gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

Improved homing of endothelial progenitor cells by the bispecific protein GPVI-CD133

Meeting Contribution

  • corresponding author T. Schönberger - Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard Karls University Tübingen, Germany
  • H. Langer - Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard Karls University Tübingen, Germany
  • A. Gauß - Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard Karls University Tübingen, Germany
  • R. Hafner - Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard Karls University Tübingen, Germany
  • J. von der Ruhr - Anatomisches Institut, Eberhard Karls University Tübingen, Germany
  • M. Schumm - Kinderklinik, Eberhard Karls University Tübingen, Germany
  • H.-J. Bühring - Medizinische Klinik II, Eberhard Karls University Tübingen, Germany
  • M. van Zandvoort - Department of Biophysics, Faculty of Medicine, University Maastricht, Netherlands
  • G. Jung - Institut für Zellbiologie, Abteilung Immunologie, Eberhard Karls University Tübingen, Germany
  • T. Skutella - Anatomisches Institut, Eberhard Karls University Tübingen, Germany
  • M. Gawaz - Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard Karls University Tübingen, Germany

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf18

DOI: 10.3205/10dgaf18, URN: urn:nbn:de:0183-10dgaf183

Published: March 23, 2011

© 2011 Schönberger et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

We designed a bifunctional protein, capable of capturing circulating endothelial progenitor cells to collagen-rich vascular lesions. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133. This construct substantially mediates EPC homing to vascular lesions. Furthermore, it augments reendothelialization of vascular lesions and reduces the extent of myocardial infarction. Therefore, this bifunctional protein could be a potential new therapeutic option in regenerative vascular medicine.


Introduction

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. Circulating endothelial progenitor cells (EPCs) contribute decisively to regeneration of vascular integrity [1]. By increased recruitment of progenitor cells healing of vascular lesion could be supported. The main component of the subendothelial matrix is collagen. To direct EPCs to endothelial lesions we developed a bifunctional protein consisting of the soluble platelet collagen receptor glycoprotein VI (GPVI) [2], [3] and a monoclonal antibody against human CD133. Progenitor cells expressing the surface antigen CD133 have a high regenerative potential [4], [5].

Aim of this study was to evaluate the in vivo functionality of this protein in different mouse models.


Methods

Isolation of CD34+ stem cells

CD34+ stem cells were were isolated from remnants of human leukapheresis transplants by MACS separation with anti-CD34+ coated microbeads as described elsewhere [6].

Intravital microscopy

C57Bl/6J mice were anesthetized and the carotids were dissected free. Via catheter 5x104 DCF-labeled CD34+ progenitor cells were injected into the V. jugularis. The cells were preincubated for 30 min with the GPVI-CD133-construct or the single components as control, respectively. A vascular lesion was induced by a 5 min lasting thread ligation of the A. carotis com. Before and after induction of the injury recording of cell-endothelium-interactions by intravital fluorescence video microscopy was performed. Subsequently, the number of transient-adherent and adherent cells was determined.

Reendothelialization after vessel injury

In male NOD/Scid mice an injury was induced as discribed above. Directly afterwards 5x104 human CD34+ progenitor cells, preincubated with construct or the single components, were injected into the tail vein. 14 days after the intervention the mice were sacrificed and the vessels were removed, paraffin-embedded, cut in sections and stained with HE. To demonstrate regeneration starting from human cells, an in situ hybridization using a DNA-Alu sequence (Alu-ISH) specific for human cells was performed [7].

Neointima formation after vessel injury

In male NOD/Scid mice a wire-induced injury of the A. carotis com was performed. At day 0 and day 3 5x105 CD34+ progenitor cells treated for 30 min with GPVI-CD133 construct (20 µg/ml) or with both single components of the construct together (20 µg/ml each), respectively, were injected intravenously. 3 weeks later the animals were killed. The carotid artery specimens were removed, embedded in paraffin blocks and cut into 5µm sections. After staining with elastic van Gieson the media and neointima areae were determined.

Myocardial ischemia and reperfusion

In anesthetized NOD/Scid mice myocardial infarction was induced by ligation of the LAD for one hour. Afterwards 5x105 CD34+ progenitor cells (± GPVI-CD133) were injected via the tail vein. One week later the mice were killed. To show the extent of the ischemia-induced damaged area the hearts were stained with TTC (triphenyltetrazolium-chloride).


Results

By intravital fluorescence microscopy in the murine carotis ligation modell we could show enhanced adhesion to injured endothelium of CD34+ endothelial progenitor cells which were preincubated with the GPVI-CD133-construct compared to control (MW±SEM: 450±92 vs. 198±58).

One week after ligation-induced injury of the A. carotis com. and application of CD34+ cells there were more EPCs integrated into the endothelium in the GPVI-CD133 group compared to control demonstrated by in situ hybridization (18.5±8 vs. 8.75±5).

In NOD/Scid mice subjected to wire-induced injury of the A. carotis 3 weeks after invention less neointima formation could be observed when treated with GPVI-CD133 preincubated cells. This resulted in a lower neointima/media rate (0.97 vs. 0.84) and a lower degree of stenosis (18% vs. 30%) in the construct group.

Similarly, after myocardial ischemia/reperfusion there was a smaller infarction area after application of GPCI-CD133 preincubated CD34+ progenitor cells.


Discussion

The focus of our study was to provide a proof of principle that circulating regenerative cells can be enriched by a bispecific molecule targeting a component of the injured vessel wall (collagen) and an epitope of circulating regenerative cells (in this case CD133), given the fact that a major and unsolved limitation to stem cell-based regenerative therapy remaining is the low grafting efficiency to the target tissue [8].

In this study, we could show in vivo that the use of GPVI-CD133 leads to increased homing of EPCs to endothelial lesions and to improved reendothelialisation of vascular lesions. Furthermore, neointima formation and the extent of myocardial infarction was reduced.

Taken together, this bispecific protein could be a new therapeutic option for cardiovascular diseases and tissue regeneration in general.


References

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