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Cardiovascular Research Advance Access originally published online on June 19, 2008
Cardiovascular Research 2008 80(1):131-137; doi:10.1093/cvr/cvn169
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

The immunoadhesin glycoprotein VI-Fc regulates arterial remodelling after mechanical injury in ApoE–/– mice

Tanja Schönberger1,*, Dorothea Siegel-Axel1, Renate Bußl1, Sabine Richter1, Martin S. Judenhofer2, Roland Haubner3, Gerald Reischl4, Karin Klingel5, Götz Münch6, Peter Seizer1, Bernd J. Pichler2 and Meinrad Gawaz1

1 DVM, Medizinische Klinik III, Abt. Kardiologie und Kreislauferkrankungen, Eberhard Karls-Universität Tübingen, Otfried-Müller-Str.10, 72076 Tübingen, Germany
2 Labor für Präklinische Bildgebung und Bildgebungstechnologie, Radiologische Klinik, Eberhard Karls-Universität Tübingen, Tübingen, Germany
3 Universitätsklinik für Nuklearmedizin, Innsbruck, Austria
4 Radiopharmazie, PET-Zentrum, Eberhard Karls-Universität Tübingen, Tübingen, Germany
5 Abt. Molekulare Pathologie, Eberhard Karls-Universität Tübingen, Tübingen, Germany
6 Corimmun, Martinsried, Germany

* Corresponding author. Tel: +49 7071 29 83688; fax: +49 7071 29 5040. E-mail address: tanja.schoenberger{at}med.uni-tuebingen.de

Received 19 July 2007; revised 6 June 2008; accepted 10 June 2008

Time for primary review: 23 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Supplementary material
 Funding
 References
 
Aims: Rupture of advanced atherosclerotic plaques initiates platelet activation and aggregation as subendothelial collagen is exposed. Platelet collagen receptor glycoprotein VI (GPVI) was found to bind preferentially to the core region of human plaques. Consequently, platelets contribute to inflammatory processes and trigger atherosclerotic lesion progression. In this study, we examined binding of soluble platelet collagen receptor GPVI-Fc to atherosclerotic lesions and its effect on platelet-triggered atheroprogression and neointima formation after wire-induced carotid injury.

Methods and results: For binding studies after ligation-induced arterial injury, the left common carotid artery of C57BL/6J mice was ligated. For binding studies at spontaneously formed atherosclerotic lesion sites, Apolipoprotein E-deficient (ApoE–/–) mice were fed a 0.25% cholesterol diet over 16 weeks. Binding of [124I]GPVI-Fc was monitored by autoradiography 48 h after intravenous injection and by immunostaining. To study the effect of GPVI-Fc on neointima formation vs. control-Fc, a wire-induced injury of the left A. carotis communis of ApoE–/–-mice was performed. Mice were treated intraperitoneally with GPVI-Fc for 8 days and neointima formation was assessed 4 weeks after intervention. [124I]GPVI-Fc preferentially bound to injury sites after carotid ligation in C57BL/6J mice and to lipid-rich atherosclerotic lesions of the carotid artery and aortic arch in uninjured ApoE–/–-mice. Histological examinations of wire-injured carotid arteries showed that neointima formation was significantly reduced in GPVI-Fc-treated ApoE–/– mice compared to ApoE–/– mice receiving control-Fc (P < 0.05).

Conclusion: GPVI-Fc preferentially bound to sites of vascular injury and was able to inhibit neointima formation after wire-induced vascular injury in ApoE–/– mice. Thus, soluble GPVI-Fc might be also a promising compound to attenuate lesion progression after plaque rupture.

