Monoclonal Antibody against α-Actinin 4 from Human Umbilical Vein Endothelial Cells Inhibits Endothelium-Dependent Vasorelaxation

Background: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. Methods: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. Results: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or- denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and alpha-actinin 4. The muscarinic M3 ACh receptor and alpha-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. Conclusions: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of a-actinin 4. Copyright (C) 2013 S. Karger AG, Basel