Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2013 Feb 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2012 Jun 21;32(8):1849–1855. doi: 10.1161/ATVBAHA.112.252510

Molecular Imaging of VEGF Receptors in Graft Arteriosclerosis

Jiasheng Zhang 1,2, Mahmoud Razavian 1,2, Sina Tavakoli 1,2, Lei Nie 1,2, George Tellides 2,3, Joseph M Backer 4, Marina V Backer 4, Jeffrey R Bender 1, Mehran M Sadeghi 1,2,*
PMCID: PMC3401339  NIHMSID: NIHMS388578  PMID: 22723442

Abstract

Objectives

Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis (GA). GA is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent (NIRF) imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors (VEGFRs) and vascular remodeling in human coronary artery grafts by molecular imaging.

Methods and Results

VEGFR-specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells (PBMCs) led to significant neointima formation in the grafts over a period of 4 weeks. NIRF imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homologue of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGFR-1, but not VEGFR-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area.

Conclusions

Molecular imaging of VEGF receptors may provide a non-invasive tool for detection of GA in solid organ transplantation.

Keywords: Imaging, Molecular Imaging, Transplantation, Vascular Remodeling

Introduction

Vascular endothelial growth factor (VEGF) promotes vascular remodeling by enhancing vascular smooth muscle cell (VSMC) migration and promoting vessel wall inflammation and angiogenesis 1–5. The effects of VEGF in the vessel wall are mediated by two receptor tyrosine kinase VEGF receptors (VEGFRs). VEGFR-1 is expressed by several cell types in the vessel wall, including endothelial cells (ECs), VSMCs, and monocyte/macrophages 6. VEGFR-2 is predominantly expressed by ECs, although other cells, including VSMCs and progenitor cells are also reported to express VEGFR-2 6, 7. Two other co-receptors, neuropilin-1 and -2 can bind VEGF165 but not the VEGF121 isoform 8.

Graft arteriosclerosis (GA) is the prototypic example of immune-mediated vascular remodeling. GA is the main cause of late organ failure after cardiac transplantation and is characterized by diffuse narrowing of coronary arteries due to concentric neointima formation 9. There is no reliable non-invasive imaging approach for tracking the remodeling process in GA. VEGF is expressed in transplanted hearts and its expression has been linked to the presence of GA 10. Inhibition of VEGF signaling inhibits vascular remodeling in transplanted arteries 4, 5, 11. Thus, alterations in VEGFR prevalence and distribution may provide useful information for tracking the remodeling process in GA. Here, we sought to investigate the use of VEGFR-targeted imaging for assessment of vascular remodeling in human coronary artery grafts. Using a chimeric model of human coronary artery transplantation to immunodeficient mice and an engineered Cy5.5-labeled single-chain VEGF probe (scVEGF/Cy) we established that scVEGF/Cy uptake in significantly increased in GA. Tissue analyses indicates that this enhanced scVEGF/Cy uptake reflects a significant increase in VEGFR-1 expression in the remodeling artery.

Materials and Methods

Reagents

All reagents were from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise. Sc VEGF/Cy and Oregon green-labeled scVEGF (scVEGF/OG) and the inactivated homologue obtained through multi-biotinylation (inVEGF/Cy) were from SibTech, Inc. (Brookfield, CT). Labeled scVEGF retains its VEGF activity and binds with high affinity (Kd of 2.8 nM) to VEGFR-2 expressing cells 12. Recombinant human VEGF121 and VEGF165 were purchased from R&D Systems (Minneapolis, MN). VEGF121 lacks the heparin-binding domain of larger VEGFs such as VEGF165, reducing VEGFR-independent binding to heparan sulfates 13.

Cell Culture

Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as described 11. Human peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation from leukocytes collected by apheresis from healthy adult volunteer donors, under protocols approved by the Yale Human Investigation Committee 14.

DNA constructs and transfection

Human embryonic kidney cells (HEK 293 cells, Invitrogen) were transiently transfected with human VEGFR-1 or VEGFR-2 expression plasmids (in pcDNA3, kindly provided by Dr. William C. Sessa, Yale University) using FuGENE 6 Transfection Reagent (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s instructions. Transfected cells were cultured in DMEM media containing 10% FBS for 48 hours before further analysis.

