Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment.
ABSTRACT Iron-derived reactive oxygen species are involved in the pathogenesis of numerous vascular disorders. One abundant source of redox active iron is heme, which is inherently dangerous when it escapes from its physiologic sites. Here, we present a review of the nature of heme-mediated cytotoxicity and of the strategies by which endothelium manages to protect itself from this clear and present danger. Of all sites in the body, the endothelium may be at greatest risk of exposure to heme. Heme greatly potentiates endothelial cell killing mediated by leukocytes and other sources of reactive oxygen. Heme also promotes the conversion of low-density lipoprotein to cytotoxic oxidized products. Hemoglobin in plasma, when oxidized, transfers heme to endothelium and lipoprotein, thereby enhancing susceptibility to oxidant-mediated injury. As a defense against such stress, endothelial cells upregulate heme oxygenase-1 and ferritin. Heme oxygenase opens the porphyrin ring, producing biliverdin, carbon monoxide, and a most dangerous product-redox active iron. The latter can be effectively controlled by ferritin via sequestration and ferroxidase activity. These homeostatic adjustments have been shown to be effective in the protection of endothelium against the damaging effects of heme and oxidants; lack of adaptation in an iron-rich environment led to extensive endothelial damage in humans.
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ABSTRACT: Evidence indicates that brain injury after intracerebral hemorrhage (ICH) is due in part to the release of iron from hemoglobin. Therefore, we examined whether such iron is cleared from the brain and the effects of ICH on proteins that may alter iron release or handling: brain heme oxygenase-1, transferrin, transferrin receptor, and ferritin. Male Sprague-Dawley rats received an infusion of 100 microL autologous whole blood into the right basal ganglia and were killed 1, 3, 7, 14, or 28 days later. Enhanced Perl's reaction was used for iron staining, and brain nonheme iron content was determined. Brain heme oxygenase-1, transferrin, transferrin receptor, and ferritin were examined by Western blot analysis and immunohistochemistry. Immunofluorescent double labeling was performed to identify which cell types express ferritin. ICH upregulated heme oxygenase-1 levels and resulted in iron overload in the brain. A marked increase in brain nonheme iron was not cleared within 4 weeks. Brain transferrin and transferrin receptor levels were also increased. In addition, an upregulation of ICH on ferritin was of very long duration. The iron overload and upregulation of iron-handling proteins, including transferrin, transferrin receptor, and ferritin, in the brain after ICH suggest that iron could be a target for ICH therapy.Stroke 01/2004; 34(12):2964-9. · 6.16 Impact Factor
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ABSTRACT: Heme arginate infusions blunt the symptoms of patients with acute intermittent porphyria without evidence of the vascular or thrombotic side effects reported for hematin. To provide a rationale for heme arginate's safety, the present study examined the effects of various ferriporphyrins to sensitize human endothelial cells to free radical injury and to induce heme oxygenase and ferritin expression. Heme arginate, unlike hematin, did not amplify oxidant-induced cytotoxicity mediated by hydrogen peroxide (5.3 +/- 2.4 versus 62.3 +/- 5.3% (51)Cr release, P <.0001) or by activated neutrophils (14.4 +/- 2.9 versus 41.1 +/- 6.0%, P <.0001). Nevertheless, heme arginate efficiently entered endothelial cells similarly to hematin, since both markedly induced heme oxygenase mRNA (more than 20-fold increase) and enzyme activity. Even with efficient permeation, endothelial cell ferritin content was only minimally increased by heme arginate compared with a 10-fold induction by hematin; presumably less free iron was derived from heme arginate despite up-regulation of heme oxygenase. Hematin is potentially vasculopathic by its marked catalysis of oxidation of low-density lipoprotein (LDL) to endothelial-toxic moieties. Heme arginate was significantly less catalytic. Heme arginate-conditioned LDL was less than half as cytotoxic to endothelial cells as hematin-conditioned LDL (P <.004). It is concluded that heme arginate may be less vasculotoxic than hematin since it is an effective heme oxygenase gene regulator but a less efficient free-radical catalyst.Blood 06/2000; 95(11):3442-50. · 9.06 Impact Factor