KEYWORDS Atherosclerosis; Remodelling; Restenosis; Platelets


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Supplementary material
 Funding
 References
 
Atherosclerosis is a systemic inflammatory disease characterized by the invasion of blood cells such as monocytes/macrophages and lymphocytes into the intima of the arterial vessel wall.1 In contrast, platelets were thought to be involved preferentially in thromboembolic events of advanced atherosclerotic lesions and after vascular injury.2 However, in the meanwhile it is well known that platelets are also major ‘players’ in the initiation and progression of the primary atherosclerotic process.3 During atherogenesis, platelets adhere to the arterial wall and trigger inflammatory and proliferative processes by the release of proinflammatory factors, finally resulting in atherosclerotic lesion formation.4,5 Thus, inhibition of platelet adhesion during this stage of atherosclerosis results in an efficient attenuation of atherosclerotic lesion formation.6

On the other hand, at the stage of advanced atherosclerotic plaque formation, platelets can be activated by subendothelial structures exposed upon plaque erosion or plaque rupture. Fibrillar collagen was identified as one of the most thrombogenic matrix components.7 Preferentially, collagen type I and type III strongly induce platelet adhesion, aggregation, and thrombus formation by the activation of the platelet collagen receptor glycoprotein, glycoprotein VI (GPVI).8 GPVI, a member of the immunoglobulin superfamily, is a 60–65 kDa type I transmembrane glycoprotein.9,10 Ex vivo studies at human and mouse platelets showed that GPVI forms a complex with the FcR {gamma}-chain at the cell surface.11,12 Previously, we could demonstrate in mice that the soluble GPVI-Fc binds preferentially to areas of vascular injury.13 Studies in vitro and in various mouse models of endothelial denudation in which GPVI was either totally absent or inhibited by antibodies showed that interactions between platelets and the injured vessel wall could be blocked efficiently.8,14,15 Consequently, thrombosis at arterial lesion sites could be prevented.14,16 These findings indicate that inhibition of GPVI interactions with the atherosclerotic vessel wall might represent a promising strategy to attenuate atherosclerotic plaque progression or neointima formation after vascular injury.

In the present study, we investigated soluble GPVI-Fc which is a fusion protein consisting of the extracellular domain of GPVI and a human C-terminal Fc fragment.16 Recently, we could show that this soluble form of human GPVI specifically bound to collagen with high affinity and attenuated platelet adhesion to immobilized collagen in vitro, as well as to sites of vascular injury in vivo.16 Here, we evaluated the binding of radiolabelled GPVI-Fc to injured arterial lesion sites as well as atherosclerotic plaques. Finally, vascular lesions were induced mechanically in the carotid artery in ApoE–/– mice and the inhibitory effect of prolonged administration of GPVI-Fc on neointima formation was studied.

This study provides proof that GPVI-Fc accumulates and binds at lesion sites after mechanical injury when collagen is exposed to blood. Furthermore, GPVI-Fc was found to inhibit neointima formation and subsequent atheroprogression. Thus, GPVI-Fc might be an attractive therapeutic tool to treat atheroprogression at sites of endothelial injury or plaque rupture.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 Supplementary material
 Funding
 References
 
2.1 Synthesis of GPVI-Fc
Synthesis of a soluble form of GPVI (GPVI-Fc) was performed as previously described.16 In brief, we fused the extracellular domain of human GPVI to the human Fc domain. Then, the adenoviral constructs, Ad-control Fc and Ad-GPVI-Fc, were generated. The resulting adenoviral plasmids were transfected into HEK 293 cells. After plaque isolation, recombinant virus particles referred to as Ad-control-Fc and Ad-GPVI-Fc were further amplified in HEK 293. The culture supernant of Ad-control-Fc and Ad-GPVI-Fc infected Hela cells was collected. Purification of control Fc and GPVI-Fc was performed by precipitation and protein A column separation. Finally, the proteins were detected using Coomassie stain or with peroxidase-conjugated goat anti-human IgG antibody. Immunoblotting of Fc, GPVI-Fc, or human platelets using the anti-GPVI monoclonal antibody 5C4 detected both adenovirally expressed GPVI-Fc fusion protein and platelet GPVI, but not the control Fc. The molecular mass of GPVI-Fc was ~80 kDa under reducing conditions and a ~160 kDa protein (homodimer) was identified under non-reducing conditions. Furthermore, binding assays using different concentrations of soluble GPVI-Fc and immobilized collagen were performed to define GPVI-Fc–collagen interactions. Bound GPVI-Fc was detected by an anti-Fc mAb antibody. GPVI-Fc was found to bind to collagen in a saturable manner which corresponds to GPVI. Binding of GPVI-Fc did not occur to BSA or vWF, supporting the specificity of GPVI-Fc binding.

