Expression of Adhesion Molecules and von Willebrand Factor in Human Coronary Artery Endothelial Cells Incubated with Differently Modified Hemoglobin Solutions

Abstract

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.

Different Faces of the Heme-Heme Oxygenase System in Inflammation

“…Several groups have reported that the efficacy of artificial blood substitutes, such as modified hemoglobin solutions, are often hampered by oxidative and inflammatory complications (Simoni et al, 1997;Goldman et al, 1998;Baldwin, 1999). This phenomenon may be related to a difference in heme-releasing capacity of the different hemoglobin solutions.…”

Artificial Oxygen Carrier With Pharmacologic Actions of Adenosine-5′-Triphosphate, Adenosine, and Reduced Glutathione Formulated to Treat an Array of Medical Conditions

“…Adenosine in HemoTech counteracts vasopressive and pro‐inflammatory properties of Hb by activating adenosine A2A and A2B receptors, which produce vasodilatation, moderation of inflammatory reactions, and prevention of platelet aggregation. Activation of the adenosine A3 receptor provides cytoprotection and induction of adenosine A1 receptor results in neuroprotection (Fig. —center).…”

“…GSH also shields heme from reactive oxygen species (ROS) and NO, further lowering the Hb pro-oxidant and vasoconstrictive potential. GSH on the HemoTech surface may form S-nitroso-GSH, thus preventing NO access to heme (15,24,(26)(27)(28)(29)33).…”

“…Such a modification procedure lowers the heme reactivity with hydrogen peroxide, thus preventing formation of ferryl species (24,27,29,34). As a consequence of this pharmacologic modification, HemoTech does not alter the cellular redox equilibrium and eliminates the activation of nuclear factor (NF)-kappa B, an inducer of many inflammatory genes (24,(26)(27)(28)(35)(36)(37). Another factor that contributes to its anti-inflammatory action is low endothelial calcium influx and low formation of 8-isoprostanes, well-known activators of protein kinases that induce NF-kappa B (12,26,37).…”

Artificial Oxygen Carriers: Exactly How Close Are We to an Ultimate Product?

“…The disadvantages are that these Hbs are potent oxidizers and activators of redox (NF‐kappa B) and oxygen (HIF‐1) regulated transcription factors . There is an established link between Hb‐mediated oxidative reactions and inflammatory responses . Hb‐induced vasoconstriction via nitric oxide (NO) and non‐NO‐mediated pathways (i.e., ET‐1, RAS, 5‐HT) is also well established .…”