Identification and functional assessment of endothelial P1H12

Monoclonal antibody P1H12 recognizes circulating endothelial cells and endothelia of all sizes of blood vessels. To identify the protein recognized by P1H12, we expressed a cDNA library in CHO cells and sequenced the cDNA from positive cells. The P1H12 sequence was identical, except at several bases, to that reported for melanoma cell surface antigen MUC18/CD146. Aggregation assays demonstrated that CD146 mediates Ca⁺⁺-independent homotypic endothelial cell adhesion. P1H12 mAb abrogated interactions between human microvascular endothelial cells (HMVECs) but not between human umbilical vein endothelial cells (HUVECs). P1H12 mAb abrogated P1H12-positive (CHO^P1H12)-association with HMVECs or HUVECs. CD146 distribution is sparser on HUVECs than on HMVECs. These data imply that HMVECs and HUVECs express the CD146 binding partner but that CD146 is functional (or at sufficient density) only on HMVECs. HMVEC monolayers treated with soluble P1H12 mAb showed increased permeability to albumin, with accompanying changes in actin, paxillin, FAK, and caveolin distribution and changes in tyrosine phosphorylation of FAK. Stimulation with P1H12 mAb led to redistribution of NF-κB to the nucleus. P1H12 mAb bound to beads inhibited closure of wounded endothelial monolayers. CD146 thus joins VE-cadherin and PECAM-1 as a molecule that mediates homotypic endothelial cell adhesion. CD146 has both structural functions and signaling functions important for endothelial monolayer integrity.