Expression profiling-defined BEC-specific proteins are expressed in vivo but lost in vitro. (A-B) In normal human skin, BECs but not LECs express MHC class II and melanoma cell adhesion molecule (MCAM)/CD146. Sections (7 μm) of snap-frozen normal human skin were subjected to triple immunolabeling using Abs specific for VE-cadherin (blue), PDPN (green), and MHC class II (red) (A) or CD146 (red) (B) and confocal laser scanning microscopy. Uniform MHC class II (A) and CD146 expression (B) by VE-cadherin⁺PDPN⁻ blood vessel ECs is shown by purple coloration of these vessels in the overlay exposures. In contrast, VE-cadherin⁺PDPN⁺ lymph vessel ECs fail to express MHC class II (A) or CD146 (B). Images shown in the upper and lower panels were taken from areas containing the superficial and the deep vascular plexus of the skin, respectively. Dotted lines in the upper panels denote the epidermodermal junction. MHC class II⁺VE-cadherin⁻ cells in panel A are epidermal LCs and DDCs. BEC-restricted protein expression in normal human skin was also verified for Duffy antigen, junctional adhesion molecule (JAM2), C1q receptor protein 1 (C1QRp1), and E- and P-selectin (data not shown). (C) Most identified BEC-restricted antigens are expressed by freshly isolated cells but are lost in cell culture. BECs (red histograms) and LECs (green histograms) either freshly isolated (left) or cultured (right) were immunostained with mAbs recognizing the indicated pan-EC (CD31) or BEC-restricted antigens (HLA-DR, Duffy antigen, JAM2, E-selectin, ICAM-1, CD146) and analyzed by flow cytometry. Cultured, nonproliferative BECs and LECs were harvested from confluent cultures at passage 4. Note that, although BEC-restricted antigen expression clearly separated freshly isolated BECs from freshly isolated LECs, this divergent expression was lost in cultured ECs mostly because of a dramatic down-regulation of antigen expression in BECs. The only notable exception is the preferential expression CD146 in BECs that is maintained in culture.