Interaction of recombinant octameric hemoglobin with endothelial cells.

Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbßG83C. We investigate the potential side effects of rHbßG83C on endothelial cells. The rHbßG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbßG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb ßG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

Angiotensin-converting enzyme inhibitor captopril reverses the adverse cardiovascular effects of polymerized hemoglobin.

AIM: Cell-free hemoglobin-based oxygen carriers (HBOCs) may increase the risk of myocardial infarction and death. We studied the effect of an angiotensin-converting enzyme (ACE) inhibitor on HBOC-induced adverse cardiovascular outcomes and elucidated the underlying mechanisms. RESULTS: With a dog cardiopulmonary bypass model, we demonstrated that a high-dose HBOC (3%, w/v) did not reduce-but aggravated-cardiac ischemia/reperfusion injury. Animals administered a high-dose HBOC experienced coronary artery constriction and depression of cardiac function. Exposure of isolated coronary arteries or human umbilical vein endothelial cells to high-dose HBOC caused impaired endothelium-dependent relaxation, increased endothelial cell necrosis/apoptosis, and elevated NAD(P)H oxidase expression (gp91(phox), p47(phox), p67(phox), and Nox1) and reactive oxygen species (ROS) production. All observed adverse outcomes could be suppressed by the ACE inhibitor captopril (100 µM). Co-incubation with free radical scavenger tempol or NAD(P)H oxidase inhibitor apocynin had no effect on captopril action, suggesting that the positive effects of captopril are ROS- and NAD(P)H oxidase dependent. ACE inhibition by captopril also contributed to these effects. In addition, bioavailable nitrite oxide (NO) reduced by high-dose HBOC was preserved by captopril. Furthermore, HBOC, at concentrations greater than 0.5%, inhibited large conductance Ca(2+)-activated K(+) channel currents in vascular smooth muscle cells in a dose-dependent manner, although captopril failed to improve current activity, providing additional evidence that captopril's effects are mediated by the endothelium, but not by the smooth muscle. INNOVATION AND CONCLUSION: Captopril alleviates high-dose HBOC-induced endothelial dysfunction and myocardial toxicity, which is mediated by synergistic depression of NAD(P)H oxidase subunit overproduction and increases in vascular NO bioavailability.

Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs.

Release of hemoglobin (Hb) into the circulation is a central pathophysiologic event that contributes to morbidity and mortality in chronic hemolytic anemias and severe malaria. These toxicities arise from Hb-mediated vasoactivity, possibly due to NO scavenging and localized tissue oxidative processes. Currently, there is no established treatment that targets circulating extracellular Hb. Here, we assessed the role of haptoglobin (Hp), the primary scavenger of Hb in the circulation, in limiting the toxicity of cell-free Hb infusion. Using a canine model, we found that glucocorticoid stimulation of endogenous Hp synthesis prevented Hb-induced hemodynamic responses. Furthermore, guinea pigs administered exogenous Hp displayed decreased Hb-induced hypertension and oxidative toxicity to extravascular environments, such as the proximal tubules of the kidney. The ability of Hp to both attenuate hypertensive responses during Hb exposure and prevent peroxidative toxicity in extravascular compartments was dependent on Hb-Hp complex formation, which likely acts through sequestration of Hb rather than modulation of its NO- and O2-binding characteristics. Our data therefore suggest that therapies involving supplementation of endogenous Hb scavengers may be able to treat complications of acute and chronic hemolysis, as well as counter the adverse effects associated with Hb-based oxygen therapeutics.

Inhaled nitric oxide enables artificial blood transfusion without hypertension.

BACKGROUND: One of the major obstacles hindering the clinical development of a cell-free, hemoglobin-based oxygen carrier (HBOC) is systemic vasoconstriction. METHODS AND RESULTS: Experiments were performed in healthy mice and lambs by infusion of either murine tetrameric hemoglobin (0.48 g/kg) or glutaraldehyde-polymerized bovine hemoglobin (HBOC-201, 1.44 g/kg). We observed that intravenous infusion of either murine tetrameric hemoglobin or HBOC-201 induced prolonged systemic vasoconstriction in wild-type mice but not in mice congenitally deficient in endothelial nitric oxide (NO) synthase (NOS3). Treatment of wild-type mice by breathing NO at 80 ppm in air for 15 or 60 minutes or with 200 ppm NO for 7 minutes prevented the systemic hypertension induced by subsequent intravenous administration of murine tetrameric hemoglobin or HBOC-201 and did not result in conversion of plasma hemoglobin to methemoglobin. Intravenous administration of sodium nitrite (48 nmol) 5 minutes before infusion of murine tetrameric hemoglobin also prevented the development of systemic hypertension. In awake lambs, breathing NO at 80 ppm for 1 hour prevented the systemic hypertension caused by subsequent infusion of HBOC-201. CONCLUSIONS: These findings demonstrate that HBOC can cause systemic vasoconstriction by scavenging NO produced by NOS3. Moreover, in 2 species, inhaled NO administered before the intravenous infusion of HBOC can prevent systemic vasoconstriction without causing methemoglobinemia.