Animal Model

All experiments were performed under protocols approved by Yale University and VA Connecticut Institutional Animal Care and Use and Human Investigation Committees. Transplantation of human coronary artery segments to immunodeficient mice was performed as described 14. Briefly, adjacent segments of human coronary artery were transplanted to the infra-renal aorta of female 8–12 week old C.B-17 SCID/beige mice (n=23, imaging: 17, immunostaining: 6). After one week, animals were inoculated intra-peritoneally with 1 ×108 human PBMCs per mouse (or control buffer) and PBMC reconstitution was verified after two weeks by flow cytometry. Animals were used for imaging studies or tissue analysis after 4 weeks as described below.

Imaging

SCID mice transplanted with human coronary artery segments and inoculated with allogeneic PBMCs for 4 weeks, and their controls (without PBMC reconstitution) were injected with scVEGF/Cy or inVEGF/cy (10 μg/mouse) through a jugular vein catheter. The animals were sacrificed after 24 hours, viscera were removed, and the abdominal aorta was exposed for in situ imaging. Animals were imaged using a Kodak 4000MM imaging system (Carestream Molecular Imaging, New Haven, CT) equipped with 12-bit monochrome CCD camera (Kodak, Rochester, NY) and an f/1.2 12.5- to 75-mm zoom lens. The following settings were used for image acquisition: λ excitation 625/emission 700nm for near infra-red imaging and λ excitation 535/emission 600nm for detection of autofluorescence. Signal intensities were quantified on regions of interest drawn over the transplant or adjacent mouse abdominal aorta (which served as background) using Kodak Molecular Imaging Software 4.0 and were expressed as background-corrected mean signal intensity in arbitrary units (AU).

Histology, Morphometry and Immunostaining

Elastica Van Gieson (EVG) staining was performed on 5-μm-thick sections according to standard techniques. Morphometric analysis was performed on digitized images of cryostat sections, as described 15. For immunohistochemistry and immunofluorescent (IF) staining, the following antibodies and their isotype-matched nonbinding controls were used: VEGFR-1 (Santa Cruz Biotechnology, Santa Cruz, CA), VEGFR-2 (Cell Signaling Technology, Danvers, MA), CD3 and CD31 (Pharmingen, San Diego, CA). Labeled secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA). For IF staining nuclei were stained with prolong antifade DAPI (Invitrogen, Eugene, OR). Images were obtained using a Zeiss LSM 510 microscope.

Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

RNA was isolated from frozen tissue sections using Absolutely RNA® Nanoprep Kit (Stratagene, La Jolla, CA) and reverse transcribed using QuantiTect® Reverse Transcription Kit (QIAGEN, Valencia, CA) following the manufacturers’ instructions. Real-time PCR was performed on cDNA in triplicates using Taqman® gene expression assays (Applied Biosystems, Foster City, CA) and an Applied Biosystems 7500 Real-Time PCR System. Because of major differences in housekeeping gene expression in the artery without or with PBMC transfer 16, mRNA expression was normalized to the template and expressed relative to PBMC-treated animals. The following primer sets were used: CD3ε (Hs99999153_m1), VEGER-1 (Hs01052936_m1), VEGFR-2 (Hs0017667_m1), NRP-1 (Hs00826128_m1), and NRP-2 (Hs00187290_m1).

Flow Cytometry

Expression of VEGFR-1 and VEGFR-2 on HUVEC and HEK 293 cells was detected by staining live cells with PE-conjugated anti-VEGFR-1, VEGFR-2 or the corresponding isotype control antibody (R&D Systems). scVEGF uptake was assessed in cells exposed with scVEGF/OG (10 nM) for 1 hour at 37°C in the absence or presence of VEGF121 (added 10 minutes before scVEGF/OG). At least 5,000 cells that satisfied a gate on forward and side scatter to eliminate aggregates and debris were acquired using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA). Data analysis was performed using FlowJo software (Tree Star Inc, Ashland, OR).

