O24	Homing of CD34+ progenitor cells to sites of angiogenic tube formation using real-time video microscopy.
1R.E.Verloop, 1M.A.Engelse, 2H.C.de Boer, 2A.J.van Zonneveld, 1V.W.M.van Hinsbergh
1Institute for Cardiovascular Research, Laboratory for Physiology, VU University medical center, Amsterdam, NL; 2Department of Nephrology, Leiden Univiversity Medical Center, Leiden, NL.

In addition to classical angiogenic sprouting as a vector for neovascularization, bone marrow-derived circulating progenitor cells have been indicated as a potential source for vascular endothelial cells in neovascularization, in a process called adult vasculogenesis. Recently published reports suggest that the CD34+ hematopoietic stem cell population contains cells that can give rise to endothelial progenitor cells (EPC) and endothelial cells, suggesting the potential use of these cells for therapeutic neovascularization. The present study was conducted to determine the effect of CD34+ progenitor cells on the formation of capillary-like tubes by human foreskin-derived microvascular endothelial cells (hMVEC) in vitro. Using real time video microscopy we studied the process of homing of progenitor cells to sites of tube formation in a fibrin-based 3D capillary tube formation assay. In this assay capillary-like tubular structures were induced by the simultaneous presence of bFGF and TNF? during a 4-day period. 24h after induction of tube formation CD34+ progenitor cells isolated from human cord blood and added to the culture. Movement of the CD34+ cells over the hMVEC monolayer was recorded by time-lapse video microscopy from 2 to 8 h after CD34+addition. During analysis of the data the areas of tubular structures were marked and the number of cells entering and leaving the areas were counted. As control the same surface areas were projected to areas of the same hMVEC culture but devoid of tubes. During movement over the endothelial monolayer the percentage of the CD34+ cells that migrated towards the sites of tube formation and remained there was 80 ± 1 of the total number of entering cells, while in the non-tubular areas this figure was 17-fold less (4.6 ± 1.2%). Immunohistochemistry confirmed that the migrated CD34+ cells were viable. Only a small fraction of the cells incorporated into the tubular structures. Blocking antibodies against ICAM-1, VCAM-1, E-selectin, and P-selectin did not inhibit this process.

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