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

Abstract

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 α-actinin 4. The muscarinic M3 ACh receptor and α-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 α-actinin 4.

A monoclonal antibody derived by inoculation of human umbilical vein endothelial cells is a potential inhibitor of acetylcholine receptor-linked vasorelaxation

“…To further investigate another monoclonal antibody associated with vascular function, we tested the effects of antibodies with specific reactivity to HUVECs other than the antibody (C7) with vascular reactivity, which is one of the seven antibodies exhibiting more than 80% specific reactivity to HUVECs in a previous study. Among the different antibodies, A-7 displayed 45.9% specific reactivity to HUVEC as reported previously (Won et al 2013) and affected vascular reactivity as shown in Figure 1. Treatment of ACh (0.01-1 μM) elicited concentration-dependent inhibition of NE (0.1 μM)-induced contraction in endothelium-intact aortic ring isolated from rats, with a maximum inhibition at 1 μM of ACh (Figure 1; n = 4).…”

“…Male Sprague-Dawley (SD) rats (7-weeks-old, 180-190 g, n = 10) were purchased from Orient Bio (Korea). The animals were euthanized using CO 2 gas inhalation and bled rapidly by severing the carotid arteries (Won et al 2013;Raffetto et al 2019). The thoracic aorta was rapidly and carefully removed and placed in a physiological salt solution (PSS) containing the following composition (in mM): NaCl 136.9; KCl 5.4; CaCl 2 1.5; MgCl 2 1.0; NaHCO 3 23.8; EDTA 0.01.…”

“…The A-7 monoclonal antibody against HUVECs was produced by injecting HUVECs into 6-week-old SD rats (Orient Bio) as described previously (Won et al 2013). Briefly, rats were immunized by injecting 3 × 10 5 HUVECs on days 0 and 7 in both hand footpads.…”

“…In a previous study, we generated 22 monoclonal antibodies that regulated the activities of vascular system and identified a vasoactive antibody (Won et al 2013). To further investigate another monoclonal antibody associated with vascular function, we tested the effects of antibodies with specific reactivity to HUVECs other than the antibody (C7) with vascular reactivity, which is one of the seven antibodies exhibiting more than 80% specific reactivity to HUVECs in a previous study.…”

“…Therefore, the identification of new functional molecules may provide basic insight into and understanding of their physiological mechanisms in targeted cells including vascular cells. Our laboratory has developed 22 monoclonal antibodies from rats inoculated with HUVECs (Won et al 2013). Although these antibodies display functional vascular responses, they may be additional antibodies possessing potential vascular reactivity.…”