN-γ levels and serum VEGF levels were determined using the Femto-High Sensitivity IFN-γ ELISA Kit (eBioscience) and a VEGF ELISA kit from Biosource.
Angiogenesis in a Hindlimb Ischemia Model
For evaluation of antiangiogenic effects induced by vaccination against VEGFR2, apoE−/− mice were vaccinated and unilateral hindlimb ischemia was introduced as described.16
Induction of Atherosclerosis
The effect of vaccination against VEGFR2 on early and late stages of atherosclerosis was studied in 2 mouse models for atherosclerosis. ApoE−/− mice and LDLr−/− were acquired from TNO-PG (Leiden, The Netherlands) and were used to study of progression and initiation of atherosclerosis, respectively.15
Induction of Postinterventional Neointima Formation
Female 3-month-old LDLr−/− mice were vaccinated (VEGFR2 vaccinated: n=15, control vaccinated: n=18) as described. Two weeks after the last immunization mice were put on a Western type diet. One week later, mice were anesthetized and endothelial denudation and analysis of the right common carotid artery was performed as described.16
Tissue Harvesting, Histology and Immunohistochemistry
Cryosections of arteries were routinely stained with hematoxylin and eosin. Staining for actin, MOMA2, CD31, VEGFR2, collagen, and IFN-γ was performed as described. Quantification of atherosclerosis and restenosis was performed as described.
Values are expressed as mean±SEM. A 2-tailed Student t test was used to compare individual groups of mice or cells. A level of P<0.05 was considered significant.
Vaccination Induces VEGFR2 Specific CD8+ Cytotoxic T Cells
Three weeks after the third vaccination of apoE−/− mice by oral administration of S typhimurium transformed with pcDNA3.1-VEGFR2 or pcDNA3.1, splenocytes and CD8+ T cells were isolated to determine their capacity to specifically kill VEGFR2-expressing cells in a standard 51Cr release assay. We observed a significant 2.4- to 3.9-fold increase in effector/target ratio dependent lysis of VEGFR2+ 3T3 cells by splenocytes from VEGFR2 vaccinated mice (Figure 1A, P<0.001) as compared with splenocytes from control vaccinated mice. Incubation with CD8+ T cells, isolated from VEGFR2 vaccinated mice, in a 50:1 effector/target cell ratio resulted in a 3.68-fold increased cytotoxicity (Figure 1B, P<0.001) as compared with incubation with CD8+ T cells from control mice, indicating that the cytotoxic effect of the spleen cells can be attributed to the CD8+ cells. A significant decrease in the number of VEGFR2+/GFP+ cells was seen on exposure to splenocytes from VEGFR2 vaccinated mice, whereas splenocytes from control mice did not affect the survival (Figure 1C through 1E). Vaccination against VEGFR2 did not affect anti-VEGFR2 IgG antibodies in serum (data not shown).
Antiangiogenic Effect of Vaccination Against VEGFR2
Next we determined the effect of lysis of VEGFR2 overexpressing cells by VEGFR2 vaccination on angiogenesis. The left femoral artery of apoE−/− mice was coagulated unilaterally 1 week after the third vaccination to induce ischemia. Two weeks later ischemia-induced angiogenesis was quantified by measuring the CD31-positive area. Vaccination against VEGFR2 resulted in a 2.0-fold decrease in CD31-positive area as compared with control vaccination (Figure 2A through 2C; 3422±581 versus 1743±168 μm2, P<0.01), whereas serum levels of VEGF were not affected after vaccination (Figure 2D; 51.2±6.4 versus 49.2±6.0 ng/mL), indicating that the antiangiogenic effect is VEGF-independent.
Effect of Vaccination Against VEGFR2 on Atherosclerosis
The effect of vaccination against VEGFR2 on advanced lesions was determined using apoE−/− mice (22 weeks of age), which had developed atherosclerotic lesions on chow diet. A group of control-vaccinated mice was euthanized directly after the last vaccination and lesion formation was quantified in the brachiocephalic artery and compared with lesion formation 12 weeks after vaccination against VEGFR2 or control vaccination. Vaccination against VEGFR2 significantly reduced the plaque size 12 weeks after vaccination by 25% compared with control vaccinated mice (Figure 3A through 3C; 283 100±15 120 versus 211 767±21 433 μm2, P<0.05), whereas cholesterol and triglyceride levels remained unchanged in all groups. However, when the initial level of atherosclerosis at the time of the last vaccination is taken into account (Figure 3, gray line [176 098 μm2]), it is clear that the progression of atherosclerosis was dramatically reduced by 66% on VEGFR2 vaccination.
