Strikingly Different Angiogenic Properties of Endothelial Progenitor Cell SubpopulationsInsights From a Novel Human Angiogenesis Assay
Daniel P. Sieveking, BSc*, ,
Andrew Buckle, PhD*,
David S. Celermajer, MBBS, PhD, DSc, and
Martin K.C. Ng, MBBS, PhD*,,,*
* Heart Research Institute, Sydney, Australia
Department of Medicine, University of Sydney, Sydney, Australia
Royal Prince Alfred Hospital, Sydney, Australia.
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Figure 1 EC–Specific Human Angiogenesis Assay
(A) Light photomicrograph (x20 original magnification) of endothelial cells (ECs) forming tubular structures (arrows) when co-cultured with MRC5 fibroblasts. (B) Immunofluorescent micrograph (x20 original magnification) of EC tubules, stained for 1,1'–dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate labeled acetylated low-density lipoprotein (Dil-AcLDL) (red), Ulex europaeus agglutinin lectin (UEA-1) (green), and counterstained with 4 , 6-diamidino-2-phenylindole (DAPI) (blue). (C) Light photomicrograph (x10 original magnification) ECs stained for CD31 (brown) (arrows) failed to form tubules when cultured on smooth muscle cells.
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Figure 2 Angiogenic Responsiveness of Angiogenesis Assay
(A) Representative binarized photomicrographs and (B) quantification of tubulogenesis in response to treatment. Data expressed as mean ± SEM. ***p < 0.001 versus control. VEGF = vascular endothelial growth factor.
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Figure 3 Late-OECs Not Early EPCs Form Tubules De Novo
Representative immunofluorescent micrographs of (A) early EPCs and (B) OECs were co-cultured with MRC5 fibroblasts and stained as in Figure 1B. EPC = endothelial progenitor cell; OEC = late-outgrowth endothelial cell.
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Figure 4 Early EPCs Do Not Incorporate Into Vascular Networks
(A) Early EPCs were pre-tagged with CellTracker 5-(((4-Chloromethyl)benzoyl)amine)tetramethyl rhodamine (red), co-cultured with differentiated ECs, then co-cultures were stained with UEA-1, fluorescein isothiocyanate (FITC) (green), and 4, 6-diamidino-2-phenylindole (blue). (B and C) Three-dimensional z-stacking micrograph showing a tagged early EPC and differentiated EC tubule in separate focal planes (white lines above and right of the images represent position in the z axis). Abbreviations as in Figures 1 and 3.
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Figure 5 OECs Incorporate Into Vascular Networks
Co-cultures were stained according to Figure 4. Pre-tagged OECs directly incorporated into developing vascular networks at (A) x20 magnification and (B) x40 magnification. (C) Three-dimensional z-stacking micrograph shows 2 tagged OECs directly incorporated into surrounding differentiated EC tubules in the same focal plane (white lines above and right of the images represent position in the z axis). Abbreviations as in Figures 1 and 3.
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Figure 6 Early EPCs Augment Angiogenesis
Early endothelial progenitor cells (EPCs) augmented angiogenesis by differentiated endothelial cells (ECs) in a dose-dependent fashion. Data expressed as the mean ± SEM. ***p < 0.001 for each concentration of early EPCs versus control. CTRL = control.
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Figure 7 Early EPCs and Not OECs Augment Angiogenesis in a Paracrine Fashion
(A) A transwell apparatus was used to assess the paracrine effects of different EPCs. (B) Early EPCs, still exerted a dose-dependent proangiogenic effect when placed in transwells (TW) (p = 0.01 by analysis of variance). (C) The OECs did not exert proangiogenic effects when placed in transwells (p = 0.3 by analysis of variance). Data expressed as the mean ± SEM. *p < 0.05 versus control. CTRL = control; other abbreviations as in Figure 3.
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