2.2 Synthesis of [124I]GPVI-Fc
124I was produced using a PETtrace cyclotron (General Electric Healthcare, Uppsala, Sweden). The 124Te(p,n)124I reaction was applied by using 12.4 MeV protons for irradiation of 200 mg/cm2 124TeO2 with a 99.8% enrichment. Under the conditions used, the production rate of [124I]Iodide was 5.5 ± 0.7 MBq/µAh (n = 25) at the end of bombardment (EOB). The only radionuclidic impurity was 123I (2.5% at EOB). Labelling was performed according to Gawaz et al.17 Briefly, in an Eppendorf tube coated with 150 µg Iodogen ca. 300 µg of a solution of the protein in PBS (approx. 1 mg/mL; pH 7.4) was incubated with 60–75 µL of [124I]Iodide (50–200 MBq) in 0.004 N NaOH for 20 min at room temperature. The product was separated from the reaction mixture using size exclusion chromatography (Micro Bio-Spin 6, Tris; Bio-Rad, Hercules, CA, USA). Quality control was carried out with thin layer chromatography [silica gel 60, F254 (Merck, Darmstadt, Germany); acetone/water/butanol/25% ammonia 65/5/20/10). Radiochemical purity was >95% and average specific activities of [124I]GPVI-Fc were 35 GBq/µmol corresponding to ca. 0.3 MBq/µg.

2.3 Animals
Male C57BL/6J mice were obtained from Charles River (Sulzfeld, Germany) and used for the experiments at an age between 8 and 12 weeks. Four-week-old male ApoE–/– (B6.129P2-ApoE tm1Unc/J) mice (The Jackson Laboratory, Bar Harbor, Maine, USA) consumed a 0.25% cholesterol diet for another 8–16 weeks. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), the guidelines for the use of living animals in scientific studies, and the German law for the protection of animals.

2.4 Adhesion of platelets prior to and after carotid injury and to atherosclerotic plaques
Murine platelets were isolated from whole blood and labelled with 5-carboxyfluorescein diacetate succinimidyl ester (DCF). The DCF-labelled platelet suspension was adjusted to a final concentration of ca. 3 x 107 platelets/200 µL. In one set of experiments, adhesion of murine platelets was assessed prior to and after carotid injury by in vivo video microscopy, in a second set after atherosclerotic plaque formation. Then, platelet adhesion was visualized at a total of six wild-type C57BL6/J or ApoE–/– mice. Animals were anesthetized by intraperitoneal injection of a solution of midazolame (5 mg/kg body weight, Ratiopharm, Ulm, Germany), medetomidine (0.5 mg/kg body weight, Pfizer, Karlsruhe, Germany), and fentanyl (0.05 mg/kg body weight, CuraMed Pharma GmbH, Munich, Germany). Endothelial denudation was induced near the carotid bifurcation by ligation of the vessel until complete cessation of blood flow occurred, as controlled by epi-fluorescence microscopy. The ligation was removed after 5 min. Prior to induction of vascular injury or after atherosclerotic plaque formation in 16-week-old ApoE–/– mice, fluorescent platelets were infused intravenously via polyethylene catheters (Portex, Hythe, UK) implanted into the left jugular vein. The fluorescent platelets were visualized in situ by in vivo video microscopy of the left common carotid artery using a Zeiss Axiotech microscope (20x water immersion objective, W 20x/0.5, Zeiss) with a 100W HBO mercury lamp for epi-illumination.

2.5 Binding of soluble GPVI-Fc after vascular injury
A total of seven C57BL/6J mice (8–12 weeks old) were anesthetized by intraperitoneal injection of a solution of midazolame (5 mg/kg body weight; Ratiopharm, Ulm), medetomidine (0.5 mg/kg body weight, Pfizer, Karlsruhe, Germany), and fentanyl (0.05 mg/kg body weight; CuraMed Pharma, Karlsruhe, Germany). The left common carotid artery was dissected free and ligated vigorously to induce vascular injury. The ligation was removed after 5 min. Approximately 15 min after the ligation, the animals received a total activity of ~7 MBq [124I]GPVI-Fc by injection into the tail vein. The animals were sacrificed 48 h after injection and the carotids were removed for autoradiography.

