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Capture of endothelial progenitor cells by a bispecific protein/monoclonal antibody molecule induces reendothelialization of vascular lesions

Abstract

Tissue injury is inevitably accompanied by disruption of the endothelium and exposure of the subendothelial matrix. To generate a guidance molecule directing progenitor cells to sites of vascular lesions, we designed a bifunctional protein. The protein consists of the soluble platelet collagen receptor glycoprotein VI and an antibody to CD133 (hereafter called GPVI-CD133). In vitro and in vivo, this construct substantially mediates endothelial progenitor cell (EPC) homing to vascular lesions. Exposure of EPCs to GPVI-CD133 did not impair their capability to differentiate toward mature endothelial cells as verified by the formation of colony-forming units, the upregulation of endothelial markers CD31 and CD146 analyzed by flow cytometry or von Willebrand factor and endoglin assessed by immunofluorescence microscopy, as well as the presence of Weibel–Palade bodies using transmission electron microscopy. In vivo, GPVI-CD133 augments reendothelialization of vascular lesions. Thus, this bifunctional protein could be a potential new therapeutic option for cardiovascular diseases.

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Acknowledgment

We acknowledge the excellent technical assistance of Sarah Gehring, Jadwiga Kwiatkowska, Heike Runge, Sandra Bundschuh, and Birgit Fehrenbacher. We thank Rupert Handgretinger for critical revision and supply of stem cells.

Funding

The study was supported by grants from the Deutsche Forschungsgemeinschaft (Graduiertenkolleg “Zellbiologische Mechanismen immunasoziierter Prozesse,” GK 794 and “Vaskuläre Medizin” [GRK 438 and MA 2186/3-1] to MG), SFB-TR19, and the Novartis Foundation and the fortüne research program of the UKT to HFL and MG. The Bio-Rad TPLSM was obtained via a grant (no. 902-16-276) from the Medical Section of the Dutch Scientific Organization. Experiments carried out by JW v.d. R, SC, and TS were financed by the Center for Regeneration Biology and Regenerative Medicine by the Carl Baresel Stiftung and Stiftung Landesbank Baden Württemberg.

Potential conflict of interest

None.

Author information

Correspondence to Thomas Skutella or Meinrad Gawaz.

Additional information

Harald F. Langer, Jürgen W. von der Ruhr, Thomas Skutella, and Meinrad Gawaz contributed equally, and T.S. and M.G. share senior authorship.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Dynamic adhesion of EPCs to GPVI-CD133 in vitro (a). Under arterial shear conditions (2,000 s−1), adhesion of EPCs to collagen additionally covered with the individual components of the construct (10 μg/mL each) is shown from minute 08:30 to minute 09:00 of perfusion. Virtually no firm adhesion of EPCs could be observed (MOV 264 kb)

Dynamic adhesion of EPCs to GPVI-CD133 in vitro (b). This sequence from minute 06:00 to minute 09:00 is representative for flow chamber experiments, in which the bispecific construct (10 μg/mL) was applied. Adherent EPCs are distributed over the complete frame (MOV 3252 kb)

Recruitment of EPCs to vascular lesions by GPVI-CD133 in vivo (a). In C57BL/6J mice, the common carotid artery was injured by ligation and DCF (green)-stained EPCs were injected intravenously. When the cells were incubated with both individual components of the construct, significantly less EPCs adhered to the injured vessel wall than in (MOV 1429 kb)

Recruitment of EPCs to vascular lesions by GPVI-CD133 in vivo (b) experiments with EPCs treated with the GPVI-CD133 construct. Both films are recorded 30 min after the induction of vascular injury (MOV 2292 kb)

Z-stack movie (see also supplemental figure 1) as imaged by TPLSM of an intact, nonvital carotid artery mounted in a flow chamber (obtained from intravital microscopy experiments) (a) (MOV 3660 kb)

EPCs were incubated with the GPVI-CD133 construct, but the carotid artery is intact. Screening starts from the luminal side of the vessel. First, the endothelial monolayer (red nuclei), then the media and the adventitia become visible. The media shows smooth muscle cell nuclei (red), the adventitia fibroblasts (red nuclei) embedded in collagen (blue, SHG). No DCF stained EPCs can be observed on the luminal side of the vessel (b) (MOV 1667 kb)

Supplemental Figure 1

(PDF 1.49 MB)

Supplemental film 1

Dynamic adhesion of EPCs to GPVI-CD133 in vitro (a). Under arterial shear conditions (2,000 s−1), adhesion of EPCs to collagen additionally covered with the individual components of the construct (10 μg/mL each) is shown from minute 08:30 to minute 09:00 of perfusion. Virtually no firm adhesion of EPCs could be observed (MOV 264 kb)

Supplemental film 1

Dynamic adhesion of EPCs to GPVI-CD133 in vitro (b). This sequence from minute 06:00 to minute 09:00 is representative for flow chamber experiments, in which the bispecific construct (10 μg/mL) was applied. Adherent EPCs are distributed over the complete frame (MOV 3252 kb)

Supplemental film 2

Recruitment of EPCs to vascular lesions by GPVI-CD133 in vivo (a). In C57BL/6J mice, the common carotid artery was injured by ligation and DCF (green)-stained EPCs were injected intravenously. When the cells were incubated with both individual components of the construct, significantly less EPCs adhered to the injured vessel wall than in (MOV 1429 kb)

Supplemental film 2

Recruitment of EPCs to vascular lesions by GPVI-CD133 in vivo (b) experiments with EPCs treated with the GPVI-CD133 construct. Both films are recorded 30 min after the induction of vascular injury (MOV 2292 kb)

Supplemental film 3

Z-stack movie (see also supplemental figure 1) as imaged by TPLSM of an intact, nonvital carotid artery mounted in a flow chamber (obtained from intravital microscopy experiments) (a) (MOV 3660 kb)

Supplemental film 3

EPCs were incubated with the GPVI-CD133 construct, but the carotid artery is intact. Screening starts from the luminal side of the vessel. First, the endothelial monolayer (red nuclei), then the media and the adventitia become visible. The media shows smooth muscle cell nuclei (red), the adventitia fibroblasts (red nuclei) embedded in collagen (blue, SHG). No DCF stained EPCs can be observed on the luminal side of the vessel (b) (MOV 1667 kb)

Supplemental film 3

Instead, an obvious enrichment of green (DCF) cells with red nuclei can be observed in the area of denudation on the luminal side of the elastica interna, where denudation was carried out and cells treated with GPVI-CD133 were applied. A zoomed z-stack of this experiment is shown in film (c) (MOV 737 kb)

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Langer, H.F., von der Ruhr, J.W., Daub, K. et al. Capture of endothelial progenitor cells by a bispecific protein/monoclonal antibody molecule induces reendothelialization of vascular lesions. J Mol Med 88, 687–699 (2010). https://doi.org/10.1007/s00109-010-0614-5

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Keywords

  • Regenerative medicine
  • Endothelialization
  • Progenitor cells
  • Stem cells
  • Vascular injury
  • Guidance molecule
  • Vascular disease
  • Therapy