Statistical analysis

All values are expressed as mean±SEM. Two-tailed ratio t-test (for paired non-parametric values) or one-way ANOVA with the Tukey post-hoc analysis were used to assess the significance of differences. Two-tailed Spearman correlation was used to test the association between 2 variables. P <0.05 was considered as significant.

Results

Cellular uptake of scVEGF

scVEGF is a fusion protein containing two aa 3–112 fragments of human VEGF121 cloned head to tail and an N-terminal Cys-tag for site-specific conjugation of imaging and therapeutic moieties 12. To investigate whether similar to VEGF, scVEGF is internalized upon binding to VEGFRs, human ECs were exposed to scVEGF/OG. Using flow cytometry we detected considerable scVEGF/OG uptake by ECs. This uptake was inhibited in the presence of excess VEGF121 or VEGF165 in a concentration-dependent manner, indicating binding specificity (Fig 1a). Microscopic analysis of ECs exposed to scVEGF/Cy confirmed scVEGF uptake and its localization in intracellular compartments (Fig 1b). Transient overexpression of either VEGFR-1 or VEGFR-2 (but not a control plasmid) in HEK 293 cells promoted scVEGF/OG uptake by transfected cells, indicating that both receptors can mediate scVEGF uptake (Fig. 1c).

Figure 1.

Figure 1

Figure 1

Figure 1

Open in a new tab

scVEGF uptake and specificity. a) Representative histograms of scVEGF/OG uptake by HUVECs in the absence or presence of VEGF121 (left) or VEGF165 (right) assessed by flow cytometry. b) Representative photomicrograph of scVEGF/Cy (in red) uptake by HUVECs. Cell membrane and nuclei are visualized respectively by CD31 (in green) and DAPI (in blue) immunofluorescent staining. Scale bar: 10 μm. c) Representative histograms of VEGFR-1 and VEGFR-2 expression (top row) and scVEGF/OG uptake (bottom row) in HEK293 following transient overexpression of VEGFR-1 (left column) or VEGFR-2 (right column) assessed by flow cytometry.

Near-infrared fluorescent imaging of VEGFRs in GA

To detect VEGFRs in GA, we used an established model of immune-mediated vascular remodeling 14. In this model, transplantation of segments of human coronary artery (which often express some degree of native atherosclerosis or diffuse intimal thickening) to SCID mice followed by adoptive transfer of allogeneic human PBMCs leads to considerable vascular remodeling of the transplanted arteries over a period of 4 weeks. scVEGF/Cy was intravenously injected to transplant recipients at 4 weeks after PBMC inoculation. Transplant recipients without PBMC transfer were used as control. In situ NIRF imaging at 24 hours after scVEGF/Cy injection demonstrated high focal uptake of scVEGF/Cy in artery grafts following PBMC transfer (Fig. 2a and supplemental figure Ia). In contrast, human artery autofluorescence was readily detectable independent of the presence of PBMCs. The specificity of scVEGF/Cy uptake in coronary transplants was addressed in an additional group of 3 animals at 4 weeks after PBMC transfer. NIRF imaging following intravenous injection of an inactivated homologue of scVEGF/Cy (inVEGF/Cy) demonstrated no visually detectable focal uptake in artery grafts (Fig. 2a and supplemental figure Ib). Imaging-derived quantitative analysis of tracer uptake demonstrated significantly higher background-corrected mean fluorescence intensity in PBMC-reconstituted, as compared to control animals (respectively 192.9 ± 70.3 vs 16.2 ± 4.7 arbitrary units, n=7 in each group, p<0.001, Fig. 2b). Tracer uptake specificity was confirmed by quantitative analysis of the mean fluorescence intensity of inVEGF/Cy which was as low as the level seen in control animals (19.5 ± 13.2, n=3, p<0.05 compared to PBMC-reconstituted animals injected with scVEGF/Cy, Fig. 2b). There was no difference in autofluorescence between the two groups of animals (respectively 17.7 ± 4.2 vs 19.0 ± 2.6 arbitrary units, n=7 in each group, p=0.36, Fig. 2c).

Figure 2.