2.6 Binding of soluble GPVI-Fc after spontaneous lesion formation in ApoE–/– mice ex vivoand in vivo
Aortic arches of 20-week-old ApoE–/– were perfusion fixed with 4% paraformaldehyde, removed, and processed for histology according to standardized protocols as follows. After embedding in paraffin and cutting into 5 µm sections, the slides were stained with haematoxylin-eosin, elastica van Gieson, and Masson Goldner reagent according to standard protocols. For the assessment of GPVI-Fc binding by immunohistochemistry, serial sections were blocked with H2O2 and 3% BSA (Sigma, Steinheim, Germany). Afterwards, they were incubated with GPVI-Fc for 1 h. Sections incubated with the Fc-fragment and PBS, respectively, served as controls. After vigorous washing, bound GPVI-Fc was stained with peroxidase-conjugated goat anti-human IgG antibody Fc{gamma} fragment specific (Dianova, Hamburg, Germany).

A total of seven transgenic ApoE–/– mice (20 weeks old) and four wild-type (8–12 weeks old) control mice were used for evaluation of GPVI-Fc uptake on plaque in the aortic arch and carotid. The mice received an activity of approximately 7 MBq [124I]GPVI-Fc by injection into the tail vein. The animals were sacrificed 48 h after injection and the carotids and the aortic arches were removed for morphological staining and autoradiography.

2.7 Lipid staining and autoradiography
The vessels were fixed with 4% paraformaldehyde and stained with Sudan III (Sigma, Steinheim, Germany) for en face vessel size and plaque extension measurement with an image analysis program (Carl Zeiss AxioVision Rel. 4.5, Oberkochen, Germany). Plaque area was calculated as percentage of total specimen area. The high resolution autoradiograms were performed with a storage phosphor imager (STORM, Amersham, USA)18 exposing the phosphor screens with the isolated carotids and aortic arches for approximately 8–12 h. The spatial resolution of the phosphor imager was set to 50 µm. The same vessels were used for both morphological staining and autoradiography allowing a correlation of the stained plaques and the enhanced tracer uptake seen in the autoradiography. The autoradiograms were used for quantification by placing equally sized, rectangular, regions of interest around both carotids, the injured and the control vessel as well as the aortic arch. The software package ImageQuant 5.1 (Molecular Dynamics, Urbana, IL, USA) was used to calculate the average counts within the region of interests. After autoradiography, the isolated blood vessels were used for quantitative measurements in a calibrated gamma-counter (Wallac, Perkin-Elmer, Finland). A standard activity was measured along with the specimens in the gamma-counter allowing for calculation of the percentage of tracer uptake in the dedicated vessels with respect to the total injected dose. To account different sizes of the blood vessels, the size of the carotids and the aortic arches were measured and the reported activity in percent injected dose (% ID) was normalized to the size of the vessels in % ID/mm2.

2.8 Evaluation of plasma levels of GPVI-Fc after intraperitoneal application
Eight-week-old cholesterol fed ApoE–/– mice received 200 µg GPVI-Fc (10 mg/kg) twice weekly by intraperitoneal injection. At indicated time points, approximately 100 µL blood was collected in a tube containing 10% (v/v) 0.1 mol/L sodium citrate under isofluran anaesthesia from the retro-orbital plexus. The blood collection was performed prior to the next injection. GPVI-Fc levels were determined in the plasma using a commercial available ELISA (IgG ELISA Kit, Immunotek, ZMC) according to manufacturer's instruction.

