Published online before print January 15, 2004, doi: 10.1161/01.STR.0000110790.05859.DA
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(Stroke. 2004;35:590.)
© 2004 American Heart Association, Inc.
Original Contributions |
Human Serum Albumin and its N-Terminal Tetrapeptide (DAHK) Block Oxidant-Induced Neuronal Death
From the Departments of Neurology (E.T.G., R.A.S., C.A.) and Neurosurgery (J.L., S.H., P.R.W., S.S.P.), San Francisco Veterans Affairs Medical Center and University of California at San Francisco.
Correspondence to Dr S. Scott Panter, Veterans Affairs Medical Center, Neurology (127), 4150 Clement St, San Francisco, CA 94121. E-mail sspanter{at}hotmail.com
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Abstract |
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Background and Purpose— Studies using animal models of stroke have shown that human serum albumin (HSA) significantly ameliorates cerebral ischemic injury after both transient and permanent ischemia, even when administered after the onset of ischemia or reperfusion. The mechanism of this effect remains uncertain, and prior studies suggest both indirect hemodynamic and direct cytoprotective effects. HSA is a potent antioxidant, in part because of its strong copper-binding capacity. Here we examined the effect of HSA on oxidant-induced neuronal death in a cortical cell culture system.
Methods— Murine cortical cultures were exposed to oxidative stress generated by hydrogen peroxide and by a mixture of copper plus ascorbic acid. We examined the ability of HSA and a tetrapeptide occupying its N-terminus (DAHK) to prevent neuronal death after these challenges.
Results— H2O2 and CuCl2/ascorbic acid were used at concentrations that, in the absence of HSA, killed >90% of the neurons. HSA provided complete protection at a concentration of 37.5 µmol/L and 50% protection at 3.75 µmol/L. The copper-binding tetrapeptide DAHK had nearly identical potency and efficacy. HSA and DAHK were also equally effective in preventing neuronal death induced by CuCl2/ascorbic acid.
Conclusions— HSA has potent antioxidant properties, probably due to binding of copper and other transition metals. HSA extravasation into ischemic brain may provide neuroprotection by limiting metal-catalyzed oxidant stress. The tetrapeptide DAHK may be an effective, small-molecular-weight alternative to HSA as a therapeutic agent for stroke.
Key Words: albumins • antioxidants • copper • ischemia • neurons
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Introduction |
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Arecent series of studies has explored the efficacy of human serum albumin (HSA) as a therapeutic agent in experimental models of stroke.1–7 HSA was administered intravenously to treat permanent or transient cerebral ischemia and was more effective in transient ischemia. HSA significantly reduced infarct size, edema, and sodium accumulation and improved neurological outcome, even when administered up to 2 hours after onset of ischemia. While these studies demonstrated significant efficacy for HSA in the treatment of stroke, the mechanism for this robust neuroprotection remains undetermined. A number of possible mechanisms have been examined, including the effect of HSA on local cerebral perfusion, blood-brain barrier disruption, systemic fatty acid responses, and microvascular patency. While many of these mechanisms probably contribute, none appear robust enough to account for the large neuroprotective effect of HSA.2–5,8,9
HSA is a unique molecule. It maintains colloidal osmotic pressure in the vasculature and has a number of important functional properties.10 It strongly binds fatty acids, some drugs, and drug metabolites, and it has a number of cation and anion binding sites.8,10 HSA is also a potent antioxidant, acting both as a free radical scavenger and as a chelator of transition metals and heme.10 A metal binding site on HSA, the 4–amino acid terminal sequence of the molecule, is unique to human albumin and may play a prominent role in its neuroprotective effects.11–15
The 4 amino acids occupying the N-terminus of HSA—aspartate, alanine, histidine, and lysine (DAHK)—constitute a relatively high-affinity binding site for a number of cations, specifically nickel, cobalt, and copper.11 In recent series of reports, it was demonstrated that a synthetic peptide composed of these 4 amino acids could inhibit copper-induced oxidative DNA double-strand breaks and telomere shortening in cell cultures.12 It was also determined that DAHK prevented lipid oxidation in a copper-catalyzed oxidant system.13 The protective effects of DAHK may also be due in part to the fact that, in addition to acting as a copper chelator, the copper-DAHK complex is a potent superoxide dismutase mimetic, thereby increasing the antioxidant potential of the tetrapeptide or HSA itself.13 It was recently demonstrated that a novel synthetic analogue of DAHK could improve recovery of rat hearts from an ischemia/reperfusion insult,16 suggesting that this approach may similarly be useful in the treatment of stroke.
We hypothesized that the antioxidant properties of this 4–amino acid terminus of HSA might explain, in part, its neuroprotective effects after ischemia/reperfusion injury in brain. To test this hypothesis, we utilized HSA and DAHK in a neuronal cell culture model of oxidative injury. Both HSA and DAHK were neuroprotective in cell culture models of hydrogen peroxide– and copper ascorbate–induced neurotoxicity.