Figure 2

Figure 2

Figure 2

Open in a new tab

VEGFR imaging in GA. a) NIRF imaging of human coronary arteries transplanted in SCID mice without (top row) or with (middle and bottom rows) adoptive transfer of human PBMCs. While there is no difference in tissue autofluorescence (second column), NIRF imaging (third column) shows considerable probe uptake in PBMC reconstituted animals following injection with scVEGF/Cy (middle row), but not with inVEGF/Cy, the non-binding probe (lower row). Arrows point to human coronary arteries transplanted end to end into murine abdominal aorta. b–c) Background-corrected mean cy5.5 signal (b) and autofluorescence (c) intensity of coronary transplants (in arbitrary units) in animals without or with adoptive transfer of PBMCs and imaged following either scVEGF/Cy or control inVEGF/Cy administration. n= 7 in each group in animals injected with scVEGF/Cy and 3 in the inVEGF/Cy group. *: p<0.001 vs no PBMC group and <0.05 vs inVEGF/Cy group.

VEGFRs in remodeling human coronary arteries

Histological analysis of coronary transplants showed that, as expected, adoptive transfer of allogeneic human PBMCs to transplanted animals had led to significant neointima formation and expansive remodeling over a period of 4 weeks, with the intimal area increasing from 0.16 ± 0.05 mm2 in control animals to 0.71 ± 0.09 mm2 at four weeks after PBMC inoculation (n=7, p<0.001, Fig. 3). Similarly the total vessel area significantly increased (from 0.60 ± 0.10 to 1.10 ± 0.12 mm2, p<0.001) and the lumen area significantly decreased (from 0.17 ± 0.02 to 0.03 ± 0.01 mm2, p<0.05) following PBMC reconstitution. Immunostaining of transplanted human coronary arteries, in the presence or absence of PBMCs, demonstrated that VEGFR-1 expression predominantly localizes in the tunica media (Fig. 3c). Following adoptive transfer of PBMCs, VEGFR-1 was also expressed in the neointima (Fig. 3c). VEGFR-2 expression predominantly localized to the luminal endothelium in both groups of animals (Supplemental Figure II). NIRF imaging of coronary grafts following in vivo scVEGF/Cy administration showed that the Cy5.5 signal localized in VEGFR-1 positive areas of the neointima in PBMC reconstituted animals (Fig. 3c). The co-localization of the Cy5.5 signal with neointimal CD3 positive cells implicated T lymphocytes in scVEGF/Cy uptake in vivo (Supplemental Figure III). Little Cy5.5 signal could be detected in the absence of PBMC transfer or following inVEGF/Cy administration (Fig. 3c).

Figure 3.

Figure 3

Figure 3

Figure 3

Open in a new tab

Histological analysis of human coronary artery grafts. a) Examples of elastic Van Gieson staining of human coronary artery grafts without or with PBMC transfer, scale bar: 200 μm. b) Morphometric analysis of the grafts. N=7 in each group. *: p<0.05, **: p<0.001. c) Representative VEGFR-1 immunohistochemistry (IHC, top) and NIRF imaging of scVEGF/Cy and inVEGF/Cy uptake in coronary artery grafts without or 4 weeks after human PBMC transfer. Arrowheads point to Cy5.5 positive areas. Elastic laminae are visualized through their autofluorescence. L: lumen, I: intima, M: media, A: adventitia. Scale bar: 50 μm.

Biological correlates of scVEGF/Cy uptake in transplanted arteries

VEGFR expression in artery grafts was quantified by real-time RT- PCR. There was no significant difference in VEGFR-2 expression between control and PBMC-reconstituted animals. However, VEGFR-1 was significantly higher in human coronary artery transplants 4 weeks after adoptive transfer of human PBMCs (Fig. 4). Expression of VEGF co-receptors, neuropilins-1 and -2 was also significantly higher in human coronary artery grafts after PBMC transfer (Fig. 4).

Figure 4.

Figure 4

Open in a new tab

Relative CD3ε and VEGFR expression in arterial transplants quantified by real time RT- PCR and normalized to PBMC-reconstituted transplants. n=5–7 in each group.