2.9 Evaluation of neointima and plaque formation in injured carotid arteries after inhibition of platelet adhesion
Wire-induced injury of the carotid artery was performed as described before.8 In brief, 8-week-old cholesterol fed ApoE–/– mice were anesthetized by intraperitoneal injection of a solution of midazolame (5 mg/kg body weight; Ratiopharm, Ulm, Germany), medetomidine (0.5 mg/kg body weight, Pfizer, Karlsruhe, Germany), and fentanyl (0.05 mg/kg body weight; CuraMed Pharma, Karlsruhe, Germany). The left carotid artery was exposed and the common, external, and internal carotid arteries were identified. A transverse arteriotomy was made in the internal carotid artery and a 0.014-in flexible angioplasty guidewire was inserted into the common carotid artery. The denudation was performed by triple withdrawal of the wire. After removal of the wire, the internal carotid artery was tied off. The mice were randomly assigned to receive either soluble dimeric GPVI-Fc (200 µg, n = 6) or control-Fc (200 µg, n = 5) for 8 days. Approximately 15 min before carotid injury, the first injection was carried out intravenously, all seven following daily injections were intraperitoneal. The mice lived for further 3 weeks, then, carotid arteries and the aortic arch were removed and processed for histomorphometry according to standardized protocols as follows. The right carotids and aortic arches were fixed in 4% paraformaldehyde and stained with Sudan III to assess en face plaque extension quantified as described above. The left carotid arteries were embedded in paraffin blocks and cut into 5 µm sections and stained with haematoxylin-eosin and elastica van Gieson reagent according to standard protocols. Six of the elastica van Gieson stained sections upstream of the carotid bifurcation were used for quantification of neointima formation. The area of the lumen and the areas bounded by internal elastic lamina (IEL) and external elastic lamina (EEL) were measured planimetric with NIS-Elements Imaging Software (Nikon GmbH, Düsseldorf, Germany). Neointimal area was calculated by subtracting lumen area from the IEL, and medial area by subtracting the EEL from the IEL area. The degree of stenosis was calculated from neointimal area and the original lumen area defined as area bounded by the IEL.

2.10 Statistical analysis
All quantitative results were presented as mean ± SEM (standard error of the mean). A two tailed t-test result with a P-value of less than 0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 Supplementary material
 Funding
 References
 
3.1 Imaging and quantitative analysis after vascular injury of the carotid artery
Autoradiography studies showed an accumulation of [124I]GPVI-Fc at the injured carotid artery (Figure 1A). Quantitative analysis of the autoradiograms (Figure 1B, top) showed a factor of 4.5 increased GPVI-Fc uptake in the injured carotid compared to the control vessel (P = 0.002). The autoradiogram data were confirmed by gamma counter measurements, showing an increased tracer uptake by a factor of 3.4 for the injured vessels compared to the control carotid.


Figure 1
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  		Figure 1 Binding of [124I]GPVI-Fc at carotid arteries of wild-type mice after mechanical injury compared with uninjured control vessels: (A) intensive uptake of [124I]GPVI-Fc in the injured carotid artery (top) compared with control carotid artery (bottom) of the same mouse in autoradiography. (B) Quantitative analysis of the autoradiograms (top) showed a factor of 4.5 increased GPVI-Fc uptake in the injured carotid compared with the control vessel. Gamma counter measurements (bottom) show an increased tracer uptake by a factor of 3.4 for the injured vessels compared to the control carotid artery confirming the results obtained from autoradiography. *P < 0.05.

 
3.2 Histological examination and assessment of ex vivo GPVI-Fc binding by immuno-histochemistry
Sections of the aortic arch of 20 weeks old, cholesterol fed ApoE–/–mice show atherosclerotic plaques at predilection sites, e.g. the branches and the lesser curvature. The endothelial layer looks irregular due to erosion or fissures (Figure 2, arrows). Medial involvement, elastic fragmentation, and acellular areas within the plaque are characteristic for advanced lesions (Figure 2, triangles and broad arrows). The Masson Goldner staining shows collagen-rich plaques (Figure 2C). Binding of GPVI-Fc is congruent to the collagen staining (Figure 2C and D).


Figure 2
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  		Figure 2 Atherosclerotic plaque in the aortic arch of 20-week-old ApoE–/– mice. Representative images of sections ca. 40 µm distal the branch of the brachiocephalic artery. The haematoxylin-eosin staining shows an overview of the vessel (A). (B)–(D) show elastica van Gieson (B), Masson Goldner (C), and immunostaining for GPVI-Fc (D) of the plaque area with heightened details. Note corresponding staining for collagen and bound GPVI-Fc (C and D). Scale bars, 100 µm. Original magnification x10 (A); x20 (B–D).

