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. Author manuscript; available in PMC: 2011 Aug 6.
Published in final edited form as: Circ Res. 2010 Jun 10;107(3):408–417. doi: 10.1161/CIRCRESAHA.109.210963

VEGF blockade inhibits lymphocyte recruitment and ameliorates immune-mediated vascular remodeling

Jiasheng Zhang 1, Teresa Silva 1, Timur Yarovinsky 1, Thomas D Manes 1, Sina Tavakoli 1, Lei Nie 1, George Tellides 1, Jordan S Pober 1, Jeffrey R Bender 1, Mehran M Sadeghi 1
PMCID: PMC2929975  NIHMSID: NIHMS210137  PMID: 20538685

Abstract

Rationale

There are conflicting data on the effects of vascular endothelial growth factor (VEGF) in vascular remodeling. Furthermore, there are species-specific differences in leukocyte and vascular cell biology and little is known about the role of VEGF in remodeling of human arteries.

Objective

We sought to address the role of VEGF blockade on remodeling of human arteries in vivo.

Methods and Results

We used an anti-VEGF antibody, bevacizumab, to study the effect of VEGF blockade on remodeling of human coronary artery transplants in severe combined immunodeficient mice. Bevacizumab ameliorated peripheral blood mononuclear cell (PBMC)-, but not interferon-γ-, induced neointimal formation. This inhibitory effect was associated with a reduction in graft T cell accumulation without affecting T cell activation. VEGF enhanced T cell capture by activated endothelium under flow conditions. The VEGF effect could be recapitulated when a combination of recombinant ICAM-1 and VCAM-1, rather than endothelial cells, was used to capture T cells. A subpopulation of CD3+ T cells expressed VEGF receptor (VEGFR)-1 by immunostaining and FACS analysis. VEGFR-1 mRNA was also detectable in purified CD4+ T cells and Jurkat and HSB-2 T cell lines. Stimulation of HSB-2 and T cells with VEGF triggered downstream ERK phosphorylation, demonstrating the functionality of VEGFR-1 in human T cells.

Conclusions

VEGF contributes to vascular remodeling in human arteries through a direct effect on human T cells that enhances their recruitment to the vessel. These findings raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling.

Keywords: Vascular endothelial growth factor, Bevacizumab, Vascular remodeling, Transplantation, T lymphocytes

Introduction

Vascular endothelial growth factor (VEGF, also referred to as VEGFA) is produced in response to hypoxia, growth factors (e.g., epidermal growth factor, platelet-derived growth factor) and pro-inflammatory cytokines (e.g., interleukin-1α and interleukin-6) by endothelial cells (ECs), leukocytes (monocyte/macrophages and T cells) and a number of other cell types1. In addition to its well-known role in promoting angiogenesis, VEGF plays an important role in leukemic cell growth and inflammatory disorders. VEGF contributions to the pathogenesis of vascular pathology include promoting angiogenesis, re-endothelialization, vascular smooth muscle cell (VSMC) migration and inflammation in the vessel wall2.

Vascular remodeling, as in graft arteriosclerosis and post-angioplasty restenosis, is a common feature of many vascular diseases. There are conflicting data on the role of VEGF in vascular remodeling3–8. As such, VEGF may promote neointima, inhibit neointima, or have opposing effects based on its endogenous or exogenous origin2. Given this controversy on the role of VEGF in vascular remodeling and species-specific differences in leukocyte and vascular cell biology between mice and humans, it is difficult to speculate what role, if any, VEGF plays in remodeling of human arteries. In this study, we sought to address the role of human VEGF in remodeling of human arteries in vivo using chimeric human/mouse models of vascular remodeling and a specific anti-human (but not anti-mouse) VEGF antibody9, 10. We demonstrate that specific blocking of human VEGF ameliorates neointima formation in transplanted human coronary arteries, and that this effect is, at least in part, through direct effects on T cell trafficking. Finally, we demonstrate that VEGF enhances human T cell binding to the endothelium under flow, and identify a subpopulation of T cells which express potentially functional VEGF receptor (VEGFR)-1.