Enhanced uptake of scVEGF/Cy in remodeling coronary arteries in conjunction with VEGFR-1 upregulation suggests that VEGFR-1 may be the primary target for scVEGF/Cy in this model. Indeed, there was a significant correlation between scVEGF/Cy uptake and VEGFR-1 (as well as neuropilin-1 and neuropilin-2, but not VEGFR-2) expression in GA samples (Table 1). Morphological assessment of transplanted coronary arteries demonstrated a significant correlation between scVEGF/Cy uptake in vivo and the neointima, lumen and total vessel areas, but not the media area following PBMC transfer (Table 2), suggesting that VEGFR imaging can detect vascular remodeling in GA.

Table 1.

Correlation Between scVEGF/Cy Uptake and VEGF Receptor Expression

Variable R Value P Value
VEGFR-1 0.56 0.046
VEGFR-2 −0.19 0.51
Neuropilin-1 0.55 0.043
Neuropilin-2 0.71 0.006
Open in a new tab

Table 2.

Correlation Between scVEGF/Cy Uptake and Morphometric Indices

Variable R Value P Value
Intima Area 0.86 <0.0001
Media Area 0.42 0.14
Lumen Area −0.85 0.0001
Total Vessel Area 0.75 0.002
Open in a new tab

Discussion

In this study we demonstrate that a tracer with specificity for VEGFRs, scVEGF/Cy 12, specifically accumulates in remodeling human coronary arteries in GA, and its uptake in vivo correlates with indices of vascular remodeling. VEGF, the prototypic growth factor for ECs, plays an important role in normal development, homeostatic response to ischemia, cancer, inflammation, and immune response 6, 13. VEGF effects are mediated by its binding to VEGFRs, including VEGFR-1 and VEGFR-2 6, 17. VEGFR-2 is expressed by ECs and mediates the pro-angiogenic effects of VEGF on ECs. More recently, VEGFR-2 expression has been described in other cells, including VSMCs and T cells 7, 18. Less is known about VEGFR-1, which is expressed by a wide variety of primary cells, including ECs, VSMCs, monocytes and lymphocytes 6, 18. The role of VEGF and VEGFRs in vascular remodeling remains controversial. While a number of studies point to a causal role of VEGF in vascular remodeling, other studies have reached an opposite conclusion 1–4, 19, 20. Recently, using a VEGF-blocking antibody we demonstrated that VEGF plays a key role in the pathogenesis of vascular remodeling in GA and linked this effect to VEGFR-1 on T cells 11. These finding suggested that VEGFR-targeted imaging may provide important information on the development of vascular remodeling in GA.

VEGF imaging was introduced as a promising approach for detection of cancer and angiogenesis, and a number of VEGF-based tracers have been developed and evaluated in animal models for these applications 21–25. One example is scVEGF labeled with Cy5.5, 99mTc, 64Cu, or 68Ga which can detect tumor angiogenesis by NIRF, SPECT or PET imaging. The scVEGF/Cy signal has been linked to receptor-mediated endocytosis and subsequent intracellular retention of the highly charged Cy5.5 moiety 12. While much of the focus of VEGF imaging has been on targeting VEGFR-2 expressed by ECs, here we demonstrated that both VEGFR-1 and VEGFR-2 can mediate scVEGF/Cy uptake by target cells. This allowed us to investigate the expression and functionality of these receptors as targets for imaging vascular remodeling in GA.

Normal arteries express both VEGF receptors. While VEGFR-2 predominantly localizes to luminal endothelium, VEGFR-1 is mostly expressed in the media where VSMCs constitute the majority of cells. Immune-mediated vascular remodeling is associated with expansion of the intima, which in this model of GA primarily consists of CD3+ T cells and matrix components 14, 26. There is little, if any change in the area of the media in GA. Here, we showed that in addition to its expression in the media, VEGFR-1 is also expressed in the neointima of remodeling arteries in GA. Quantitative assessment of VEGFR expression demonstrated a significant increase in VEGFR-1 (but not VEGFR-2) mRNA transcripts in GA. Interestingly, the development of GA was also associated with significant upregulation of neuropilin-1 and -2, two VEGFR co-receptors.

