Introduction There is compelling evidence that accumulating monocyte-derived macrophages are pivotally involved in driving liver fibrogenesis. It remains unclear which molecules mediate transmigration of these cells across hepatic sinusoidal endothelial cells (HSEC). VAP-1 is an atypical adhesion molecule with enzymatic monoamine oxidase activity that is predominantly expressed in the liver microvasculature. It possesses key function in the recruitment of various lymphocyte subsets. The aim of this study was to decipher VAP-1 contribution to monocytic transendothelial transmigration.
Methods Primary human HSEC were isolated from explanted and grown to confluence in flow chambers. After activation with TNF-α/IFN-γ for 24h HSEC were treated with VAP-1 antibody and enzyme inhibitors. Monocytes were enriched from peripheral blood by using OptiPrep gradient. Monocyte subsets (CD14++CD16−, CD14++CD16+, CD14+CD16++) were isolated by FACS-sorting. Isolated monocytes were perfused over HSEC monolayers under constant flow simulating physiological shear stress (0.05 Pa). Adhesion and transmigration was studied using phase contrast microscopy. Transwell assays were used to study the phenotype of transmigrated monocytes by flowcytometry.
Results HSEC pretreatment with VAP-1 antibody (TK8-14) or enzyme inhibitor Semicarbizide equally reduced monocyte transmigration by ∼50%. VCAM-1 blockade had a similar but redundant effect whereas CLEVER-1-antibody or LOX-inhibitor (β-APN) did not alter monocyte transmigration. VAP-1 antibody acted in a time-dependent manner with influence on monocyte adhesion only after short-term application (15 min). Inhibiting VAP-1 led to profound reduction of proinflammatory nonclassical CD14+CD16++ monocyte transmigration but also affected classical CD14++CD16− whereas the intermediate CD14++CD16+ subtype was not affected. Under static conditions VAP-1 enzymatic or antibody inhibition was significantly blunted suggesting flow to be a mandatory prerequisite for the biological function of VAP-1 on monocytes. Increased expression of HLA-DR and the M2 macrophage marker CD206 on monocyte subsets after endothelial transmigration was not altered by VAP-1 inhibition.
Conclusion Endothelial VAP-1 differentially modulates monocyte recruitment under flow conditions in a time-dependent fashion and .favours transmigration of a proinflammatory monocyte subset. The critical role of VAP-1 enzyme function renders small molecules as a promising therapeutic approach in combating liver inflammation and subsequent fibrosis.
Competing interests None declared.
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