Material and Methods

An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.

Animal Models

Human coronary artery transplantation in immunodeficient mice was performed as described11. Briefly, adjacent segments of human coronary artery were implanted into the infra-renal aortae of 8–12 week old C.B-17 SCID/beige mice. A group of animals received 1 ×108 human PBMCs per mouse (or control buffer), injected intra-peritoneally one week after transplantation. Other animals were injected with replication incompetent Ad5.CMV-human IFN-γ or Ad5.CMV-LacZ through the jugular vein. Bevacizumab or control human immune globulins were administered at a dose of 5 mg/kg, ip, three times per week, starting with PBMC transfer or injection of adenovirus. Animals were sacrificed at 4 weeks after PBMC transfer or adenovirus injection (5 weeks after coronary artery transplantation), and transplanted arteries were removed, and frozen in OCT for further analysis. All experiments were performed under protocols approved by Yale University Institutional Animal Care and Use and Human Investigation Committees.

Results

VEGF and VEGFR expression in immune-mediated vascular remodeling

Transplantation of segments of human coronary artery to the abdominal aorta of SCID mice followed by adoptive transfer of allogeneic human PBMCs (of which only memory T cells reconstitute the host) led to significant neointima formation and expansive remodeling over a period of 4 weeks as previously described11. The intima and total vessel areas increased from 0.21 ± 0.05 mm2 and 0.62 ± 0.12 mm2 in control animals that were not given PBMC to 0.67 ± 0.16 mm2 and 1.12 ± 0.17 mm2 at four weeks after PBMC inoculation (n=5, p=0.004 and 0.005, respectively)12 (Fig. 1a and Online Fig. I). Both VEGF and its receptor, VEGFR-1 were readily detectable by immunostaining in transplanted coronary arteries, whether in the presence or absence of PBMC transfer, and predominantly localized to the neointima and media. VEGFR-2 expression was less apparent, and mainly localized to the luminal endothelium (Fig. 1b). There was no significant difference in 18S rRNA-normalized VEGFR-1 mRNA levels detected by quantitative RT-PCR between the two groups of animals (n=5 per group). Normalized VEGFR-2 levels were slightly, but significantly decreased in transplanted arteries following PBMC transfer (n=5, p<0.05, Fig. 1c)

Figure 1.

Figure 1

Figure 1

Figure 1

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VEGF and VEGFR expression in PBMC-induced remodeling of human coronary arteries. a) Representative example of Elastica-van Gieson staining and morphometric analyses of adjacent segments of human coronary artery transplanted to SCID/beige mice in the absence of, or 4 weeks (WKS) after human PBMC transfer. There is a significant increase in the intima and total vessel areas in PBMC-treated animals. n=5 per group, *: p<0.05. Scale bar: 100μm. b) Representative examples of VEGF, VEGFR-1, and VEGFR-2 immunofluorescent staining (in red) of transplanted human coronary arteries in the absence of, or 4 weeks after human PBMC transfer, demonstrating VEGF and VEGFR-1, both predominantly expressed in the media and neointima, and VEGFR-2 expressed along the lumen. Elastic membrane autofluorescence is overlaid in green. Staining with control antibody is shown in the inset. Scale bar: 50μm. The figure is representative of 5 independent experiments. c) Quantitative RT-PCR analysis of human VEGFR-1 and VEGFR-2 expression in transplanted arteries in the absence of, or 4 weeks after human PBMC transfer. VEGFR mRNA levels were normalized to 18S rRNA and no PBMC samples. n= 5 per group. A: Adventitia, M: Media, I: Intima, L: Lumen.

