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Strikingly Different Angiogenic Properties of Endothelial Progenitor Cell Subpopulations

Insights 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.

Manuscript received June 27, 2007; revised manuscript received August 15, 2007, accepted September 10, 2007.

^_* Reprint requests and correspondence: Dr. Martin K. C. Ng, Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, Sydney, NSW 2050, Australia. (Email: mkcng{at}med.usyd.edu.au).

Objectives: An endothelial cell (EC)-specific angiogenesis assay was developed to functionally characterize angiogenic properties of 2 distinct putative endothelial progenitor cells (EPCs): early EPCs and late outgrowth endothelial cells (OECs).

Background: Endothelial progenitor cells promote revascularization of ischemic tissue. However, the nature of different EPCs and their role in angiogenesis remains debated.

Methods: Tubulogenesis was assessed by immunohistochemistry in co-cultures of differentiated ECs (including human umbilical vein, coronary artery, and microvascular ECs) or non-ECs with monolayers of human fibroblasts (MRC5). Using adaptations of the co-culture assay, early EPCs and OECs, isolated from peripheral blood mononuclear cells, were assessed by 3-dimensional immunofluorescence microscopy for their capacity for: 1) independent tubulogenesis; 2) incorporation into pre-existing vascular networks; and 3) paracrine angiogenic effects using transwell cultures.

Results: Branched interconnecting EC-specific tubules formed with all differentiated ECs after 72 h. Proangiogenic and antiangiogenic agents modulated tubulogenesis appropriately (vascular endothelial growth factor 10 ng: +142 ± 13%, 1 µM anti-vascular endothelial growth factor: –44 ± 7% vs. control, p < 0.001). In contrast, early EPCs, along with nonendothelial cell types, failed to independently form tubules or incorporate into differentiated EC tubules. Nevertheless, early EPCs indirectly augmented tubulogenesis by differentiated ECs even when physically separated by transwells (+115 ± 4% vs. control; p < 0.001). By contrast, OECs independently formed tubules and incorporated into differentiated EC tubules but exerted no significant paracrine angiogenic effects.

Conclusions: A novel EC-specific tubulogenesis assay highlights strikingly different angiogenic properties of different EPCs: late OECs directly participate in tubulogenesis, whereas early EPCs augment angiogenesis in a paracrine fashion, with implications for optimizing cell therapies for neovascularization.

Abbreviations and Acronyms   ANOVA = analysis of variance   Dil-AcLDL = 1,1'–dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate labeled acetylated low-density lipoprotein   EC = endothelial cell   eNOS = endothelial nitric oxide synthase   EPC = endothelial progenitor cell   MNC = mononuclear cell   OEC = late-outgrowth endothelial cell   VEGF = vascular endothelial growth factor

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