Recently discovered angio-associated migratory cell protein (AAMP) was shown to reduce neoatherosclerosis (NA) formation in an ApoE knockout mouse model of arterial injury. Reconstitution of the endothelial layer afterarterial injury is accompanied by an inflammatory reaction and originates from both resident endothelial cell recovery as well as recruitment of bone marrow-derived endothelial progenitor cells (EPC). However, expression of AAMP in EPC and NA-associated monocytes is unknown. Here, we assess EPC and monocyte AAMP expression and impact of AAMP blockade upon migratory activity of human umbilical vein endothelial cells (HUVEC).Methods
An affinity-purified recombinant AAMP antibody (anti-rAAMP-ab) was generated by immunization of rabbits. HUVEC were isolated from umbilical cords and cultured in endothelial growth medium. Peripheral blood-derived mononuclear cells were isolated from human buffy coats and cultured to enable differentiation towards an EPC phenotype; EPC status was confirmed by DiIac-LDL uptake and UEA-lectin binding of EPC. A human acute monocytic leukemia cell line, THP1, was assayed for inflammatory contribution. AAMP expression was detected using fluorescence-activated cell sorting (FACS), immunofluorescence, and computed quantitative western blot analysis. Impact of AAMP blockade by anti-rAAMP-ab upon EPC and THP1 adhesion to HUVEC as well as upon HUVEC migratory capacity was evaluated. Expression of AAMP after balloon dilation was analysed in an abdominal aorta model of arterial injury.Results
Intense early AAMP expression in EPC comparable to AAMP expression in HUVEC was demonstrated. Subcellular immunfluorescence breakdown revealed strong and primarily membrane-localized AAMP expression in both EPC and HUVEC; concordantly, HUVEC were predominantly positive for AAMP as assessed by FACS. AAMP blockade reduced HUVEC migratory activity dose-dependently, as evaluated by awound healing assay. Also, AAMP was upregulated in endothelial cells afterarterial injury as compared to uninjured control in vivo. Furthermore, AAMP blockade dose-dependently reduced both EPC and THP1 adhesion on TNF-alpha activated HUVEC.Conclusions
For the first time, expression of NA development-involved AAMP by EPC is demonstrated, which, notably, is time-dependent and membrane-bound. Also, an intense antimigratory effect is executed upon HUVEC by AAMP blockade. Moreover, AAMP blockade reduces HUVEC adhesion of both EPC and monocytes. Taken together, these date indicate a complex differential contribution of AAMP during arterial healing after injury.