NIRF imaging of transplanted arteries was remarkably consistent with histological findings. Since inherent depth limitation of NIRF imaging precluded in vivo imaging of transplanted coronary arteries in the mouse, we relied on in situ imaging to investigate scVEGF/Cy uptake in artery grafts. There was only a minimal probe uptake in control animals in the absence of vascular remodeling. After allogeneic PBMC transfer and in conjunction with the development of GA, scVEGF/Cy uptake significantly increased in artery grafts with the uptake correlating directly with neointimal and total vessel areas and inversely with luminal area. The lack of inVEGF/Cy uptake in PBMC-reconstituted animals excluded the possibility that the scVEGF/Cy signal is merely due to neointima mass as opposed to VEGFR expression in the neointima. Tracer uptake in the transplanted arteries localized to VEGFR-1 and CD3 positive areas of the neointima, implicating T lymphocytes in the uptake of scVEGF/Cy in this model. Recently, we identified a population of VEGFR-1 expressing CD3+ T cells and demonstrated that VEGFRs play a role in T cell endothelial adhesion 11. Therefore, it is likely that intimal inflammatory cells mediate much of the enhanced scVEGF/Cy uptake in GA. Interestingly, despite considerable VEGFR-1 expression in the media, scVEGF/Cy uptake in the media is minimal. This may be explained by the probe’s limited access to the media, e.g., due to the barrier function of elastic laminae. Alternatively, it is possible that there are differences in VEGFR functionality, e.g., due to neuropilin expression 8 in the neointima, which lead to enhanced scVEGF/Cy uptake in GA in vivo.

Imaging VEGFRs appears to be a promising approach for detection of vascular remodeling in GA. In addition to its potential as an investigational tool for vascular biology research, our findings suggest that scVEGF-based imaging can serve as a clinical tool for tracking the development of vascular remodeling and response to therapy in cardiac transplantation. Although the clinical application of fluorescent probes in clinical cardiovascular medicine is probably limited to invasive approaches (such as intravascular imaging), radiolabeled homologues of scVEGF may be used for non-invasive imaging. While imaging coronary arteries in humans is complicated by the small size of coronary arteries and cardiac motion, the diffuse nature of GA would facilitate cardiac imaging and the clinical application VEGFR imaging in GA. Finally, the development of VEGFR-1 and VEGFR-2-specific probes based on characterized mutations in VEGF 27 can further enhance the specificity of imaging information.

Supplementary Material

Supplementary figures

Acknowledgments

Funding: This work was supported by NIH Program Project HL70295, RO1 HL085093, R01 HL043331 and a Department of Veterans Affairs Merit Award to MMS.

Footnotes

Disclosures: JMB has equity in Sib Tech, Inc.

