MODIFIED HEMOGLOBIN SOLUTION, WITH DESIRED PHARMACOLOGICAL PROPERTIES, DOES NOT ACTIVATE NUCLEAR TRANSCRIPTION FACTOR NF-KAPPA-B IN HUMAN VASCULAR ENDOTHELIAL-CELLS

The aim of the present study was to evaluate the role of hemoglobin (H b) and the contribution of chemically modified Hb solutions on the act ivation of nuclear transcription factor, NF-kappa B, and propagation o f oxidative stress within human vascular endothelial cells. The activa tion of an oxidative stress-sensitive NF-kappa B can be linked with th e propagation of an inflammatory state via rapid induction of genes fo r several pro-inflammatory mediators. Human coronary artery endothelia l cells (HCAEC) were cultured on glass coverslips or cell culture plat es to confluence. Then, the cells were incubated for up to 18 hours wi th endothelial basal medium (EBM) supplemented with 5% FBS and test ag ents 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 co ntrol for the NF-kappa B activation study included a treatment of the cells with: 1) endotoxin; IL-1; TNF; and H2O2. Results indicate that H b's pro-oxidant potential was influenced by the type of chemical modif ication procedure. The GLUT-Hb autoxidation rate, peroxidase-like acti vity and reactivity with H2O2/ferryl species formation were higher as compared to UHb, by 15%, 35% and 30%, respectively. However, pro-oxida nt potential of Hb-PP-GSH was significantly lower than that of UHb (by 22%, 12% and 28%, respectively). The extent of oxidative stress of th e HCAEC's was found to be the Hb modification-type and concentration d ependent. 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 compa red to the control cells. The UHb generated a moderate oxidative stres s to the endothelium. The immunofluorescent and EMSA results indicate a correlation between the type of Hb chemical modification and the ind uction of NF-kappa B nuclear translocation. We found that GLUT-Hb rapi dly activated NF-kappa B and induced nuclear translocation. Treatment of the cells with an increasing amount of UHb leads to the partial nuc lear induction of NF-kappa B. However, Hb-PP-GSH did not acrivate NF-k appa B directly. In this study, the positive control cells treated wit h endotoxin, IL-1 or TNF demonstrated full nuclear translocations, whe reas H2O2 caused only partial induction. In conclusion, nuclear transl ocation of NF-kappa B by Hb solutions might be dependent on Hb's pro-o xidant potential and extent of Hb-mediated endothelial oxidative stres s. 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 t he anti-inflammatory properties of adenosine which is used in our nove l modification procedure. In this study, only the Hb-PP-GSH, crosslink ed intramolecularly with a-adenosine triphosphate and intermolecularly with o-adenosine, and combined with reduced glutathione, was shown to be non-toxic to the endothelium and promises to be an effective free- Hb based blood substitute.