Fig. 4. 6ß4-integrin-positive endothelial cells of the vasa vasorum contribute to angiogenesis in explant cultures of human saphenous vein. Human saphenous vein was treated with collagenase to strip the endothelial lining from the main lumen. (A,B) Cross sections of collagenase-treated explants were stained with pAb to PECAM-1 (A) and 3E1 to ß4 (B). Endothelial cells of the vasa vasorum maintained PECAM-1 and ß4 expression (black arrowheads); however, expression of PECAM-1 was not observed on the lumenal surface (open arrowhead), indicating the endothelial cells had been successfully removed. (C,D) Phase contrast images of endothelial outgrowth from collagenase-treated (D) or untreated (C) segments of saphenous vein after 14 days in culture in fibrinogen gels. Little angiogenic outgrowth occurred in the collagenase-treated lumen (D); however, robust endothelial outgrowth was observed from the vasa vasorum (E). (F-I) Cross sections of collagenase-treated saphenous vein explants were immunostained after 14 days in culture with either pAb to PECAM-1 (F,G), or with 3E1 to ß4 (H), indicating the outgrowing endothelial cells (arrow) do not express ß4. A region of outgrowth stained for PECAM-1 is shown at higher magnification in G. (I) A phase contrast image of H. (F,H) The perimeter of the explant is outlined by the dotted blue line. Controls with secondary antibody alone showed insignificant staining similar to the controls for immunostainings in Fig. 2 (data not shown). (J) Day-12 explant of human neonatal foreskin double stained with AE1/AE3 to human epidermal keratins (red) and 439-9B to ß4 (green), demonstrating that keratinocytes do not downregulate ß4 in explant cultures. Asterisks indicate vessel lumen. Plus signs indicate original explanted tissue. Arrows indicate outgrowing endothelial cells or keratinocytes. Scale bars: (A,B,F,H,I) 100 µm; (G) 20 µm; (J) 50 µm.

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