Collar-induced atherosclerosis in LDLr−/− mice fed a Western type diet was used to determine the effect of vaccination on the initiation of atherosclerosis. Vaccination against VEGFR2 reduced the initiation of atherosclerosis by 77% (Figure 4D, P<0.05), which translated into a marked 2.7-fold reduction in intima/media ratio (Figure 4E, P<0.01) and a 3.0-fold decreased intima/lumen ratio (Figure 4F, P<0.01). In addition VEGFR2 vaccination significantly diminished media area, whereas we did not observe significant differences in macrophage, SMC, collagen, or IFN-γ content (Figure 5G through 5H) or serum IFN-γ levels (supplemental Results and supplemental Figure I).
VEGFR2+ Cells and Neovessels in Atherosclerosis
The presence of neovessels in atherosclerosis-affected arteries sections was assessed by CD31 staining. Apart from endothelial cells covering the atherosclerotic plaque (Figure 5A), CD31+ cells were not found within the neointima, but CD31+ vessels were found in the media and adventitia underlying the atherosclerotic lesion (Figure 5B) and were more abundant as compared with arteries in which atherosclerosis was not induced. Many of the latter microvessels were positive for VEGFR2 (Figure 5C and 5D), whereas VEGFR2+ cells were frequently identified in the adventitia without association with microvessels (Figure 5E). Apart from occasional VEGFR2+ endothelial cells covering the atherosclerotic lesion, no VEGFR2+ cells were found within the atherosclerotic plaques of carotid or brachiocephalic arteries (Figure 5F).
Effect of VEGFR2 Vaccination on Postinterventional Neointima Formation
The endothelial layer of carotid arteries was denudated in LDLr−/− mice and vascular wall or neointima formation was analyzed 1 and 5 weeks later to evaluate the effect of vaccination against VEGFR2 on postinterventional neointima formation. One week after denudation the degree of endothelial replacement was quantified by counting the luminal flat-shaped hematoxylin stained nuclei of CD31-positive–stained cells at the site of maximal media area (supplemental Figure II) and a 31% reduction in endothelial cells after vaccination against VEGFR2 was observed (43.8±4.3 versus 30.2±3.3, P<0.05; supplemental Figure IIC). Furthermore, vaccination against VEGFR2 resulted in a 1.4-fold increase in media area 1 week after denudation (34 026±1020 versus 47 657±4690 μm2, P<0.05; supplemental Figure IID). Five weeks after denudation neointimal lesions were stained for SMCs and showed a typical restenotic phenotype in which SMCs form the main cellular component and a significant 2.7-fold increase in lesion size on vaccination against VEGFR2 (16 028±3968 versus 43 591±8943 μm2, P<0.01; supplemental Figure III).
Despite the use of lipid lowering, antiinflammatory, or plaque stabilizing treatments, atherosclerosis remains the major cause of cardiovascular disease, which encourages the development of alternative strategies to reduce atherosclerosis. Blockade of the recruitment, the removal of cells that contribute to atherosclerosis or the administration of antiatherosclerotic cell types, may contribute to a reduction in lesion formation.17–19
In the present study, we aimed to reduce atherosclerosis by targeting those cells that overexpress VEGFR2 by a novel DNA vaccination technique. Cell-mediated immunity against VEGFR2-overexpressing cells was induced in atherosclerosis prone mice by oral administration of S typhimurium carrying a plasmid encoding mouse VEGFR2.13
We confirmed that splenocytes and specifically CD8+ T cells, from VEGFR2 vaccinated mice specifically lyse cells overexpressing VEGFR2, whereas control splenocytes and control CD8+ T cells were unable to do so. A humoral response against VEGFR2 was not observed on vaccination, indicating that cellular immunity via the induction of VEGFR2-specific CD8+ T cell cytotoxicity is responsible for the observed effects. In addition, a potential VEGF-dependent mechanism is unlikely to play a role in the antiangiogenic effects of VEGFR2 vaccination because serum levels of VEGF did not change on VEGFR2 vaccination. The fact that vaccination against VEGFR2 overexpressing cells, that normally contribute to angiogenesis,3–5 inhibited hindlimb ischemia induced angiogenesis by 50% shows that the functional role of VEGFR2-overexpressing cells is impaired in vivo on VEGFR2 vaccination. These results are in line with the study of Niethammer et al in which VEGFR2 vaccination was used to block tumor growth by inhibiting tumor-related angiogenesis. In his study CD8+ T cells colocalised with the target site, the tumor vasculature.13