Selenium reduces hemoglobin-induced epithelial damage to intestinal mucosa.

Modified hemoglobins are being considered as possible "blood substitutes." Experiments were performed to determine whether diaspirin cross-linked hemoglobin (DBBF-Hb) produces epithelial damage and whether this is reduced by selenium (Se). Anesthetized Sprague-Dawley rats, half of which received 2 x 10(-6) g/ml Se, daily for 3 weeks, in their drinking water, were injected with a 5 ml bolus of 10 mg/ml DBBF-Hb. Control animals received saline (5 animals per group). After 30 minutes, the intestine was perfusion-fixed for light and electron microscopy. Eighty villi per rat were assigned an epithelial integrity index (E.I.), ranging from 1 (intact) to 3 (some cell-cell and cell-basement membrane separation). In non-Se rats, E.I. was significantly compromised by DBBF-Hb, compared to HBS-BSA (2.47+/-0.57 (SD) vs. 1.36+/-0.49, p<0.001). In Se rats, neither injection with DBBF-Hb or HBS-BSA caused epithelial damage (1.03+/-0.17 vs. 1.07+/-0.26). Mast cell degranulation per villus (MCD) was measured in 60 villi per rat. In non-Se rats, MCD was significantly greater after DBBF-Hb than after HBS-BSA injection (1.83+/-1.42 vs. 0.2+/-0.4). Supplementary Se did not reduce this effect. In fact, MCD was significantly increased in both sets of rats compared to their non-Se counterparts (3.27+/-2.40 and 1.48+/-1.70 for DBBF-Hb and HBS-BSA, respectively). Since mast cell mediators damage cells, Se must protect the mucosal epithelium in some way.

Estrogens attenuate neuronal injury due to hemoglobin, chemical hypoxia, and excitatory amino acids in murine cortical cultures.

A growing body of evidence supports the hypothesis that estrogens may be beneficial in Alzheimer's disease and other neurodegenerative processes. Less is known of their therapeutic potential in acute CNS insults. In this study, we assessed the effect of estrogens in three injury paradigms that may be relevant to CNS hemorrhage, trauma, and ischemia. Supraphysiologic concentrations of 17beta-estradiol, estrone, or equilin attenuated neuronal loss due to prolonged exposure to the pro-oxidant hemoglobin, with complete protection at 10 microM. Most of this effect persisted despite concomitant treatment with the antiestrogen ICI 182,780 or the protein synthesis inhibitor cycloheximide. In contrast, the non-estrogenic steroid methylprednisolone, which is currently in clinical use in spinal cord injury, reduced neuronal loss by only about 30%. High concentrations of equilin or estrone also attenuated the submaximal neuronal injury induced by 3.5-4.5 h exposure to the cytochrome oxidase inhibitor sodium azide, with near complete protection at 30 microM. Estrogens had a weaker and somewhat variable effect on pure excitotoxic injury, reducing neuronal loss due to 24 h kainate exposure by about half, and due to 24 h NMDA exposure by 15-65%; similar neuroprotection was provided by the antioxidant 21-aminosteroid U74500A. These results suggest that estrogens may be beneficial in acute CNS injuries associated with oxidative and excitotoxic stress. Investigation of high dose estrogen therapy in in vivo models of CNS hemorrhage, trauma, and ischemia is warranted.

Liver function and morphology after resuscitation from severe hemorrhagic shock with hemoglobin solutions or autologous blood.

OBJECTIVE: To test the effects of three hemoglobin solutions on liver function and hepatic morphology after resuscitation from severe hemorrhagic shock. DESIGN: Prospective study. SETTING: Laboratory. SUBJECTS: Thirty-three beagle dogs. INTERVENTION: Hemorrhagic shock was induced in anesthetized dogs by removal of blood at a rate of 2 mL/kg/min until systolic blood pressure (BP) reached 50 mm Hg. BP was maintained at this level 2 hrs by further withdrawing 5 to 10 mL aliquots whenever BP increased > 50 mm Hg. Resuscitation was then initiated with autologous whole blood (n = 7), 4% pyridoxalated-hemoglobin-polyoxyethylene conjugate (4% PHP [n = 6]), 8% pyridoxalated-hemoglobin-polyoxyethylene conjugate (8% PHP [n = 9], or 8% stroma-free hemoglobin (n = 7). Four dogs were managed identically but were not resuscitated. Gross necropsy and histologic examination of the liver were performed on all dogs after 7 days, or earlier if death occurred. MEASUREMENTS AND MAIN RESULTS: In vitro interferences of PHP and stroma-free hemoglobin with liver function tests were determined and recommendations for interpretation of results from blood samples containing PHP and stroma-free hemoglobin were made. Blood was collected before, during, and after resuscitation from hemorrhagic shock. The dogs were then awakened and survivors were monitored daily with blood sampling until they were killed and necropsy was performed. After 7 days, the survival rate following hemorrhagic shock was 100% for whole blood and 4% PHP, 86% for stroma-free hemoglobin, and 33% for 8% PHP. Of the resuscitated dogs not surviving 7 days, all but one died within 27 hrs from coagulopathy. All dogs not resuscitated died within 1.75 hrs after 2 hrs of shock. Bilirubin, alkaline phosphatase, and lactic dehydrogenase concentrations could not be measured due to interferences of stroma-free hemoglobin and PHP. Aspartate (AST) and alanine (ALT) aminotransferase concentrations could be measured after dilution to overcome the interferences. Significant increases in AST and ALT values in all groups 24 hrs after resuscitation were attributed to hypoxic hepatocellular damage associated with the severity of the shock model rather than to the resuscitation fluid. Liver histology showed no changes attributed to toxic damage of hepatocytes in dogs resuscitated with stroma-free hemoglobin or PHP. However, changes, were less severe in dogs resuscitated with 4% PHP than in other groups. CONCLUSION: Morphologic studies at necropsy and liver function tests in dogs receiving hemoglobin solutions, compared with autologous blood, support the conclusion that the PHP and stroma-free hemoglobin solutions tested did not produce hepatic toxicity when used as resuscitation fluids in this model of severe shock.

