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Fig. 7. CD45 and CD68 positive cells in HITA wall before and after ring assay. Similar to CD34⁺ cells, CD45⁺ cells (arrows) are localized at the transitional zone between smooth muscle cell layer (SMC) and adventitial layer (ADV) (A, at higher magnification in B), but their number is significantly lower than that of CD34⁺ cells. No specific reaction is visible in endothelial cell layer (EC). Further immunohistochemical studies demonstrate in only a few cases sporadic and randomly distributed CD68⁺ cells in the adventitial layer (arrows) of untreated HITA (C). They are mostly localized distant from the mural zone where CD34⁺ and CD45⁺ cells were found. By contrast, a significant accumulation of CD68⁺ cells is visible in the adventitial layer including the zone containing the CD34⁺ and CD45⁺ cells when HITA rings were subjected to the ring assay (D). Some of these macrophages migrate into the collagen gel outside the ring (arrow) (E) and are involved in the capillary sprouts (arrow) in the collagen gel (arrow) (F). FACS-analyses of cells isolated from the collagen gel after ring assay demonstrate the existence of different cell populations such as CD133⁺ cells, CD45⁺ cells and CD105⁺ cells (G-I) among the cells migrated into the gel and/or involved in vessels sprouting from HITA. These FACS analyses show that approximately 20% of the CD133 positive cells exhibit CD45 (G). Unexpectedly, more than 60% of CD34⁺ cells co-express CD105 (H), although freshly isolated cells from the HITA wall did not express CD105 when prior to these analyses the pre-existing mature endothelial cells were removed via CD105 conjugated super paramagnetic microbeads. Furthermore, CD133⁺ cells also exhibit CD105 approximately in equal intensity (I). IgG-FITC and IgG-PE were used as control (J).

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