 
3.3 Imaging and quantitative analysis after spontaneous plaque formation in ApoE–/– mice
The aortic arches of the 20-week-old ApoE–/– mice showed an intensive Sudan-III staining compared to wild-type mice (Figure 3A, left side). In addition, the tracer uptake in the autoradiograms (Figure 3A, right side) matches the histology results. Quantitative analysis of the autoradiograms showed a significantly higher uptake of [124I]GPVI-Fc by a factor of 1.7 in the aortic arches of the transgenic mice than in the wild-type animals (Figure 3B). This finding is consistent with the 1.6 higher uptake (P = 0.03) seen in the measurements performed with the gamma counter.


Figure 3
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  		Figure 3 Binding of [124I]GPVI-Fc at the aortic arches of ApoE–/– mice compared with wild-type mice: (A) aortic arches of 20-week-old ApoE–/– mice show an intensive Sudan-III staining (left) predominantly at bifurcation sites compared with wild-type mice. (B) Quantitative analysis of autoradiograms and gamma counter measurement showing a significantly higher uptake by a factor of 1.7 and 1.6, respectively, in the aortic arches of the transgenic mice vs. wild-type animals. *P < 0.05.

 
3.4 Plasma levels of GPVI-Fc after intraperitoneal application
To estimate the blood level of GPVI-Fc after repeated intraperitoneal injection of 200 µg GPVI-Fc, a pharmakokinetik study in ApoE–/– mice was done. After 2 weeks, an average plasma level of 8.6 µg/mL was determined and after 3 weeks 8.9 µg/mL (Table 1).


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  		Table 1 Intraperitoneal injection of 200 µg GPVI-Fc in 8-week-old ApoE–/– mice twice weekly: plasma levels (mean±SEM) are shown in 1 and 7 mice, respectively, 14 h, 1, 2 and 3 weeks after first injection

 
3.5 Inhibition of plaque and neointima formation after denudation in ApoE–/– mice
To analyse the effect of GPVI-Fc on plaque extension and neointima formation, 4-week-old ApoE–/– mice received a 4-week cholesterol diet. Mechanical injury was then induced by denudation of the left carotid artery and GPVI-Fc or human-Fc was applied for another 8 days. Neointima formation could be detected in all Fc-treated mice, whereas it was totally absent or significantly reduced in GPVI-Fc treated mice (Figure 4A).


Figure 4
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  		Figure 4 Inhibition of plaque and neointima formation by GPVI-Fc after denudation in ApoE–/– mice. Histomorphometry and histochemistry analysis of carotid arteries and aortic arches 4 weeks after denudation. (A) Haematoxylin-Eosin- and elastica von Gieson stainings at lesion sites show a lumen-narrowing neointima formation in all Fc-treated mice (n = 5, bottom) which was totally absent or significantly reduced in GPVI-treated mice (n = 6, top). Bars represent 100 µm. (B) Quantification of the luminal stenosis (defined as percental lumen narrowing) revealed that GPVI-treatment reduced luminal stenosis significantly compared with control animals treated with human-Fc. (C) Determination of the neointima/media ratio shows a significant difference between the GPVI-treatment group vs. the control group. *P < 0.05.

 
Quantification of the luminal stenosis (defined as percental lumen narrowing) correlated with the measurement of absolute neointima area. Accordingly, GPVI-Fc treatment was found to reduce luminal stenosis significantly compared to control animals treated with human-Fc, respectively (63.2 ± 12.4%, n = 5, vs. 19.4 ± 6.2%, n = 6, P < 0.05, Figure 4B). Evaluation of media thickness revealed only a small, but statistically not significant reduction of media area in GPVI-Fc treated mice by 19% vs. control mice (P = 0.57, Figure 4C). Determination of the neointima/media ratio, however, provided evidence for a significant difference between the GPVI treatment group vs. the control group, respectively (0.2 ± 0.01, n = 6, vs. 1.8 ± 0.7, n = 5, P < 0.05, Figure 4C). By staining with Sudan-III, atherosclerotic plaques could be detected in both groups. Determination of relative plaque area revealed that after treatment with GPVI-Fc, the relative extension of plaques at the aortic arches were reduced by 32% vs. control mice (plaque area: 37.6 ± 5.5%, n = 5 vs. 55.3 ± 7.3%, n = 6, P = 0.07, respectively, figure not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 Supplementary material
 Funding
 References
 
The major findings of the present study are: (i) the soluble platelet collagen receptor GPVI-Fc binds at subendothelial collagenous structures preferentially exposed after vascular injury at carotid arteries of mice. (ii) An increased GPVI-Fc binding and uptake could be observed at atherosclerotic plaques of carotid arteries and aortic arches in ApoE-deficient mice compared to wild-type mice. (iii) Administration of soluble GPVI-Fc over 8 days but not control Fc attenuated neointima formation and atheroprogression in ApoE-deficient mice significantly.

