Heme Induces the Expression of Adhesion Molecules ICAM-1,VCAM-1,and E Selectin in Vascular Endothelial Cells

Abstract

Heme is an important immunostimulating agent and oxidative factor contributing to endothelial cell activation. To investigate the mechanism of heme-induced endothelial cell activation, we analyzed the effect of heme and the inflammatory cytokine, tumor necrosis factor-β (TNF-β), on the expression of the heme-degrading stress protein, heme oxygenase (HO), and adhesion molecules in human umbilical vein endothelial cells (HUVEC). Indirect immunofluorescence double labeling studies demonstrated a simultaneous increase of ICAM-1 and HO-1 after exposure of cells to heme for 24 hr. Co-expression of HO-1 and ICAM-1 was also demonstrated in TNF-β-exposed cells. Dot blot immunoassay and quantitative analysis by ELISA demonstrated that heme treatment for 24 hr caused a 2-fold increase in ICAM-1 expression (P < 0.002) compared with quiescent cells, while in cells stimulated by TNF-β for 24 hr CAM-1 gene expression increased by 5-fold. Moreover, heme exposure also resulted in a marked increase in VCAM-1 and E selectin expression (three and four times over control levels, respectively). On the other hand, TNF-β treatment showed similar expression levels for VCAM-1 and E selectin, compared with stimulation by heme (100 μM). The level of HO activity in endothelial cells exposed to heme or TNF-β was increased from 24.7 ± 5.7 pmol bilirubin/mg protein/min in control to 70.0 ± 9.5 and 36.7 ± 3.1 pmol bilirubin/mg protein/min in heme- and TNF-β-stimulated cells, respectively. These results suggest that upregulation of CAM-1, VCAM-1, and E selectin expression is associated with oxidative stress induced by hemoglobin/heme and that HO-1 may play a modulating role via its ability to degrade heme to a substance with antioxidant properties.