EXPRESSION OF ADHESION MOLECULES AND VON-WILLEBRAND-FACTOR IN HUMAN CORONARY-ARTERY ENDOTHELIAL-CELLS INCUBATED WITH DIFFERENTLY MODIFIED HEMOGLOBIN-SOLUTIONS

Previous studies have established a linkage between free Hb molecules and the production of inflammatory mediators by the reticuloendothelia l cells. An important aspect of the endothelial response to the inflam matory stimuli is the expression of adhesion molecules on the luminal surface. Therefore, the present study was designed to investigate the effects of various free-Hb based oxygen carrying solutions on the intr acellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion mol ecule-1 (VCAM-1) and also von Willebrand factor(vWF) expression by hum an endothelium. Human coronary artery endothelial cells (HCAEC) were c ultured on glass coverslips until they reached confluence, then incuba ted for 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and a 0.1 mmol or 0.2 mmol of the bovine Hb solutions: 1) pure unmodified bovine Hb (UHb); 2) modified bovine Hb solution (Hb-PP-GSH ) prepared according to our newly developed procedure (U.S. Patent No. 5,439,882); and 3) modified bovine Kb solution polymerized with gluta raldehyde (GLUT-Hb). The HCAEC's were also incubated with EBM (negativ e control) and EBM containing bacterial endotoxins in a concentration of 50 EU/ml (positive control). After treatment, cells were exposed to primary antibodies: anti-human ICAM-1, anti-human VCAM-1 or anti-huma n vWF, and consequently to the secondary antibody (fluorescein isothio cyanate-conjugated F(ab')(2)). Immunofluorescence analysis revealed di fferent expressions of ICAM-1 and VCAM-1 on the surface membranes of v ariously treated cells. Although negative control cells had an undetec table level of adhesion molecules, the positive control cells, activat ed by endotoxin, exhibited high immunoreactivity for ICAM-1 and VCAM-1 . The Hb's treated cells demonstrated differing degrees of activation. An insignificant expression of ICAM-1 was observed in HCAEC, followin g treatment with a 0.1 or 0.2 mmol of Hb-PP-GSH and 0.1 mmol of UHb. C ell treated with 0.2 mmol of UHb and both concentrations of GLUT-Hb de monstrated a massive expression of this adhesion molecule. A similar e ffects was observed during induction of VCAM-1. While a lack of expres sion was noted with both concentrations of Hb-PP-GSH and 0.1 mmol of U Hb, the GLUT-W stimulated significant VCAM-1 induction at all tested c oncentrations. Immunofluorescence analysis confirmed the expression of vWF uniformly in HCAEC from the different experimental groups. The da ta suggest, VWF expression was unaffected by all but the GLUT-Hb treat ment. In conclusion, the Hb stimulatory activity toward ICAM-1 and VCA M-1 inductions were related with the type of Hb chemical modification method. Although modification of Hb with glutaraldehyde potentiates ad hesion molecules expression, our novel Hb modification procedure, whic h comprises intramolecular cross-linking with o-adenosine triphosphate and intermolecular with o-adenosine, and combined with reduced glutat hione, apparently prevents these inflammatory events.