The aim of the present study was to evaluate the role of hemoglobin (Hb) and the contribution of chemically modified Hb solutions on the activation of nuclear transcription factor. NF-kappa B, and propagation of oxidative stress within human vascular endothelial cells. The activation of an oxidative stress-sensitive NF-kappa B can be linked with the propagation of an inflammatory state via rapid induction of genes for several pro-inflammatory mediators. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips or cell culture plates to confluence. Then, the cells were incubated for up to 18 hours with endothelial basal medium (EBM) supplemented with 5% FBS and test agents in a concentration of 0.1 and 0.2 mmol: 1) unmodified bovine Hb (UHb): 2) modified Hb solution polymerized with glutaraldehyde (GLUT-Hb), and 3) a novel modified Hb solution (Hb-PP-GSH) prepared according to our patented procedure (U.S. Patent No. 5,439,882). The positive control for the NF-kappa B activation study included a treatment of the cells with: I) endotoxin: IL-1; TNF; and H2O2. Results indicate that Hb's pro-oxidant potential was influenced by the type of chemical modification procedure. The GLUT-Hb autoxidation rate, peroxidase-like activity and reactivity with H2O2/ferryl species formation were higher as compared to UHb, by 15%, 35% and 30%, respectively. However, pro-oxidant potential of Hb-PP-GSH was significantly lower than that of UHb (by 22%, 12% and 28%, respectively). The extent of oxidative stress of the HCAECs was found to be the Hb modification-type and concentration dependent. Although the highest endothelial lipid peroxidation and the largest depletion of intracellular GSH was associated with 0.2 mmol of GLUT-Hb, the Hb-PP-GSH did not produce significant changes when compared to the control cells. The UHb generated a moderate oxidative stress to the endothelium. The immunofluorescent and EMSA results indicate a correlation between the type of Hb chemical modification and the induction of NF-kappa B nuclear translocation. We found that GLUT-Hb rapidly activated NF-kappa B and induced nuclear translocation. Treatment of the cells with an increasing amount of UHb leads to the partial nuclear induction of NF-kappa B. However, Hb-PP-GSH did not activate NF-kappa B directly. In this study, the positive control cells treated with endotoxin, IL-1 or TNF demonstrated full nuclear translocations, whereas H2O2 caused only partial induction. In conclusion, nuclear translocation of NF-kappa B by Hb solutions might be dependent on Hb's pro-oxidant potential and extent of Hb-mediated endothelial oxidative stress. Besides the low oxidative potential of Hb-PP-GSH, the observed lack of NF-kappa B activation by this Hb solution can be also related to the anti-inflammatory properties of adenosine which is used in our novel modification procedure. In this study, only the Hb-PP-GSH, cross-linked intramolecularly with o-adenosine triphosphate and intermolecularly with o-adenosine, and combined with reduced glutathiore, ...

Previous studies have established a linkage between free Hb molecules and the production of inflammatory mediators by the reticuloendothelial cells. An important aspect of the endothelial response to the inflammatory 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 intracellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and also von Willebrand factor (vWF) expression by human endothelium. Human coronary artery endothelial cells (HCAEC) were cultured on glass coverslips until they reached confluence, then incubated 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 Hb solution polymerized with glutaraldehyde (GLUT-Hb). The HCAECs were also incubated with EBM (negative 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-human vWF, and consequently to the secondary antibody (fluorescein isothiocyanate-conjugated F(ab)2). Immunofluorescence analysis revealed different expressions of ICAM-1 and VCAM-1 on the surface membranes of variously treated cells. Although negative control cells had an undetectable level of adhesion molecules, the positive control cells, activated 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, following treatment with a 0.1 or 0.2 mmol of Hb-PP-GSH and 0.1 mmol of UHb. Cell treated with 0.2 mmol of UHb and both concentrations of GLUT-Hb demonstrated a massive expression of this adhesion molecule. A similar effects was observed during induction of VCAM-1. While a lack of expression was noted with both concentrations of Hb-PP-GSH and 0.1 mmol of UHb, the GLUT-Hb stimulated significant VCAM-1 induction at all tested concentrations. Immunofluorescence analysis confirmed the expression of vWF uniformly in HCAEC from the different experimental groups. The data suggest, vWF expression was unaffected by all but the GLUT-Hb treatment. In conclusion, the Hb stimulatory activity toward ICAM-1 and VCAM-1 inductions were related with the type of Hb chemical modification method. Although modification of Hb with glutaraldehyde potentiates adhesion molecules expression, our novel Hb modification procedure, which comprises intramolecular cross-linking with o-adenosine triphosphate and intermolecular with o-adenosine, and combined with reduced glutathione, apparently prevents these inflammatory events.

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