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Materials and Methods |
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HSA was obtained from Centeon L.L.C. as a 25% solution, and DAHK was obtained from Genemed Synthesis Incorporated. All other reagents were obtained from Sigma-Aldrich, except where otherwise noted. Adventitious metals were removed from HSA by applying it to a column of iminodiacetic acid attached to cross-linked polystyrene, after which it was reconcentrated to a 25% solution.
Cell Cultures
The studies were conducted in accordance with National Institutes of Health guidelines and with the use of protocols approved by the local institutional committee on animal studies. Mice were anesthetized with isoflurane before being killed for harvesting of brains. Cortical cell cultures were prepared in a 2-step process, which has previously been described in detail.17,18 Astrocytes were plated at 1.5x105 cells per well. Neurons were plated on the astrocyte layers at a density of 6x105 cells per well.
Experimental Procedures
Experiments were begun by replacing the culture medium with a balanced salt solution (BSS), as previously described.17,18 The pH of the BSS was adjusted to pH 7.2 and during equilibration in a 5% CO2 atmosphere. Osmolarity was measured with a Wescor vapor pressure osmometer and adjusted with H2O or NaCl when necessary to achieve 280 to 320 mOsm. Test compounds were prepared as x100 stock solutions in distilled deionized water and were diluted to working concentrations in BSS before use. The test compounds were added to the cultures in BSS, and the cultures were then replaced in the 37°C, 5% CO2 incubator. In studies in which H2O2 was used, the medium was replaced with BSS after 60 minutes of H2O2 exposure. All other treatment combinations were maintained for 20 to 22 hours. Control wells received only medium exchanges. In each experiment, all comparisons were made with the use of sister cultures derived from single plating.
Assessment of Neuronal Survival
Neuronal survival was assessed by the propidium iodide exclusion method.18,19 Propidium iodide was added at 0.03 mg/mL to each well. Dead (fluorescent) and live (nonfluorescent) neurons were counted in 4 optical fields chosen randomly in each well, with the use of a Nikon fluorescence microscope with phase-contrast optics. Neurons were easily distinguished from the underlying astrocyte layer by their phase-bright, process-bearing morphology (Figure 3). Results from each well were expressed as percent neuronal survival, calculated as (live cellsx100)/(live cells+dead cells). In a subset of the experiments, cell counts were also performed by a second observer in a blinded fashion to exclude observer bias.
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Statistical Analysis
Data are presented as mean±SEM. Statistical significance was assessed with the use of 1-way ANOVA followed by the Dunnett post hoc test for multiple comparisons against a control group. Differences were considered significant at P<0.05.
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Results |
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To determine the antioxidant capacity of HSA and DAHK, we first used H2O2 to generate oxidative stress. Cortical cultures were exposed for 1 hour to a range of H2O2 concentrations, and neuronal survival was assessed by propidium iodide staining after an additional 22 to 24 hours in BSS. The resulting concentration-response curve showed a threshold effect on neuronal survival (Figure 1), similar to previous reports.18,20 Exposure to 100 µmol/L caused near-complete neuronal death, and this concentration was used for all subsequent studies.
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We evaluated the efficacy of HSA as a neuroprotective agent by adding various concentrations of HSA to the culture simultaneously with H2O2. HSA had a dose-dependent neuroprotective effect, with neuronal death reduced to a level comparable to control conditions at concentrations of 
15 µmol/L (Figure 2A). To determine whether the chelating tetrapeptide DAHK would also prevent neuronal death resulting from H2O2, cultures were incubated with 100 µmol/L H2O2 in the presence of a range of DAHK concentrations. These studies showed a potent, dose-dependent effect of DAHK on H2O2-induced neuronal death (Figure 2B). Photomicrographs showing the effect of HSA on H2O2-induced neuronal death are shown in Figure 3.
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We tested the ability of HSA to protect neurons against the mixture of 25 µmol/L CuCl2 and 50 µmol/L ascorbic acid (Figure 4), which in oxygenated solutions generates oxygen-derived free radical species.21,22 In the absence of HSA, this exposure killed >95% of the neurons, but in the presence of 150 µmol/L HSA neuronal death was reduced to control values. HSA has a high-affinity binding site for copper and other transition metals at its N-terminus in the form of a DAHK tetrapeptide.21–23 Synthetic DAHK tetrapeptide alone also completely blocked Cu/ascorbic acid–dependent neurotoxicity when added at concentrations of 50 µmol/L, although the potency of DAHK was >100-fold greater than HSA when the concentrations of both were expressed as percent weight/volume. H2O2 requires interaction with a transition metal to produce reactive oxygen species.24
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The antioxidant effect of HSA could, in principle, occur in either the extracellular or the intracellular compartments, since HSA can be internalized by neurons under some conditions.25 As a possible way to determine the compartment in which the neuroprotective effect of HSA was exerted, we assessed the ability of HSA to prevent N-methyl-D-aspartate (NMDA)–induced neuronal death, a process that is mediated in part by intracellular production of oxygen free radicals and is blocked by cell-permeant oxygen free radical scavengers.26,27 HSA had no effect on NMDA neurotoxicity. This suggests that the action of HSA occurs in the extracellular space (Figure 5).