References

  • 1.Asahara T, Bauters C, Pastore C, Kearney M, Rossow S, Bunting S, Ferrara N, Symes JF, Isner JM. Local delivery of vascular endothelial growth factor accelerates reendothelialization and attenuates intimal hyperplasia in balloon-injured rat carotid artery. Circulation. 1995;91:2793–2801. doi: 10.1161/01.cir.91.11.2793. [DOI] [PubMed] [Google Scholar]
  • 2.Hutter R, Carrick FE, Valdiviezo C, Wolinsky C, Rudge JS, Wiegand SJ, Fuster V, Badimon JJ, Sauter BV. Vascular endothelial growth factor regulates reendothelialization and neointima formation in a mouse model of arterial injury. Circulation. 2004;110:2430–2435. doi: 10.1161/01.CIR.0000145120.37891.8A. [DOI] [PubMed] [Google Scholar]
  • 3.Khurana R, Zhuang Z, Bhardwaj S, Murakami M, De Muinck E, Yla-Herttuala S, Ferrara N, Martin JF, Zachary I, Simons M. Angiogenesis-dependent and independent phases of intimal hyperplasia. Circulation. 2004;110:2436–2443. doi: 10.1161/01.CIR.0000145138.25577.F1. [DOI] [PubMed] [Google Scholar]
  • 4.Lemstrom KB, Krebs R, Nykanen AI, Tikkanen JM, Sihvola RK, Aaltola EM, Hayry PJ, Wood J, Alitalo K, Yla-Herttuala S, Koskinen PK. Vascular endothelial growth factor enhances cardiac allograft arteriosclerosis. Circulation. 2002;105:2524–2530. doi: 10.1161/01.cir.0000016821.76177.d2. [DOI] [PubMed] [Google Scholar]
  • 5.Raisky O, Nykanen AI, Krebs R, Hollmen M, Keranen MA, Tikkanen JM, Sihvola R, Alhonen L, Salven P, Wu Y, Hicklin DJ, Alitalo K, Koskinen PK, Lemstrom KB. VEGFR-1 and -2 regulate inflammation, myocardial angiogenesis, and arteriosclerosis in chronically rejecting cardiac allografts. Arterioscler Thromb Vasc Biol. 2007;27:819–825. doi: 10.1161/01.ATV.0000260001.55955.6c. [DOI] [PubMed] [Google Scholar]
  • 6.Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003;9:669–676. doi: 10.1038/nm0603-669. [DOI] [PubMed] [Google Scholar]
  • 7.Greenberg JI, Shields DJ, Barillas SG, Acevedo LM, Murphy E, Huang J, Scheppke L, Stockmann C, Johnson RS, Angle N, Cheresh DA. A role for VEGF as a negative regulator of pericyte function and vessel maturation. Nature. 2008;456:809–813. doi: 10.1038/nature07424. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Pellet-Many C, Frankel P, Jia H, Zachary I. Neuropilins: structure, function and role in disease. Biochem J. 2008;411:211–226. doi: 10.1042/BJ20071639. [DOI] [PubMed] [Google Scholar]
  • 9.Tellides G, Pober JS. Interferon-gamma axis in graft arteriosclerosis. Circ Res. 2007;100:622–632. doi: 10.1161/01.RES.0000258861.72279.29. [DOI] [PubMed] [Google Scholar]
  • 10.Reinders ME, Fang JC, Wong W, Ganz P, Briscoe DM. Expression patterns of vascular endothelial growth factor in human cardiac allografts: association with rejection. Transplantation. 2003;76:224–230. doi: 10.1097/01.TP.0000071363.55007.D0. [DOI] [PubMed] [Google Scholar]
  • 11.Zhang J, Silva T, Yarovinsky T, Manes TD, Tavakoli S, Nie L, Tellides G, Pober JS, Bender JR, Sadeghi MM. VEGF blockade inhibits lymphocyte recruitment and ameliorates immune-mediated vascular remodeling. Circ Res. 2010;107:408–417. doi: 10.1161/CIRCRESAHA.109.210963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Backer MV, Levashova Z, Patel V, Jehning BT, Claffey K, Blankenberg FG, Backer JM. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med. 2007;13:504–509. doi: 10.1038/nm1522. [DOI] [PubMed] [Google Scholar]
  • 13.Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25:581–611. doi: 10.1210/er.2003-0027. [DOI] [PubMed] [Google Scholar]
  • 14.Lorber MI, Wilson JH, Robert ME, Schechner JS, Kirkiles N, Qian HY, Askenase PW, Tellides G, Pober JS. Human allogeneic vascular rejection after arterial transplantation and peripheral lymphoid reconstitution in severe combined immunodeficient mice. Transplantation. 1999;67:897–903. doi: 10.1097/00007890-199903270-00018. [DOI] [PubMed] [Google Scholar]
