Recent research focusing on the effects of intravascular free Hb on NO bioavailability led to the speculation that sequestration and compartmentalization of Hb by Hp may attenuate the intrinsic hypertensive activity of Hb (4
). Furthermore, the potential of manipulating the physiologic functions of Hp and/or its pharmacologic application to counter the vasoconstrictive and oxidative toxicities of intravascular free Hb has not been experimentally investigated to date. We have recently demonstrated that Hp site specifically binds and protects Hb in the presence of oxidants, such as H2
). Here, we conclusively show
that Hp complex formation is effective in suppressing Hb-mediated hemodynamic imbalance as well as heme-mediated oxidative toxicity in vivo.
To better understand the role of Hb-Hp complex in ameliorating vascular toxicity, we infused free Hb in 2 different animal models to mimic intravascular hemolysis. In the course of these investigations, we made the following observations: (a) pharmacological induction of endogenous Hp synthesis or application of purified Hp results in retention of infused Hb within the intravascular compartment; (b) Hp blunts the systemic hypertensive response to cell-free Hb infusion through high-affinity complex formation; and (c) intravascular retention of Hb by Hp prevents peroxidation-related toxicity in extravascular compartments such as the kidney.
The canine model was recently proposed for the study of Hb-induced acute vascular toxicity (28
). In comparison to most laboratory animals that display very low baseline Hp levels, dogs contain Hp plasma concentrations within the range found in human plasma (29
). Although speculative at this point, the similar, high Hp concentrations found in dog and human may be related to a similar extent of mechanical intravascular hemolysis in the 2 species and thus comparable kinetics of plasma Hp consumption under baseline conditions. Ektacytometry data from blood samples from beagle dogs used in our study show a very similar rbc deformability/fragility as found in a human control population (Supplemental Figure 1; supplemental material available online with this article; doi:
). This observation adds further value to the beagle dog model for investigating intravascular hemolysis. Additionally, the specific glucocorticoid responsiveness of the dog HP
gene promotes the pharmacologic induction of supraphysiologic Hp plasma concentrations (21
). The dog is therefore a unique model, in which the effects of intravascular Hb can be analyzed at a range of targeted endogenous Hp plasma concentrations. The guinea pig, on the other hand, allowed us to analyze both vascular and extravascular effects of extracellular Hb in the presence or absence of exogenously administered, purified human Hp in a nonascorbate-producing species, with blood and tissue antioxidant status comparable to that of humans (30
In our experiments, the hypertensive response in Hb-infused dogs similarly occurred at a plasma Hb concentration of more than 100 μM and reached a maximum level comparable to the canine model of intravascular hemolysis reported earlier (28
). We can thus confirm that intravascular Hb infusion in dogs is a robust and reproducible system to study the acute cardiovascular toxicity of Hb at different plasma Hp levels. Modulation of local NO synthesis by glucocorticoids cannot be excluded as a Hp-independent mechanism that may blunt the Hb response in our prednisone-treated animals (31
). However, the congruent observation made with pharmacologic Hp treatment in nonglucocorticoid-treated guinea pigs strongly suggests that overexpressed Hp is directly involved in limiting Hb-induced hypertension in dogs. To confirm that stable Hb complexation is an essential mechanism by which Hp can counter Hb toxicity in vivo, we examined the effects of Hp on hypertension mediated by chemically modified Hb, which is unable to bind Hp as a result of covalent α-globin subunit cross-linking (26
). Our experiments clearly show that upon infusion into animals, αα-DBBF did not form a complex with Hp, and the hypertensive response was not attenuated, even in the presence of excess Hp. Based on data shown in Figure A, a complex multidimensional mechanism of blood pressure control appears to be at work. Since NO binding was not changed by Hb sequestration within the Hp complex, it is possible that the small blood pressure increase seen in guinea pigs transfused with Hb-Hp may be due to NO scavenging within the vascular compartment. However, it appears likely that yet-to-be-determined mechanisms of blood pressure control, regarding the Hb-Hp complex, are functioning.
Several mechanisms may contribute to Hb-mediated hypertension and vascular toxicity. NO consumption by the dioxygenase reaction of Hb, leading to a state of vascular depletion of NO, is the most widely investigated mechanism to date (32
). Under normal circumstances, the endothelial cell layer, rbc-free plasma zone, unstirred plasma layer around rbc, and erythrocyte membrane provide diffusion barriers, which impair rapid, unhindered interactions between rbc Hb and endothelial NO (34
). Upon hemolysis or infusion of cell-free Hb, these diffusion barriers are no longer effective in preventing free Hb from reaching and reacting with endothelial NO. Indeed, the vasoactivity of cell-free Hb blood substitutes is attributed to both NO scavenging and premature delivery of oxygen to systemic arterioles, both of which trigger vasoconstriction and subsequent elevation of blood pressure (36
). Heme, the redox-active prosthetic group of Hb, generates and interacts with oxygen-free radicals (ROS), such as superoxide ion (O2•–
) and H2
). These radicals increase the vascular tone, either through direct modulation of vasoconstrictive pathways or inactivation of NO, providing another possible mechanism of Hb-induced vasoactivity (43
). Hb-derived free radicals can potentially activate platelets, and markers of platelet activation were indeed found to be increased in patients with sickle-cell disease (46
). Platelet-released mediators like serotonin could therefore be another NO-independent cause of vascular complications in hemolytic anemias (48
). Following extensive examination of the effects of Hp complex formation on the redox and ligand-binding properties of Hb, we observed that neither NO dioxygenation, autoxidation, nor H2
redox cycling differed between free Hb and the Hb-Hp complex. Consistent with these in vitro observations, the level of methemoglobin (a form of Hb in which the iron molecules are in the Fe3+
state) was very similar in plasma of Hb-infused animals, regardless of their Hp plasma concentrations. In addition, oxygen binding to the Hb-Hp complex as well as its cooperativity were comparable to those of uncomplexed Hb. Thus, it appears unlikely that the reduced hypertensive activity of the Hb-Hp complex is solely due to altered ligand-binding or enzymatic properties of circulating Hb.