VEGF inhibition and modulation of immune-mediated vascular remodeling

To address the role of VEGF in the pathogenesis of immune-mediated vascular remodeling, we investigated the effect of bevacizumab, a function-blocking anti-human (but not murine) VEGF antibody9, 10, on PBMC-induced vascular remodeling in SCID mice transplanted with human coronary arteries. Bevacizumab (5 mg/kg, i.p., three times a week) significantly reduced neointima (0.74 ± 0.07 and 0.52 ± 0.09 mm2 respectively for the control and bevacizumab-treated groups, n=10, p=0.004) and increased lumen areas (0.17 ± 0.06 and 0.26 ± 0.07 mm2, respectively for the control and bevacizumab-treated groups, n=10, p=0.049) at four weeks after PBMC reconstitution, demonstrating a non-redundant causal role of human VEGF in this model of immune-mediated human vascular remodeling (Fig. 2 and Online Fig. I).

Figure 2.

Figure 2

Figure 2

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Effect of VEGF blockade on PBMC-induced vascular remodeling. Representative examples of Elastica-van Gieson staining and morphometric analyses of human coronary artery transplants in SCID/beige mice reconstituted with human PBMCs, and treated with either control human IgG or bevacizumab for 4 weeks. Each pair represents animals transplanted with adjacent segments of the same coronary artery. VEGF blockade with bevacizumab led to a significant decrease in the neointima and increase in lumen area. n=10 per group. Scale bar: 100μm.

T cell recruitment, activation, and IFN-γ secretion are key steps in PBMC-induced vascular remodeling13. Therefore, to address the mechanism(s) of the modulatory effect of VEGF inhibition, we assessed the effect of bevacizumab on T cell infiltration, activation, and cytokine production in human coronary artery transplants. There was a significant reduction in the number of intimal human CD45 positive cells (leukocytes) in animals treated with bevacizumab (824 ± 152 and 367 ± 121, respectively for the control and bevacizumab-treated groups, n=7, p=0.02, Fig. 3a–b), indicating that VEGF blockade inhibits leukocyte accumulation in transplants. This was confirmed by quantitative RT-PCR that demonstrated a significant reduction in GAPDH-normalized human CD3ε transcripts in animals treated with bevacizumab (to 45 ± 15%, n=7, p=0.039, Fig. 3c). Similarly, a trend toward reduction of the number of CD69+ cells was detected in animals treated with bevacizumab (Fig. 4a–b). However, bevacizumab did not affect leukocyte activation beyond its effect on reducing leukocyte infiltration (ratio of infiltrating CD69+ to CD45+ cells: 0.18 ± 0.04 and 0.35 ± 0.12, respectively for the control and bevacizumab-treated groups, n=7, p=NS, fig 4c). There was no clear difference in CD31 (EC), α-actin (VSMC), VEGF or VEGFR expression pattern in arteries from control and bevacizumab-treated animals (data not shown).

Figure 3.

Figure 3

Figure 3

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Effect of VEGF blockade on T cell infiltration in PBMC-induced vascular remodeling. a) Representative examples of human CD45 immunofluorescent staining (in green) of human coronary artery transplants in SCID/beige mice reconstituted with human PBMCs, and treated with either the control human IgG or bevacizumab for 4 weeks. Nuclei are stained with DAPI in blue. The inset represents a high-magnification photomicrograph of the area shown by the arrow. b) Quantitative analysis of intimal human CD45+ cells, demonstrating a significant reduction in the number of infiltrating human CD45+ cells in animals treated with bevacizumab, n=7 per group. c) Quantitative RT-PCR analysis of relative human CD3ε expression, demonstrating a significant reduction of GAPDH-normalized human CD3ε transcripts in animals treated with bevacizumab relative to control animals. n=6 per group. A: Adventitia, M: Media. I: Intima. Scale bar: 50μm.

Figure 4.

