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Live monitoring of small vessels during development and disease using the flt-1 promoter element

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Abstract

Vessel formation is of critical importance for organ function in the normal and diseased state. In particular, the labeling and quantitation of small vessels prove to be technically challenging using current approaches. We have, therefore, established a transgenic embryonic stem (ES) cell line and a transgenic mouse model where the vascular endothelial growth factor receptor VEGFR-1 (flt-1) promoter drives the expression of the live reporter eGFP. Fluorescence microscopy and immunostainings revealed endothelial-specific eGFP labeling of vascular networks. The expression pattern recapitulates that of the endogenous flt-1 gene, because small and large vessels are labeled by eGFP during embryonic development; after birth, the expression becomes more restricted to small vessels. We have explored this in the cardiovascular system more in detail and found that all small vessels and capillaries within the heart are strongly eGFP+. In addition, myocardial injuries have been induced in transgenic mice and prominent vascular remodeling, and an increase in endothelial cell area within the peri-infarct area could be observed underscoring the utility of this mouse model. Thus, the transgenic flt-1/eGFP models are powerful tools to investigate and quantify vascularization in vivo and to probe the effect of different compounds on vessel formation in vitro.

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Acknowledgments

We thank A. Nagy (Mount Sinai Hospital Toronto) and M. Gertsenstein (Toronto, Canada) for providing the G4 ES cell line, N. Copeland (NCI, Frederick, USA) for providing SW105 cells, R. Schneider-Kramann (University of Aachen) for help with teratoma analysis and Y. Matuschek (University of Bonn) for assistance in vector cloning. Funding was provided to the junior research group of D.W. by the Ministry of Innovation, Science, Research and Technology of the State of North Rhine-Westphalia. This work was further supported EU FP7 consortium CardioCell Grant No. 223372 (to BKF).

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Authors and Affiliations

  1. Institute of Physiology I, University of Bonn, Sigmund-Freud-Str. 25, 53127, Bonn, Germany

    Katia Herz, Jan C. Heinemann, Michael Hesse, Annika Ottersbach, Caroline Geisen, Christopher J. Fuegemann, Bernd K. Fleischmann & Daniela Wenzel

  2. Department of Cardiac Surgery, University of Bonn, Bonn, Germany

    Wilhelm Röll

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  1. Katia Herz
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Correspondence to Daniela Wenzel.

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K. Herz and J. C. Heinemann contributed equally to the work.

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Suppl. Fig. 1: Re-formation of vascular networks after dissociation of flt-1/eGFP EBs. a) After enzymatic dissociation of day 5 (d 5) EBs and re-plating on gelatine single eGFP+ cells were visible one day later. b) After 3 days eGFP+ sprouts have formed (arrow). c) The sprouts generated network-like vascular structures 7 days after dissociation. d) Re-formed vascular networks were found through day 12, bar= 300 μm.

Suppl. Fig. 2: EGFP+ vessels in adult organs. a-f) Immunohistochemistry revealed endothelial-specific eGFP expression in vessels of the thymus of chimeric (a-c) and of the kidney of heterozygous mice (d-f), inset: also vessels of the glomeruli were eGFP+, green=eGFP, red=PECAM, blue=Hoechst, magenta=autofluorescence, bars=50 μm.

Suppl. Fig. 3: EGFP expression in the endothelium of large adult vessels. a-i) Immunohistochemistry showed that adult aorta displayed no eGFP labeling in endothelial cells (a-c), this was also true for the carotid artery (d-f). In contrast, the endothelium of the murine jugular vein was highly eGFP+ (g-i), green=eGFP, red=PECAM, bar=50 μm.

Suppl. Fig. 4: EGFP+ structures and leukocytes invading the peri-infarct zone after cryoinfarction. a-c) Immunohistochemistry showed that eGFP+ vascular structures in the peri-infarct zone 3 days after cryoinfarction were CD45- (green=eGFP, magenta=CD45, blue=Hoechst), bar=50 μm.

Suppl. Video file: The video shows sprouting angiogenesis within an flt-1/eGFP BAC EB on day 5+5. Vascular sprouts elongated and connected to early vascular networks. Video covered app. 12 h (acquisition at 4 frames per hour (fph), display rate at 10 fps) and was recorded with a 20× objective.

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Herz, K., Heinemann, J.C., Hesse, M. et al. Live monitoring of small vessels during development and disease using the flt-1 promoter element. Basic Res Cardiol 107, 257 (2012). https://doi.org/10.1007/s00395-012-0257-5

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