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ABSTRACT: Numerous pathologies may involve toxic side effects of free heme and heme-derived iron. Deficiency of the heme-catabolizing enzyme, heme oxygenase-1 (HO-1), in both a human patient and transgenic knockout mice leads to an abundance of circulating heme and damage to vascular endothelium. Although heme can be directly cytotoxic, the present investigations examine the possibility that hemoglobin-derived heme and iron might be indirectly toxic through the generation of oxidized forms of low-density lipoprotein (LDL). In support, hemoglobin in plasma, when oxidized to methemoglobin by oxidants such as leukocyte-derived reactive oxygen, causes oxidative modification of LDL. Heme, released from methemoglobin, catalyzes the oxidation of LDL, which in turn induces endothelial cytolysis primarily caused by lipid hydroperoxides. Exposure of endothelium to sublethal concentrations of this oxidized LDL leads to induction of both HO-1 and ferritin. Similar endothelial cytotoxicity was caused by LDL isolated from plasma of an HO-1-deficient child. Spectral analysis of the child's plasma revealed a substantial oxidation of plasma hemoglobin to methemoglobin. Iron accumulated in the HO-1-deficient child's LDL and several independent assays revealed oxidative modification of the LDL. We conclude that hemoglobin, when oxidized in plasma, can be indirectly cytotoxic through the generation of oxidized LDL by released heme and that, in response, the intracellular defense-HO-1 and ferritin-is induced. These results may be relevant to a variety of disorders-such as renal failure associated with intravascular hemolysis, hemorrhagic injury to the central nervous system, and, perhaps, atherogenesis-in which hemoglobin-derived heme may promote the formation of fatty acid hydroperoxides.Blood 09/2002; 100(3):879-87. · 9.06 Impact Factor
Forum review article
HEME, HEME OXYGENASE AND FERRITIN: HOW THE VASCULAR ENDOTHELIUM
SURVIVES (AND DIES) IN AN IRON-RICH ENVIRONMENT
József Balla1, Gregory M. Vercellotti2, Viktória Jeney1, Akihiro Yachie3, Zsuzsa Varga1, Harry S.
Jacob2, John W. Eaton4 and György Balla1
1Departments of Medicine and Neonatology, University of Debrecen, Debrecen 4012, Hungary
2Department of Medicine, University of Minnesota, Minneapolis, MN 55455, USA
3Department of Laboratory Sciences, Kanazawa University, Kanazawa 920-8641, Japan
4James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA
Running title: Cytoprotective systems of endothelium
Corresponding author: József Balla, M.D., D.Sc.
Nagyerdei krt. 98.
Phone/Fax: (36)-52-413 653
Iron-derived reactive oxygen species are involved in the pathogenesis of numerous vascular
disorders. One abundant source of redox active iron is heme, which is inherently dangerous when
escapes from its physiological sites. Here, we present a review of the nature of heme-mediated
cytotoxicity and of the strategies by which endothelium manages to protect itself from this clear
and present danger. Of all sites in the body, the endothelium may be at greatest risk of exposure
to heme. Heme greatly potentiates endothelial cell killing mediated by leukocytes and other
sources of reactive oxygen. Heme also promotes the conversion of low-density lipoprotein to
cytotoxic oxidized products. Hemoglobin in plasma, when oxidized, transfers heme to
endothelium and lipoprotein, thereby enhancing susceptibility to oxidant-mediated injury. As a
defence against such stress, endothelial cells upregulate heme oxygenase-1 and ferritin. Heme
oxygenase opens the porphyrin ring, producing biliverdin, carbon monoxide and a most
dangerous product redox active iron. The latter can be effectively controlled by ferritin via
sequestration and ferroxidase activity. These homeostatic adjustments have been shown effective
in the protection of endothelium against the damaging effects of heme and oxidants; lack of
adaptation in an iron-rich environment led to extensive endothelial damage in human.
Free heme and heme iron are toxic to endothelium
Hemoglobin-, heme- and iron-mediated oxidation of low-density lipoprotein
How endothelium adapts and survives in an iron-rich environment: heme oxygenase-1
Hemoglobin, heme and iron: Possible importance in LDL oxidation and atherogenesis
Importance of ferritin in cytoprotection and cell proliferation
Cytoprotective effects of heme oxygenase
An unfortunate experiment of nature: Congenital deficiency of heme oxygenase-1
Heme is the most important iron complex in the human body and is responsible for oxygen and
electron transport among other functions. Unfortunately, in certain situations, heme may be
released from heme proteins and both the heme and iron released from it may subsequently
catalyze free radical reactions. Of all sites in the body, the vasculature and in particular the
endothelial lining may be at greatest risk of exposure to free heme. This is because erythrocytes
contain heme in a concentration of 20 mmol/L and are vulnerable to unexpected lysis.