Figure 4

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Effect of VEGF blockade on T cell activation and TH1 polarization in PBMC-induced vascular remodeling. a) Representative examples of CD69 immunofluorescent staining (in red) of human coronary artery transplants in SCID/beige mice reconstituted with human PBMCs, and treated with either the control human IgG or bevacizumab for 4 weeks. Nuclei are stained with DAPI in blue. The inset represents a high-magnification photomicrograph of the area shown by the arrow. b) Quantitative analysis of intimal CD69 cells, demonstrating a non-significant trend towards reduction in the number of infiltrating CD69+ cells in animals treated with bevacizumab. n=7 per group. c) Quantitative analysis of T cell activation. There is no significant difference in the ratio of infiltrating CD69+ to CD45+ cells, indicating that bevacizumab did not affect T activation, n=7 per group. d) Quantitative RT-PCR analysis of relative human IFN-γ expression. There is no significant difference in the ratio of IFN-γ to CD3ε transcripts between animals treated with bevacizumab and those treated with the control IgG, indicating that VEGF blockade does not alter TH1 polarization of T cells in this model. n=6 per group. Scale bar: 50μm.

Next, we assessed the effect of VEGF inhibition on IFN-γ production in the arterial transplants by quantitative RT-PCR. There was no significant difference in the ratio of IFN-γ to CD3ε transcripts between animals treated with bevacizumab and those treated with the control IgG, indicating that VEGF inhibition does not alter IFN-γ expression by T cells in this model (IFN-γ to CD3ε transcript ratio: 0.21 ± 0.04 and 0.19 ± 0.07, respectively for the control and bevacizumab-treated groups, (n=6, p=NS, fig 4d).

VEGF inhibition and IFN-γ-induced vascular remodeling

IFN-γ is a key mediator of PBMC-induced vascular remodeling which can induce remodeling in transplanted arteries in the absence of allogeneic PBMC transfer14. Unlike the neointima of PBMC-induced remodeled arteries (which is mainly composed of infiltrating human memory T cells), the neointima in IFN-γ-induced arterial remodeling is rich in VSMCs13, 15, 16. To establish whether the observed effect of bevacizumab on reducing vascular remodeling is dependent on its modulatory effect on leukocyte recruitment, we assessed the effect of VEGF blockade on IFN-γ-induced arterial remodeling. As expected, infection with ad-IFN-γ in SCID mice transplanted with human coronary artery led to a significant increase in the transplant intima and total vessel areas over a period of 4 weeks (intima area: 0.25 ± 0.02 vs 0.12 ± 0.02 mm2, and total vessel area: 0.71 ± 0.03 vs 0.43 ± 0.05 mm2, respectively for IFN-γ and control, Lac Z adenovirus-treated animals, n=4 in each group, p=0.037 and 0.02, respectively) (Fig. 5a and Online Fig. I). A similar pattern of VEGF and VEGFR expression was detected in IFN-γ-induced remodeled arteries (data not shown). However, contrary to its effect on PBMC-induced vascular remodeling, bevacizumab treatment failed to demonstrate any inhibitory effect on IFN-γ- induced vascular remodeling (intima area 0.38 ± 0.06 vs 0.34 ± 0.07 mm2, and total vessel area: 0.93 ± 0.08 vs 0.83 ± 0.10 mm2 , respectively for the control and bevacizumab-treated groups, n=5 in each group, p=NS, Fig 5b), suggesting that the observed inhibitory effect of bevacizumab on vascular remodeling is dependent on the presence of T cells.

Figure 5.

Figure 5

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Effect of VEGF blockade on IFN-γ-induced vascular remodeling. a) Representative examples of Elastica-van Gieson staining and morphometric analyses of adjacent segments of human coronary artery transplanted to SCID/beige mice 4 weeks (Wks) after intravenous injection of either AD-LacZ or AD-IFN-γ, demonstrating a significant increase in the intima and total vessel areas in animals injected with IFN-γ adenovirus. n=4 per group. *: p<0.05. b) Representative examples of Elastica-van Gieson staining and morphometric analyses of human coronary artery transplants in SCID/beige mice injected with AD-IFN-γ, and treated with either the control human IgG or bevacizumab for 4 weeks. VEGF blockade with bevacizumab had no significant effect on IFN-γ-induced vascular remodeling. n=5 per group. Scale bar: 100μm.

