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Fig. 3. Capillary formation by and in vitro maturation of CD34+
VW-EPC. Immunostaining on sections of paraffin wax-embedded HITA rings
after being subjected to ring assay demonstrates that intraluminar new
capillaries exhibit both CD34 (A) and CD31 (B), whereas
capillary outgrowth within the wall of and outside the HITA rings stains only
positive for CD34 (arrows) (C). CD31 is detectable in vasa vasorum
(arrows) (D) but not in new vessels at this stage of vascular
formation. Capillarization through the entire vascular wall, as indicated by
positive staining for CD34, occurs when rings were cultured for more than 14
days (E). Cells located in the CD34+ vasculogenic zone
(arrows) and cells forming new tubes within the HITA lumen (arrowheads)
exhibit immunostaining for VEGFR2 (F) and TIE2, the receptor for Ang1
and Ang2 (G). The endothelial marker VE-cadherin is present in mature
endothelial cells lining the vascular lumen and in the newly formed vessels in
the lumen (arrowheads) (H). Remarkably, VE-cadherin is also visible at
the cell-cell contacts (arrowheads) of new vessel sprouts outside the rings
(I). Immunostaining for 
-smooth muscle actin after the ring
assay revealed only few positive cells within the adventitial layer
(arrowhead) (J) and also within the collagen gel outside the rings
(arrowheads) (K) that mostly tightly associated to the endothelial
tubes (indicated by broken lines) or to single cells (arrow). (C,D,E,H,I,J,K)
Counterstaining with Calcium Red; (A,B,F,G) counterstaining with
Hematoxylin.
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