We now show that VEGFR2 vaccination inhibited the progression of preexisting atherosclerotic lesions in apoE−/− mice for 66%. In a second experiment we determined that vaccination against VEGFR2 reduced the initiation of atherosclerosis in LDLr−/− for 77%, which indicated that VEGFR2 vaccination already interfered with a very initial stage of atherosclerosis. We conclude that cells that highly express VEGFR2 are involved in the development of atherosclerosis and conclude that these cells regulate atherosclerosis already in a very early stage. Because several cell types overexpress VEGFR2, the effect of vaccination on atherosclerosis may be exerted by a several mechanisms. Proliferating endothelial cells show a high expression of VEGFR2 and contribute to angiogenesis,3–5 which explains the inhibiting effect on angiogenesis in the hindlimb ischemia by VEGFR2 vaccination. Angiogenesis may contribute to the progression of atherosclerosis,6,7 and blockade of angiogenesis by VEGFR2 vaccination may result in decreased atherosclerosis. Microvessels, indicated by positive CD31 and VEGFR2 staining, were found in adventitia and media, but we did not identify VEGFR2 or CD31+ cells within the neointima, which suggests the lack of intraplaque angiogenesis in the used models for atherosclerosis. This may be explained by the fact that adaptive hypoxia driven neovessel formation only occurs when (tumor) tissue is located >100 μm from the blood capillaries.20 Because in our mouse models for atherosclerosis, neointimal tissue in the brachiocephalic arteries is always within 100 μm of blood capillaries, this may explain the absence of intraplaque angiogenesis. Moulton et al7 show a 9-fold increase in the frequency of neovessels in atherosclerotic lesions, but these lesions were thicker than 250 μm.7 We did, however, observe VEGFR2 positive microvessels within the media at the site of the lesion, which may indicate the initiation of neovascularization of the lesions. These VEGFR2-positive vessels may have served as a target for the CD8+ VEGFR2-specific T cells, and this may diminish the optimal growth of the plaque on VEGFR2 vaccination. This is in line with the suggestion that adventitial angiogenesis contributes to atherosclerotic plaque growth because the degree of adventitial angiogenesis correlates with the extend of neointima formation.21,22 In addition, stimulation of adventitial angiogenesis, by administration of endothelial progenitor cells or adventitial application of VEGF, results in increased neointima formation.23,24
Recently, circulating hematopoietic stem cells were shown to differentiate into vascular cells that participate in atherosclerosis.25,26 Furthermore, progenitor cells, including VEGFR2+ cells, have been identified in the adventitia of murine and human arteries.25,26 These progenitor cells migrate to the intima, where they can differentiate into vascular SMCs and thus promote atherosclerosis. In the present study, we confirmed the presence of these VEGFR2+ cells, which were not associated with neovessels, in the adventitia of atherosclerosis affected arteries. Although their presence in the adventitia does not prove their role in atherosclerosis as progenitor cells in our models for atherosclerosis, this study shows that VEGFR2+ cells are at least associated with atherosclerosis. In addition, VEGFR2+ cells, isolated from embryonic stem cells, have been indicated as vascular progenitor cells that can differentiate into both endothelial and smooth muscle cells.9 A recent study confirms that, although not under atherosclerotic conditions, VEGFR2+ cells are able to differentiate into SMCs in vivo during vascular recovery after denudation.10 Taking together, vaccination against VEGFR2 may have resulted in reduced amounts of progenitor cells that contribute to atherosclerosis.
The fact that VEGFR2 is highly expressed on activated endothelial cells, which cover the atherosclerotic plaque, may indicate that vaccination against VEGFR2 removes these activated cells,8 which then become replaced by “healthy” new endothelial cells. Furthermore, VEGF has been shown to accelerate atherosclerosis in hypercholesterolemic mice and rabbits.11 Although it is still not clear what the relative contribution is of the 2 major receptors for VEGF, namely VEGFR1 or VEGFR2, to atherosclerosis development, immunomodulatory capacities of VEGFR2 support their important role in lesion formation.12 Binding of VEGF to VEGFR2 induces NF-κB activation and elevated expression levels of VCAM-1, ICAM-1, and E-selectin in endothelial cells leading to increased adherence of monocytes12 and overexpression of VEGF-A in atherosclerotic lesions enhances monocyte influx and plaque expansion,27 indicating that interference in the binding of VEGF to VEGFR2 by removing VEGFR2+ cells may lead to reduced atherosclerosis. The fact that we did not observe changes in the circulating levels of proinflammatory cytokine IFN-γ suggests that vaccination against VEGFR2 does not affect atherosclerosis via interference in the systemic inflammatory response. Furthermore, VEGFR2 has recently been identified as part of a shear stress–sensing complex on endothelial cells, which becomes activated during disturbed flow patterns, which is the case at atherosclerosis-prone/affected sites of the vasculature, leading to NF-κB activation.28
Further immunohistochemical analysis of the composition of atherosclerotic plaques in the brachiocephalic and carotid arteries did not reveal any differences in the percentage macrophage, SMC, collagen, or IFN-γ staining, indicating that the inflammatory status and stability of the lesions is not affected by VEGFR2 vaccination. The finding that the neointima did contain macrophages and SMCs but no VEGFR2+ cells indicates that macrophages and SMCs are not the direct target in the vaccination against VEGFR2.