VEGF and T cell adhesion to endothelium

T cell recruitment to transplanted arteries involves a cascade of events, including chemotaxis, adhesion to endothelial cells (ECs) and transmigration which may be targets of VEGF stimulation. The IFN-γ and VEGF-inducible IP-10 is a readily detectable chemokine in transplanted arteries and is believed to play a role in the recruitment T cells15, 17. We assessed the effect of bevacizumab treatment on IP-10 expression by quantitative RT-PCR (data not shown). Unexpectedly, bevacizumab treatment had no inhibitory effect on IP-10 expression in transplanted arteries. To address whether VEGF has a direct effect on T cell adhesion to ECs and/or trans-endothelial migration, we assessed the effect of VEGF on T cell-EC interactions under flow conditions. To dissociate a potential effect of VEGF on T cells from its known effects on ECs, TNF (1ng/ml, 20hrs)-treated ECs were pretreated with an inhibitor of VEGFR signaling, SU541618, 19, under conditions which completely inhibited VEGF-induced activation of ERK (Online Fig. II) and Akt (data not shown). VEGF (100 ng/ml, 5 min) had no effect on T cell transmigration through the endothelium under shear stress (data not shown), but significantly increased the number of T cells adhering to the ECs (Fig. 6a). The VEGF effect could be recapitulated when recombinant ICAM-1 alone or a combination of recombinant ICAM-1 and VCAM-1, rather than ECs, was used to capture T cells under shear stress (Fig. 6b). Pretreatment of T cells with SU5416 or pre-incubation of ECs with an anti-ICAM-1 antibody inhibited VEGF-induced T cell adhesion to TNF-treated ECs (Online Fig. III).

Figure 6.

Figure 6

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Adhesion of CD4+ T cells after treatment with VEGF. CD4+ T cells were treated with vehicle or 100 ng/ml VEGF for 5 minutes before flowing over HUVEC treated with 1 ng/ml TNF 20 h and 5 μM SU5416 2 h (a) or over recombinant adhesion molecules (b) for 2 minutes, followed by 8 minutes of medium only. Graphs represent mean and SEM of the number of T cells/field. Graph in (a) from one representative of 3 independent experiments. Graph in (b) from one representative of 2 independent experiments. ***, p<0.001; **, p<0.01; *, p<0.05.

VEGF receptors in T cells

VEGF-induced enhanced T cell capture by ECs is highly suggestive of direct effects of VEGF on T cells or at least a subpopulation of T cells. This would imply that T cells express a functional VEGF receptor. In fact, immunostaining of PBMC smear demonstrated the presence of a population of CD3+ T cells that express VEGFR-1 on their surface (Fig. 7a). By flow cytometry, 1.2±0.2% of CD3+ cells (n=3) were found to express VEGFR-1 (Fig. 7b). Quantitative RT-PCR confirmed expression of VEGFR-1 (but failed to convincingly detect expression of VEGFR-2) mRNA by purified CD4+ and CD4+CD45RA− memory T cells (Fig. 7c). Jurkat and HSB-2 human transformed T cell lines also expressed detectable levels of VEGFR-1 mRNA and protein but lacked VEGFR-2 (Fig. 7c and d). Using directly conjugated antibody for VEGFR-1 and flow cytometry, we confirmed surface expression of VEGFR-1 by Jurkat and HSB-2 cells (Fig. 7e). Stimulation of HSB-2 cells with VEGF triggered detectable but transient phosphorylation of ERK, demonstrating the functionality of VEGFR-1 in this cell line (Fig. 7f). Finally, consistent with the presence of a population of VEGFR-1 expressing T cells, VEGF induced ERK phosphorylation in a subset of CD3+ T cells, as detected by intracellular staining and flow cytometry (Fig. 7g and Online Fig. IV). Similarly, ERK phosphorylation could be induced in a subset of CD3+ T cells upon stimulation with placenta growth factor (PlGF), a VEGFR-1-specific ligand1, demonstrating the functionality of VEGFR-1 in human T cells (Fig. 7g).

Figure 7.

