Molecular Characterization of Hypoxia-Hypothermia–Conditioned Human Endothelial Cells and Their Interaction With Human Monocytes

This in vitro study was designed to characterize the molecular profiling of human endothelial cells (ECs) during the early phase of hypoxia-hypothermia (HH) conditioning and to evaluate their interactions with allogeneic monocytes. The HH-conditioned ECs were analyzed using real-time quantitative polymerase chain reaction (RT-PCR). A cell adhesion assay was performed to assess adhesion of purified allogeneic monocytes as well as CD4- and CD8-positive T cells to HH-conditioned ECs with or without blocking antibodies specific for CD15s and CD162. Uptake of EC membrane by monocytes with or without scavenger receptor blockade was examined using fluorescence-activated cell scanning. The RT-PCR revealed up-regulation of gene transcripts for inflammatory cytokines, monocyte-associated growth factors, costimulatory, and apoptosis-related molecules in HH-conditioned ECs. Analysis using fluorescence-activated cell scanning showed minimal CD54 up-regulation in HH-conditioned ECs. We noted low-level adhesion of CD4- or CD8-positive cells to resting and HH-conditioned ECs. High-level adhesion of monocytes to HH-conditioned ECs was observed when compared with resting ECs. Blockade of CD15s and CD162 dramatically reduced monocyte adhesion to normal and HH-conditioned ECs. Monocytes but not T cells showed uptake of EC membranes during their interactions with HH-conditioned ECs, which was inhibited by scavenger receptor blockade. These data characterized the molecular features of ECs during early HH-conditioning. The EC transcripts related to monocyte recruitment and interaction between monocytes and HH-conditioned ECs dominated the early post-HH condition. Blockade of CD15s and CD162 prevented monocyte adhesion to ECs. These findings suggest that the initial interaction between monocytes and HH-conditioned ECs has a central role during the early phase of reperfusion injury.