In this work, we could demonstrate the binding and uptake of GPVI-Fc at atherosclerotic arteries of ApoE-deficient mice with ex vivo tissue autoradiography. Moreover, histomorphological examinations showed that 1 week administration of soluble GPVI-Fc attenuated neointima formation in ApoE-deficient animals compared with control mice receiving human control Fc.

The ApoE–/–-mouse model used in this study is a well established model to induce consistent plaque formation after 16–20 weeks with a pathomorphology similar to humans.8,19 However, it is much more difficult to create a mouse model for acute plaque rupture. Earlier reports stated that spontaneous plaque rupture and thrombosis are clearly rare events in ApoE–/– mice.20,21 But, in the meanwhile, a number of studies demonstrated that spontaneous plaque rupture and secondary thrombosis do in fact occur in ApoE–/– and LDLR–/– mice although plaque ruptures and deep erosions were far less frequent than in humans.3,22–25 Previous data suggest that predominantly after long-term, high fat-feeding for more than 20 weeks and in older Apolipoprotein E knockout mice atherosclerotic plaque fissuring and plaque rupture can be observed in the aorta and predominantly in the brachiocephalic artery.3,22,23

In the light of these reports, it can be assumed that our observations of the binding and uptake of radioactively labelled GPVI-Fc at lipid-rich plaques at the aortic arch after 16 weeks cholesterol-feeding occurred at lesions sites where plaques are already fissured or ruptured, where collagenous subendothelial structures are presented, and where platelet adhesion occurs.8,19 This was supported by our additional immunhistochemical data showing atherosclerotic plaques with an inhomogeneous endothelium, high collagen expression which is presented directly to the lumen, and a strong and specific binding of GPVI-Fc to the deendothelialized, collagenous areas at the shoulder regions of the plaque. Furthermore, intravital microscopy demonstrated clearly the severe adhesion of platelets at such plaques (see Supplementary material online, Video S2). However, acute plaque rupture and primary thrombosis compared to humans occur too infrequent in mice for statistically accurate estimates of their prevalence requiring very large groups.3,25 To overcome these problems, we used also an injury model in wild-type and ApoE–/– mice to study the binding of GPVI-Fc after mechanical denudation in wild-type mice, as well as to test the antiatherosclerotic effects of GPVI-Fc after denudation in ApoE–/– mice. To study the adhesion of platelets at the injured endothelium and binding of GPVI-Fc, our more feasible ligation model was used in accordance to earlier binding studies of our group in which adhering platelets and platelet aggregates could be detected and counted (see Supplementary material online, Video S1). Ligation injury is tolerated better by animals and shows a lower risk of complications (e.g. bleedings). On the other hand, neointima formation occurs only after wire-induced injury, not after ligation-induced injury. For this reason, the effects of GPVI-Fc on neointima formation were examined after wire-injury.

Previous studies of our group could already provide proof that at the age of 6 weeks, a substantial increase in platelet adhesion to the carotid endothelium was detectable in ApoE–/– mice and that platelet-endothelium interactions preceded the development of manifest atherosclerotic lesions found in 20-week-old ApoE–/– mice. In contrast, platelet accumulation at endothelial cells was found to be virtually absent in wild-type mice. In the present study, the effects of soluble dimeric GPVI-Fc were studied because GPVI represents one of the most important receptors responsible for platelet recruitment at site of vascular injury.

Autoradiography and gamma counter measurement demonstrated that an increased binding and uptake of radioactively marked GPVI-Fc occurred preferentially at lesion-prone sites in ApoE–/– mice, predominantly in the aortic arches of the transgenic mice in contrast to wild-type animals. Accordingly, the aortic arches of the 20-week-old ApoE–/– mice showed also an intensive Sudan-III staining in contrast to wild-type mice indicating the formation of lipid-rich atherosclerotic plaques which are prone for plaque rupture and presentation of subendothelial collagen. In fact, an enhanced GPVI-Fc binding and uptake could be measured predominantly at these sites.