Figure 7

Figure 7

Figure 7

Figure 7

Figure 7

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VEGF receptor expression and signaling by T cells. a) Immunofluorescent staining of PBMC smear, demonstrating VEGFR-1 expression (stained in red) on a subset of CD3+ T lymphocytes stained in green (arrow). Nuclei are stained by DAPI in blue. Insets show a higher magnification of a CD3 positive VEGFR-1 positive cell. The figure is representative of 3 independent experiments. b) Flow cytometry of lymphocytes demonstrating surface expression of VEGFR-1 in a subset of CD3+ T cells. The figure is representative of experiments with blood from 3 different donors. c) VEGFR-1 expression in T cell subsets. Total RNA from purified CD4+, CD4+CD45RA−, Jurkat and HSB-2 cells was isolated and used to detect VEGFR-1 mRNA by quantitative RT-PCR. The levels of VEGFR-1 mRNA expression were normalized to the levels of GAPDH. The figure is representative of two independent experiments. d) Lysates from Jurkat and HSB-2 cells (50 μg/lane) and MVEC (30 μg/lane) were analyzed for expression of VEGFR-1 and VEGFR-2 by immunoblotting. HSP90 was used as a loading control. The figure is representative of three independent experiments. e) Surface expression of VEGFR-1 in Jurkat and HSB-2 cells is shown following staining with PE-conjugated VEGFR-1 or isotype control antibody and by flow cytometry. The figure is representative of four experiments. f) Phosphorylation of ERK in HSB-2 cells that were incubated with 50 ng/ml VEGF for the indicated time was detected by immunoblotting. Bar graph represents the data from three independent experiments. *: p<0.05 compared to time 0. g) Phosphorylation of ERK detected by intracellular staining and flow cytometry in a subset of CD3+ PBMCs incubated for 10 minutes with VEGF (50 ng/ml) or PlGF (100 ng/ml). Phorbol 12-myristate 13-acetate (PMA, 50 nM) was used as positive control for ERK phosphorylation. The figure represents one of three experiments with similar results.

Discussion

In this study we provide new information on the role of VEGF in vascular remodeling, demonstrating that human VEGF inhibition with bevacizumab ameliorates immune-mediated arteriosclerosis. While early studies suggested that VEGF may prevent vascular remodeling by enhancing re-endothelialization3, other studies pointed to a more nuanced picture where, depending on the species and source of VEGF, it may enhance or inhibit neointima formation5–8. The VEGF effect on vascular remodeling is probably multi-faceted and may include modulation of VSMC migration20, 21, angiogenesis, and immune and inflammatory responses4, 17, 22–25. The immune-modulatory effects of VEGF have been linked to modulation of monocyte4, 22, 25, 26 and/or lymphocyte recruitment17, 25 via regulation of angiogenesis25, chemokine production (IP-10, Monocyte chemotactic protein-1)17, 22, 25, EC activation27 and dendritic cell maturation28. In parallel, through induction of nitric oxide production by ECs, VEGF can function as an anti-inflammatory agent and limit VSMC proliferation29, 30. An extra layer of complexity in interpreting these observations is raised by established differences in VEGF biology between mice and humans. For example, it is reported that the VEGF effect on chemokine production by ECs is species-specific. VEGF alone can induce IP-10 in murine, but not human ECs, where it can potentiate the IFN-γ effect17.

The chimeric human/mouse models of immune-mediated vascular injury provided us with a unique opportunity to investigate the effect of human VEGF blockade on vascular remodeling in human coronary arteries. Because of the species-specificity of the bevacizumab effect10, one can be reasonably confident that human VEGF, presumably produced by human vascular cells and/or adoptively transferred human PBMCs, is involved in the pathogenesis of remodeling in this model. Although we did not directly investigate the source of human VEGF in this model, ECs, VSMCs31, and leukocytes (monocyte/macrophages and activated T lymphocytes)32–34 have been shown to produce VEGF under specific experimental conditions. VEGF was detectable by immunostaining in transplanted arteries. While it is possible that VEGF is produced elsewhere and deposited in the vessel wall4, its non-uniform distribution suggests that it is locally produced by vascular cells exposed to ischemia/reperfusion, cytokines or growth factors35. Interestingly, VEGF protein is detected in transplanted arteries 5 weeks after transplantation, presumably long after the initial exposure to ischemia, even in the absence of PBMC transfer. The molecular mechanisms of this sustained VEGF expression in the transplanted arteries remains to be determined.