  • 15.Zhang J, Krassilnikova S, Gharaei AA, Fassaei HR, Esmailzadeh L, Asadi A, Edwards DS, Harris TD, Azure M, Tellides G, Sinusas AJ, Zaret BL, Bender JR, Sadeghi MM. Alphavbeta3-targeted detection of arteriopathy in transplanted human coronary arteries: an autoradiographic study. Faseb J. 2005;19:1857–1859. doi: 10.1096/fj.05-4130fje. [DOI] [PubMed] [Google Scholar]
  • 16.Bustin SA. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol. 2002;29:23–39. doi: 10.1677/jme.0.0290023. [DOI] [PubMed] [Google Scholar]
  • 17.Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. VEGF receptor signalling - in control of vascular function. Nat Rev Mol Cell Biol. 2006;7:359–371. doi: 10.1038/nrm1911. [DOI] [PubMed] [Google Scholar]
  • 18.Basu A, Hoerning A, Datta D, Edelbauer M, Stack MP, Calzadilla K, Pal S, Briscoe DM. Cutting edge: Vascular endothelial growth factor-mediated signaling in human CD45RO+ CD4+ T cells promotes Akt and ERK activation and costimulates IFN-gamma production. J Immunol. 2010;184:545–549. doi: 10.4049/jimmunol.0900397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Walter DH, Cejna M, Diaz-Sandoval L, Willis S, Kirkwood L, Stratford PW, Tietz AB, Kirchmair R, Silver M, Curry C, Wecker A, Yoon YS, Heidenreich R, Hanley A, Kearney M, Tio FO, Kuenzler P, Isner JM, Losordo DW. Local gene transfer of phVEGF-2 plasmid by gene-eluting stents: an alternative strategy for inhibition of restenosis. Circulation. 2004;110:36–45. doi: 10.1161/01.CIR.0000133324.38115.0A. [DOI] [PubMed] [Google Scholar]
  • 20.Ohtani K, Egashira K, Hiasa K, Zhao Q, Kitamoto S, Ishibashi M, Usui M, Inoue S, Yonemitsu Y, Sueishi K, Sata M, Shibuya M, Sunagawa K. Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lineage cells. Circulation. 2004;110:2444–2452. doi: 10.1161/01.CIR.0000145123.85083.66. [DOI] [PubMed] [Google Scholar]
  • 21.Li S, Peck-Radosavljevic M, Kienast O, Preitfellner J, Hamilton G, Kurtaran A, Pirich C, Angelberger P, Dudczak R. Imaging gastrointestinal tumours using vascular endothelial growth factor-165 (VEGF165) receptor scintigraphy. Ann Oncol. 2003;14:1274–1277. doi: 10.1093/annonc/mdg344. [DOI] [PubMed] [Google Scholar]
  • 22.Lu E, Wagner WR, Schellenberger U, Abraham JA, Klibanov AL, Woulfe SR, Csikari MM, Fischer D, Schreiner GF, Brandenburger GH, Villanueva FS. Targeted in vivo labeling of receptors for vascular endothelial growth factor: approach to identification of ischemic tissue. Circulation. 2003;108:97–103. doi: 10.1161/01.CIR.0000079100.38176.83. [DOI] [PubMed] [Google Scholar]
  • 23.Chan C, Sandhu J, Guha A, Scollard DA, Wang J, Chen P, Bai K, Lee L, Reilly RM. A human transferrin-vascular endothelial growth factor (hnTf-VEGF) fusion protein containing an integrated binding site for (111)In for imaging tumor angiogenesis. J Nucl Med. 2005;46:1745–1752. [PubMed] [Google Scholar]
  • 24.Blankenberg FG, Mandl S, Cao YA, O’Connell-Rodwell C, Contag C, Mari C, Gaynutdinov TI, Vanderheyden JL, Backer MV, Backer JM. Tumor imaging using a standardized radiolabeled adapter protein docked to vascular endothelial growth factor. J Nucl Med. 2004;45:1373–1380. [PubMed] [Google Scholar]
  • 25.Rodriguez-Porcel M, Cai W, Gheysens O, Willmann JK, Chen K, Wang H, Chen IY, He L, Wu JC, Li ZB, Mohamedali KA, Kim S, Rosenblum MG, Chen X, Gambhir SS. Imaging of VEGF receptor in a rat myocardial infarction model using PET. J Nucl Med. 2008;49:667–673. doi: 10.2967/jnumed.107.040576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Burns WR, Wang Y, Tang PC, Ranjbaran H, Iakimov A, Kim J, Cuffy M, Bai Y, Pober JS, Tellides G. Recruitment of CXCR3+ and CCR5+ T cells and production of interferon-gamma-inducible chemokines in rejecting human arteries. Am J Transplant. 2005;5:1226–1236. doi: 10.1111/j.1600-6143.2005.00892.x. [DOI] [PubMed] [Google Scholar]
  • 27.Gille H, Kowalski J, Li B, LeCouter J, Moffat B, Zioncheck TF, Pelletier N, Ferrara N. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem. 2001;276:3222–3230. doi: 10.1074/jbc.M002016200. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary figures
NIHMS388578-supplement-Supplementary_figures.pdf (436.7KB, pdf)

RESOURCES