To investigate the effect of vaccination against VEGFR2 on postinterventional neointima formation, a mouse model of the common carotid artery guide wire denudation was used, which shows characteristics of a restenotic process. Shortly after denudation endothelial replacement was significantly reduced in mice that were vaccinated against VEGFR2. This delayed replacement is likely to be caused by the specific attack of proliferating endothelial cells which overexpress VEGFR2 by the CD8+ T cells. Furthermore, reduced levels of circulating endothelial progenitor cells may have contributed to delayed endothelial replacement, which is in line with a recent study in which VEGFR2+ cells in the peripheral blood have been shown to contribute to reendothelialization of balloon injured femoral arteries of mice.10 Already 1 week after denudation vaccination against VEGFR2 led to an increase in media area, and 5 weeks after denudation neointima formation in VEGFR2 vaccinated mice was 2.7-fold increased. Most likely, this increase in postinterventional neointima formation on vaccination against VEGFR2 is caused by the prolonged activation of the media, exposed to the blood stream and adhesive platelets, attributable to the delayed reendothelialization.
Very recently, the group of Curtiss29 elegantly confirmed our initial data, presented at the scientific sessions of the American Heart Association30 concerning the effect of VEGFR2 vaccination on atherosclerosis. Petrovan et al further underline the antiatherosclerotic effect of VEGFR2 vaccination by quantifying the initiation of atherosclerosis at another site in the vasculature, namely the aortic root, and also show that adventitial angiogenesis is reduced.29 Our data extend these studies and we show that also plaque progression is affected by VEGFR2 vaccination. In addition, we evaluated the presence of atherosclerosis-associated VEGFR2+ (progenitor) cells and determined the effect of vaccination on media thickness. The reduced adventitial angiogenesis in the aortic root on vaccination against VEGFR2 nicely extrapolates the previously observed effects on angiogenesis at other sites to the adventitia13,30 and supports the hypothesis that the effect on atherosclerosis may be mediated by interference in adventitial angiogenesis. In addition we extended these studies by showing that vaccination against VEGFR2 negatively affects postinterventional neointima formation presumably by delaying endothelial replacement.
Recently, several vaccination strategies revealed promising effects on atherosclerosis. Vaccination against modified LDL, apoB100, cholesterol ester transfer protein (CETP), and interleukin (IL) 12 resulted in reduced atherosclerosis by inducing antibodies against these factors.31–34 In contrast to the induction of humoral responses by vaccination against soluble proteins such as IL-12, which significantly downregulates atherosclerosis,34 this study shows that vaccination induced cell-mediated immunity against cells that potentially contribute to atherosclerosis results in reduced lesion formation. These results suggest that specific targeting of cells that contribute to atherosclerosis may form a new lead for the development of immunotherapies against atherosclerosis. The relatively low costs and the long duration of the therapeutic efficacy, which was also shown for this vaccination strategy (>10 months after the last administration of the vaccine),13 make vaccination an attractive tool in establishing protection against atherosclerosis. The only known adverse effect of vaccination against VEGFR2 comprises a slightly but significantly delayed wound healing (13.3 versus 14.7 days closure time of wounds of 3 mm in diameter). On the other hand, body weight, fertility, neuromuscular performance, and hematopoiesis are not affected by vaccination against VEGFR2.13 However, because vaccination against VEGFR2 not only results in attenuated atherosclerosis but at the same time increases postinterventional neointima formation, vaccination against VEGFR2 overexpressing cells may only be suitable for a limited patient population that does not qualify for transluminal angioplasty.
In summary, our study demonstrates that vaccination against VEGFR2 induces cell-mediated immunity against VEGFR2 leading to strongly attenuated atherosclerosis, but enhanced postinterventional neointima formation in hypercholesterolemic mice, which suggests an important role of VEGFR2+ cells in atherosclerosis and restenosis. These results indicate that cell-based therapies can be of potential use in the treatment of vascular disorders and that induction of cell-mediated immunity by vaccination can be a useful tool in targeting specific atheroma-associated cell types to reduce atherosclerosis.
Sources of Funding
J.K., N.P., and G.H.M.v.P. were supported by grant 2000D040, A.D.H. by grant 2000.198 from the Netherlands Heart Foundation, E.J.A.v.W. by a grant from the Netherlands Organization for Scientific Research (NWO 050-10-014). The authors belong to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community’s Sixth Framework Program for Research Priority 1 (Life Science, Genomics, and Biotechnology for Health; contract LSHM-CT-2003-503254).
The major findings of this study were presented at the scientific sessions of the American Heart Association in 2004.
Original received March 13, 2007; final version accepted June 4, 2007.
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