Bevacizumab ameliorated PBMC-induced, but not the closely related IFN-γ-induced, vascular remodeling. In addition, VEGF blockade had no apparent effect on T cell activation and polarization. Memory CD3+ T lymphocytes expressing the CD45RO marker constitute the bulk of transplant-infiltrating human leukocytes in this model, where very few CD68+ macrophages and dendritic cells are detectable15, while VSMCs constitute the major cells of neointima in IFN-γ-induced vascular remodeling13, 16. This led us to identify leukocyte recruitment as the critical step in the observed modulatory effect of bevacizumab on GA. While we did not directly address this possibility here, VEGF might facilitate lymphocyte recruitment through induction of endothelial adhesion molecules, ICAM-1 and VCAM-127. The direct effects of VEGF on monocytic cells36, 37 are well recognized. However, much less is known about the direct VEGF effect on T cell responses. It is reported that VEGF is involved in T cell recruitment in alloimmunity17, 38. While IP-10 production plays a part in this VEGF effect, other yet to be discovered mechanisms appear to play a major role in the VEGF effect in alloimmunity17. Similar to our own findings, previous studies have failed to demonstrate an effect of VEGF on lymphocyte activation17. It is reported that VEGF modifies rat T cell cytokine secretion profile towards a Th1 phenotype39. However, we did not detect any effect of VEGF in vitro (data not shown) or its blockade in vivo on IFN-γ production. Searching for potential additional mechanisms, and in the absence of a demonstrable inhibitory effect of bevacizumab on IP-10 production, we hypothesized that VEGF may directly act on T lymphocytes and modulate their recruitment to the vessel wall. Therefore, to investigate whether VEGF can affect other critical steps in T cell recruitment to the artery, namely T cell adhesion to the endothelium and trans-endothelial migration, we studied the effect of VEGF under both static and flow conditions. CD4+ effector memory cells expressing integrin receptors for endothelial adhesion molecules readily migrate to the sites of inflammation and mediate allogeneic responses in reconstituted SCID/beige mice40. To dissociate the VEGF effects on ECs from its potential effects on T cells, VEGF signaling in TNF-activated ECs was inhibited with SU541618. VEGF had little effect on T cell binding to ECs under static conditions (data not shown). However, it significantly enhanced T cell capture by activated ECs under flow conditions. This effect was recapitulated when a combination of recombinant ICAM-1 and VCAM-1 replaced activated ECs.

VEGFA, the prototypic member of the VEGF family of glycoproteins, interacts with several receptor tyrosine kinase VEGFRs. VEGFR-2 is the main mediator of mitogenic effects of VEGF in ECs, where VEGFR-1 may serve as a decoy receptor to fine-tune VEGF responses1. VEGF binding to VEGFR-2 triggers a cascade of signaling events, including p44/42 MAPK and AKT phosphorylation which regulate EC proliferation and migration41. VEGFR-1 is the predominant VEGF receptor in a number of non-endothelial cells, including VSMCs and monocytes20, 36. Compared to VEGFR-2, less is known about signaling events triggered by VEGFR-1 activation1, 21, 42. Our data demonstrate that at least a subset of human T cells (as well as two human T cell lines), express VEGFR-1. While we are yet to fully characterize this population, another group of investigators recently reported that activated murine T cells express VEGFR-1 and can migrate in response to VEGF43. VEGF- and PlGF-induced ERK phosphorylation demonstrated the functionality of VEGFR-1 in human T cells in our study.