Synthesis and properties of polymerized, diaspirin cross-linked hemoglobins.

During the course of our studies it became clear that there were therapeutic applications for which a polymeric hemoglobin having an extended half-life in circulation would be appropriate. Therefore, a process for the glutaraldehyde-polymerization of diaspirin cross-linked hemoglobin (DCLHb) was developed and used to prepare glutaraldehyde-polymerized DCLHb (GP-DCLHb) in lactated Ringer's solution in sufficient quantities for biological testing. Both isovolemic exchange-transfusion and "top-load" studies (rats; primates and swine, respectively) were completed in which a broad spectrum of physiologic, histopathologic and analytical parameters were monitored and assessed. In general, GP-DCLHb in lactated Ringer's solution was well-tolerated physiologically. When compared to DCLHb, GP-DCLHb offers the advantages of reduced renal clearance of hemoglobin and an extended half-life in circulation. GP-DCLHb has the disadvantages that (1) glutaraldehyde is an ineffective virucidal agent under the conditions of the polymerization reaction and a separate virus inactivation step is required; (2) low-endotoxin (LAL-negative) GP-DCLHb solutions are pyrogenic (rabbits); and (3) unusual deposition of hemoglobin-containing material in the small arterioles of the liver and kidney (rats) was sometimes seen even after a period of time (2 weeks) during which treatment-related organ pathologies are usually resolved, a finding peculiar to GP-DCLHb among the various hemoglobin derivatives we have tested.

A preclinical screening test for modified hemoglobin to bridge the gap between animal safety studies and use in human.

The infusion of large amount of modified hemoglobin as blood substitute can potentially result in hypersensitivity and anaphylactic reactions, antibody-antigen reactions and others. Animal safety studies are important. However, response in animals may not be the same as in human. Before injecting into human, we may need to use an in-vitro screening procedure. One approach is based on testing the effects of modified Hb on complement activation (C3a) of human plasma. This paper describes this screening test. It also discusses how this may potentially be used. For instance using this to test for contamination from trace membrane fragments with blood group antigen or lipids, antibody-antigen complexes, endotoxin, trace fragments of microorganisms, residual amounts of some polymers, emulsifying agents, and organic solvents. There is also the possibility of obtaining plasma from a very large human population and analyse each of these to study the epidemiology of adverse reactions in different groups and types of patients.

Evaluating new red cell substitutes: a critical analysis of toxicity models.

Because red cell substitutes (RCS) will improve oxygen delivery to tissues rendered hypoxic or anoxic they are presumed to be effective. Prior to clinical application the safety--toxicity of any proposed solution must be established. Models used to demonstrate efficacy do not necessarily evaluate toxicity. Infusion of human hemoglobin (Hb) solutions into animals raises one issue of immune response but obviates looking at the human response to similar materials. Differentiating the cause of an immunologic effect may be difficult. Effects seen in acute models may not have chronic implications; they may be transients of no consequence. Differentiating the effects of volume load intravascularly from the effects of the solution is also a problem. Identification of proper controls is essential for evaluation. However, the control solution may have problems of its own that must be defined. For any proposed RBS a menu of models exploring cellular, tissue, organ and organism responses to the solution, its residual modifiers and the products of metabolism are needed to define primary and secondary effects.

Intraoperative blood salvage: medical controversies.

Although a great deal has been learned about the medical aspects of intraoperative blood salvage, several fundamental medical issues remain controversial. As pressure increases to maximize the use of IBS, more research will be needed on the application of salvage techniques in cancer surgery and in the presence of bacterial contamination. The reintroduction of the use of devices that do not wash salvaged blood have reopened investigations into the effects of reinfusion of partially hemolyzed and partially clotted salvaged blood on coagulation, renal function, and cardiopulmonary performance. More studies are also needed so that empirically based standards of practice for the collection and storage of salvaged blood can be established. No longer confined to a few pioneering surgical departments, IBS is now widely practiced and likely to continue to grow rapidly. Knowledge and research of the medical issues surrounding its use will become increasingly valuable in transfusion medicine.