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J.M. Runnels, M. Poureshagh, J. Novak, P. D'Amore, C.P. Lin; Detection of Circulating Endothelial Precursor Cells by in vivo Flow Cytometry . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3229.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: : Angiogenesis and its consequences are devastating in diseases such as proliferative diabetic retinopathy and age related macular degeneration. Postnatal angiogenesis involves release of endothelial precursor cells (EPC) from the stem cell reservoir of bone marrow into the circulation. Like mature endothelial cells which are incorporated into the vasculature, EPC express endothelial–specific receptor tyrosine kinase (Tek or Tie2), but unlike mature endothelial cells, they can be found in the circulation for a limited time. Using FACS analyses of multiple blood samples, EPC have been identified and followed in organisms undergoing angiogenesis. We set out to develop a method to noninvasively identify these cells directly within the circulation of live mice. Methods:Circulating EPC were detected and counted using an in vivo flow cytometer developed in this laboratory. This method offers the advantage of following cell populations over time within an individual without repeated blood sampling. Like FACS analysis, in vivo flow cytometry makes use of laser illumination of a narrow stream of moving cells; specifically, cells circulating through an arteriole. Mice that express green fluorescent protein (GFP) under the control of the Tie2 gene were used to measure bone marrow derived EPC in the circulation before and after intravenous injection of recombinant mouse VEGF (vascular endothelial growth factor). VEGF is a potent angiogenic factor, which has been shown to mobilize VEGFR2, Tie2 positive EPC from the bone marrow. Results: No GFP positive cells were detected in the circulation before VEGF injection. Tie2–GFP positive cells became apparent 40 minutes after a single injection of 1 ug VEGF, and persisted for up to 2 hrs. The peak cell count was about 5 cells/min in a 30 micron arteriole, corresponding to approximately 50,000 cells per ml of blood. Conclusions: In vivo flow cytometry can be used to detect circulating EPC when they are responding to a limited VEGF signal. The response to VEGF was rapid and short–lived. This method may be useful in the study of many conditions in which postnatal angiogenesis is a factor as in conditions involving retinal neovascularization.
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