A possible mechanism that may have contributed to the control of vascular Hb effects in our models involves the molecular configuration of the complex, which can potentially hinder the transendothelial passage of Hb (50
). An inverse, nonlinear relationship between Hb molecular size and vasoactivity exists in the case of chemically modified Hb, with a markedly reduced hypertensive effect of multitetrameric Hb compared with tetrameric Hb (51
). This relationship, though controversial, may be attributed to the more restricted diffusion/transition of large (polymeric or conjugated) Hbs into the subendothelial space (38
). Consistent with data obtained with chemically modified Hbs of different molecular sizes (50
), in our experiments, the most visible effect of Hb sequestration by Hp was the retention of infused Hb within the circulation, as reflected by the complete absence of renal Hb excretion in animals with high levels of endogenous or exogenously administered Hp. However, it is unlikely that the molecular weight of the Hb-Hp complex (>150 kDa) is the sole determining factor in preventing Hb-induced vasoactivity, since some larger polymeric Hbs with little or no tetramers induced a hypertensive response in animals and humans. Therefore, altered surface charge, Hp glycosylation patterns, and molecular shape of the complex may be other important factors contributing to the limited effect of the Hb-Hp complex on vascular tone. For instance, the rod-shaped Hb-Hp complex has a maximal molecular diameter of ~180–200 Å, corresponding to more than 6 times the diameter of the Hb tetramer (~30 Å) or more than 10 times the diameter of the Hb dimer (~15 Å) (55
). A globular Hb polymer with an equivalent molecular diameter as the Hb-Hp complex would consist of approximately 30 Hb tetramers with a molecular weight of 2 MDa. Thus, considering all the available physiologic, biochemical, and structural evidence, we propose that compartmentalization of cell-free Hb within the intravascular space and away from extravascular spaces is a potential mechanism underlying Hp-induced attenuation of the hypertensive activity of Hb.
In addition to hypertension, oxidative stress–related tissue damage is a central mechanism of Hb-mediated toxicity (6
). In mouse knockout models, the plasma Hb and heme scavengers, Hp and hemopexin, respectively, are essential for protection against heme-mediated oxidative damage (19
). While the redox reactions initiated at heme iron are well characterized from a chemical perspective, there is limited evidence that these reactions occur
in vivo during hemolysis. Evaluating animal models of hemoglobinuria and exchange transfusion with a long circulating HBOC, we recently obtained evidence of globin and heme oxidative modifications, including oxidation of key β-chain amino acids and protein cross-linking, in animal blood and urine (9
). Here, we found no evidence of heme oxidation within the circulation, as less than 2% of free intravascular Hb was observed in the oxidized Fe3+
state and no Hb was observed in the higher oxidation ferryl (Fe4+
) state throughout the infusion time of up to 8 hours. However, in the absence of Hp, non-modified Hb can rapidly escape the intravascular compartment and might be exposed to more oxidant (or less antioxidant) environments within certain extravascular regions, such as the subendothelial space or the renal tubular system. Therefore, while Hp effectively restricts access of Hb to the extravascular compartment, it may also prevent the participation of Hb in potentially hazardous oxidative reactions within extravascular spaces. This is consistent with our observation that Hp is effective in preventing iron deposition and related lipid peroxidation (4-HNE reactivity) in renal tubular cells. The presence of Hb oxidation products in urine of non-Hp–treated guinea pigs indicates that peroxidative Hb reactions indeed occur within the kidneys of Hb-infused animals, and may be the actual source of iron accumulation and kidney oxidative damage in the absence of Hp.
In conclusion, our studies support the potential therapeutic application of Hp in preventing toxicities and end-organ injuries associated with circulating extracellular Hb. Alternatively, pharmacological stimulation of endogenous Hp synthesis or administration of Hp analogs that mimic Hb binding and the antioxidant properties of Hp are feasible strategies to attenuate Hb-mediated toxicity. High-dose glucocorticoid treatment has been shown to have beneficial effects in conditions associated with accelerated hemolysis. Glucocorticoids attenuate the severity of sickle-cell vaso-occlusive pain crisis, the acute chest syndrome, and the hyperhemolysis syndrome. It will be interesting to investigate in clinical studies whether enhanced Hp synthesis in these patients is among the mechanisms involved in the protective glucocorticoid effect (58
). Additionally, our data demonstrate that the Hb-Hp complex retains oxygen-binding characteristics with prolonged circulating time. This presents an intriguing possibility that the Hb-Hp complex could be developed further as a safe and effective blood substitute.