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To test whether it is the DAHK moiety of HSA that is primarily responsible for its antioxidant effects, we compared HSA with several other proteins that do not contain this tetrapeptide at the N-terminus: lactalbumin, 
-globulin, bovine serum albumin, and casein. Somewhat surprisingly, each of these proteins also protected neurons against H2O2 toxicity with potencies roughly similar to that of HSA (Figure 6).
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Discussion |
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These results show that the neurotoxic oxidant stress induced by either hydrogen peroxide or copper/ascorbic acid can be blocked by HSA and by a tetrapeptide that is the same sequence as the N-terminus of HSA. These results may be relevant to the recent series of articles that describe the protective effect of HSA in animal models of stroke and traumatic brain injury.2–5,9
In vivo, the concentration of HSA in cerebrospinal fluid is approximately 3.7 µmol/L.28 This concentration of HSA produced an approximately 40% reduction in H2O2-induced neuronal death under the conditions used in the cell culture studies described here. Additional HSA may enter the brain from the plasma compartment after stroke through an open blood-brain barrier.25 Since the concentration of HSA in serum is approximately 588 µmol/L,29 even a small movement of serum proteins across the blood-brain barrier could substantially raise HSA concentrations in the extracellular space surrounding postischemic neurons and increase resistance to oxygen free radicals in the extracellular space.
For the purpose of testing the neuroprotective effects of HSA and DAHK in cultured neurons, 2 different models of oxidant-mediated neuronal injury were developed. The first uses hydrogen peroxide as the stressor, and the second uses a copper/ascorbic acid–driven free radical–generating system. Both oxidants were used under conditions that, in the absence of HSA or DAHK, killed nearly 100% of the neurons in the cultures. Copper-ascorbic acid–driven stress involves a transition metal, and it is almost certain that the hydrogen peroxide neurotoxicity also requires transition metals. In the absence of metals, hydrogen peroxide is extremely stable, with a calculated half-life for its uncatalyzed unimolecular homolysis at 30°C of 1011 years.24 H2O2 interaction with organic molecules requires transition metals, and chelation of transition metals prevents this interaction.
Transition metals (in particular, iron and copper) are capable of cycling between their reduced and oxidized states and, in the process, generating an electron that can create a free radical. In many cases, transition metals themselves may be bound by a lipid, protein, or nucleic acid molecule, and a free radical causes site-specific damage at or near its binding site.21–23 It is this type of activity that is most likely responsible for copper-dependent cell death in culture. If copper and/or iron is decompartmentalized by ischemia/reperfusion, the neurotoxic effects may be accelerated, and the presence of HSA or its N-terminal tetrapeptide may prevent its binding to sites where it could contribute to cellular injury. Alternatively, ambient, normal levels of transition metals in the extracellular space may have no deleterious effects under normal conditions but may become highly deleterious in the presence of H2O2 formed by the superoxide generated during ischemia/reperfusion.
The N-terminus DAHK of HSA is not the only amino acid sequence that can bind transition metals or copper specifically.30–33 In the present studies DAHK was found to be equipotent with HSA in preventing H2O2 or copper/ascorbic acid neurotoxicity, despite the fact that HSA is 126-fold larger than DAHK. This suggests that the DAHK N-terminal tetrapeptide is the primary locus of HSA antioxidant activity. However, the other proteins examined had effects similar to those of HSA, despite absence of the DAHK domain. It is possible that relatively weak interactions between sulfhydryl groups and/or amino acids (particularly tryptophan or histidine34,35) on these proteins and transition metals produce a chelating effect that, in aggregate, is comparable to that achieved with HSA or DAHK. The relatively high-affinity, nonspecific binding of copper to proteins has been previously reported to inhibit its capacity to generate hydroxyl radicals,34,35 which may explain in part the neuroprotective effects of proteins other than HSA. Regardless of the mechanism of this effect, these results suggest that other proteins may also contribute to brain antioxidant effects during blood-brain barrier breakdown. This possibility has not been tested in vivo.
In summary, we have demonstrated that HSA and its N-terminal tetrapeptide DAHK can block oxidant-driven neuronal injury produced with the use of 2 different oxidant-generating systems: hydrogen peroxide and copper/ascorbic acid. The fact that the peptide and HSA can block the neurotoxicity of the latter generating system in a stoichiometric fashion implies that they are both binding copper and can stop its redox cycling. The efficacy of DAHK in the hydrogen peroxide–driven system suggests that copper is also involved in oxidant-driven neurotoxicity in vitro and may be involved in tissue injury after ischemia and reperfusion in vivo. DAHK may be a useful alternative to HSA for the treatment of stroke.
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Acknowledgments |
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This work was supported by a Department of Veterans Affairs REAP grant and National Institutes of Health grant RO1-HL53040 (to Dr Panter). We gratefully acknowledge the technical assistance of Angelo Zegna and the advice of Dr Weihai Ying.
Received July 23, 2003; revision received September 24, 2003; accepted October 14, 2003.
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