However, injury of the carotid arteries of wild-type mice after ligation of the carotid artery resulted in qualitatively and quantitatively increased tracer uptake in the injured vessels compared to the control carotids in autoradiography studies.

Together, the present data demonstrate that both vascular lesions induced by spontaneous plaque formation in ApoE–/– mice as well as after mechanical injury of carotid arteries in wild-type mice are prone for GPVI-Fc binding and uptake. In addition, previously published data of our group have already shown that intravenous administration of 1 or 2 mg/kg GPVI-Fc in mice reduced firm platelet adhesion by 49 and 65%, as determined by intravital microscopy. To achieve inhibition of platelet adhesion after intraperitoneal administration, 200 µg GPVI-Fc was applied in the present study which corresponds to a 5-fold higher dose of 10 mg/kg GPVI-Fc. Further, in vitro studies have shown a half maximal binding of GPVI-Fc to immobilized collagen at 6.0 µg/mL GPVI-Fc. The levels of ~8 µg/mL measured here correspond to the effective half-maximal dose determined for the inhibition of collagen-binding in vitro. Potential species differences (application of human GPVI-Fc into mice) are not relevant because there is a high sequence homology between human and mouse collagens. Collagens are highly conserved structures in evolution which do not differ significantly between species. In addition, previous binding studies of our group between human GPVI-Fc and collagens I and III of mouse, rat, rabbit, and human origin showed that GPVI-Fc binding did not differ significantly between different species (unpublished data).

To further address whether plaque progression after mechanical denudation of atherosclerotic plaques can be slowed by the inhibition of platelet adhesion receptors, the effects of the soluble immunadhesin GPVI-Fc were studied. Previously, a reduction of platelet adhesion to the atherosclerotic vessel wall and a decrease of atherosclerotic lesion formation could be observed after treatment of ApoE-deficient mice with a monoclonal anti-GPIb{alpha} antibody or in GPIIb–/–ApoE–/– mice.4,6 Aortic arch and carotid lesion formation could be reduced by about 35 and 81%, respectively, after long-term treatment with the GPIb{alpha}-antibody for 12 weeks4 and in GPIIb–/–ApoE–/– mice 20 and 66% less aortic arch and carotid lesions were observed, respectively, after 12 weeks.6

In the present study, we assessed the area of neointima and media, the vessel lumen and determined the stenotic degree and plaque burden after systemic treatment with GPVI-Fc for only 8 days vs. using human control-Fc. Notably, GPVI-Fc but not control-Fc treated mice showed a reduction of plaque area by 22% and a significantly lower neointima formation compared with untreated control animals by 88% after only 1 week. Furthermore, neointima/media ratio was significantly lower in GPVI-Fc-treated mice vs. Fc-treated mice. As a result, luminal stenosis was found to be reduced in GPVI-Fc-treated mice by 69%.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
Supplementary material
 Funding
 References
 
In summary, GPVI-Fc attenuated atheroprogression when applied for only 1 week after vascular injury in carotid arteries of ApoE–/– mice. In contrast, in ApoE–/– mice treated with human control-Fc atheroprogression in the carotid artery was neither accelerated nor retarded.

Together, the present data demonstrate that inhibition of GPVI-binding and uptake at sites of atherosclerotic lesions after wire-induced injury attenuates neointima formation significantly and results in an attenuation of atheroprogression. In the light of these results, inhibition of platelet adhesion via GPVI-Fc might also have a crucial influence on atheroprogression in humans.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Supplementary material
 Funding
References
 
The study was supported in part by the Deutsche Forschungsgemeinschaft (Graduiertenkolleg GK794, MA121/2-1), Novartis-Stiftung, the Karl & Lore Klein Stiftung and the Karl Kuhn Stiftung to M.G.


   Acknowledgements
 
We acknowledge the excellent technical assistance of Funda Cay, Daniel Bukala, and Sandra Bundschuh.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 Supplementary material
 Funding
 References
 

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