VEGF-induced T cell capture by recombinant ICAM-1 alone or to a greater degree to a combination of ICAM-1 and VCAM-1 (which unlike ICAM-1 supports rolling) suggests that VEGF may alter integrin expression and/or activation of T cells. VEGF signaling through VEGFR-2 can activate β1, β3 and β5 integrins on ECs44. It is reasonable to speculate that a similar mechanism may exist for VEGFR-1 in T cells. The flow requirement in the VEGF-induced augmented adhesion suggests an additive or synergistic effect on leukocyte integrin activation and/or expression. Endothelial cells express mechanosensory complexes that contain VEGFR-2, PECAM-1, VE-cadherin and αvβ345. Flow-induced VEGFR-2 activation (in the absence of ligand) has been demonstrated46, as has an altered cellular pattern of αvβ3 in its active conformation45. Although these responses have not been demonstrated in leukocytes exposed to flow, it is likely that an analogous mechanosensory complex exists in T cells, linking VEGF responses and integrin activation. We are currently studying the effects of VEGF and flow on T cell β1 and β2 integrin function. Together, our data strongly support a role for VEGF in regulating T cell recruitment in immune-mediated vascular remodeling, possibly through regulation of VEGFR-1 signaling. A similar phenomenon may exist in other forms of vascular remodeling. While we have not directly assessed the effect of T cell VEGFR-1 blockade on neointima formation in our model, given the challenges of T cell-specific growth factor receptor targeting in vivo, it is interesting to note that VEGFR-1 tyrosine kinase deficient mice display reduced neointima formation after cuff placement47. This effect may also be, at least in part, mediated by T cells. These VEGF effects raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling. Given the important role of T cells in many aspects of immunity48, the implications of our findings go beyond vascular diseases and may extend to other immune and inflammatory disorders.

Novelty and Significance.

What is known?

  • Vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, leukemic cell growth and inflammation.

  • Vascular remodeling is a common feature of many vascular diseases, including graft arteriosclerosis and post-angioplasty restenosis.

  • Interferon-γ plays a non-redundant role in the development of graft arteriosclerosis.

What new information does this article contribute?

  • VEGF blockade inhibits neointima formation in immune-mediated vascular remodeling, at least in part, through a reduction in T cell accumulation in the neointima.

  • A subset of T cells express functional VEGF receptor-1.

  • VEGF enhances T cell capture by activated endothelium under flow conditions.

There are conflicting data on the role of VEGF in vascular remodeling, with some data indicating that VEGF promotes neointima formation while others point to an opposite effect. Transplantation of human coronary artery segments to infrarenal aorta of severe combined immunodeficient mice followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells or treatment with interferon-γ leads to significant remodeling of the vessel graft. We show that bevacizumab, a blocking anti-human VEGF antibody inhibits peripheral blood mononuclear cell, but not interferon-γ-induced neointima formation. Human VEGF blockade leads to a reduction in T cell accumulation in the neointima, without changes in T cell activation or polarization. This led us to identify a novel subset of T cells that express functional VEGF receptor-1 and demonstrate that human T cell capture by activated endothelium under flow conditions is enhanced by VEGF. Our findings establish a novel role for VEGF in inflammation and immunity and raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling in leukocytes.

Supplementary Material

Materials and Methods
Supplementary figures

Acknowledgments

Sources of Funding This work was supported by NIH Program Project HL70295, RO1 HL085093 and a Department of Veterans Affairs Merit Award to MMS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Non-standard Abbreviations and Acronyms

EC

Endothelial cell

ICAM-1

Inter-cellular adhesion molecule 1

IFN-γ

Interferon-γ

PBMC

Peripheral blood mononuclear cell

PMA

Phorbol 12-myristate 13-acetate

SCID

Severe combined immunodeficient

VCAM-1

Vascular cell adhesion molecule-1

VEGF

Vascular endothelial growth factor

VEGFR

Vascular endothelial growth factor receptor

VSMC

Vascular smooth muscle cell

Footnotes

Disclosures: None

References

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Supplementary Materials

Materials and Methods
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Supplementary figures
NIHMS210137-supplement-Supplementary_figures.pdf (496KB, pdf)

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