Extracellular hemoglobin is easily oxidized from ferro- to ferri-hemoglobin (methemoglobin)
which, in turn, will readily release heme. Given the hydrophobic nature of heme, it is no surprise
that it easily crosses cell membranes and can synergistically enhance cellular oxidant damage.
The present review concerns the involvement of heme in vascular endothelial cell injury and the
strategies used by endothelium to minimize such damage.
Free heme and heme iron are toxic to endothelium
Cell and organ damage provoked by reactive oxygen species can be greatly amplified by free
redox active iron (1). For example, iron-rich Staphylococcus aureus are three orders of magnitude
more susceptible to killing by hydrogen peroxide than are iron-poor staphylococci (2).
Conversely, depletion of cellular iron powerfully protects eukaryotic and prokaryotic cells
against oxidant challenge (3). We have shown that one critical feature required for iron-mediated
damage to endothelium is intrusion of the metal into cells. Chelation of iron by certain lipophilic
chelators, such as 8-hydroxyquinoline, results in the accumulation of catalytically active
lipophilic iron chelates in endothelial lipid compartments; endothelium pretreated with the 8-
hydroxyquinoline-iron chelate is exquisitely sensitive to both endogenous and exogenous oxidant
One abundant source of potentially toxic iron is iron protoporphyrin-IX or heme which is also
hydrophobic. Heme, a ubiquitous iron-containing compound, is present in large amounts in many
cells (5) and is also inherently dangerous, particularly when it escapes from intracellular sites (6-
9). Heme greatly amplifies cellular damage arising from activated oxygen (Fig. 1A) (6-8).
The potential toxicity of free heme derives from the ease with which this highly hydrophobic
compound can enter and cross cell membranes; heme readily concentrates within the hydrophobic
milieu of intact cells (6, 7). Heme uptake by endothelial cells can exacerbate their damage by
activated polymorphonuclear leukocytes (PMNs) (Fig. 1A) cells that tend to marginate along
endothelial surfaces in the presence of diverse inflammatory mediators (6, 8). Intriguingly, heme
was shown by Graca-Souza and colleagues to induce PMN activation as well (10). Moreover,
Wagener et al. reported that heme can enhance endothelial cell adhesion molecule expression,
increasing PMN adhesion and provoking inflammation (11, 12).
The uptake of heme is required for this synergistic toxicity and the hydrophobicity of heme is
critical for entry into endothelial cells. The spontaneous uptake of heme and the associated
amplification of cellular oxidant sensitivity are both inhibited by the heme-binding protein,
hemopexin (Fig. 1A) (6, 7) which blocks the catalytic (i.e., peroxidase) activity of heme and
promotes its clearance (6, 7, 13, 14). Hemopexin is certainly not the sole factor in plasma that
protects against heme-amplified oxidant damage to endothelium. Albumin may also limit the
intrusion of extracellular heme into cells and lessen its pro-oxidant effects.
Once within the cell, heme can promote oxidative damage either directly or, perhaps more
importantly, via the release of iron which can occur either through non-enzymatic oxidative
degradation of heme (6, 7) or enzymatic, heme oxygenase catalyzed heme cleavage. In either
case, the iron may initially lodge within the hydrophobic interstices of the phospholipid bilayer.
Within this highly oxidizable matrix, iron acts as an especially active catalyst of oxidation of cell
membrane constituents (6).
Given the protective effects of plasma constituents such as hemopexin and albumin, we
wondered whether heme could sensitize endothelial cells to oxidative challenge in the presence of
plasma (8). In fact, exposure of endothelium to heme in the presence of whole human plasma also
synergizes cellular damage caused by exogenous oxidants, with an optimal heme-exposure
duration of 60 minutes (8). Interestingly, cytotoxicity studies showed little toxicity to endothelium
if water soluble heme arginate is added instead of heme (8). This is so despite the fact that
exposure of endothelium to heme arginate in plasma free medium increases endothelial cell heme
content to an extent similar to what is observed after heme treatment. Comparable heme uptake is
also observed in the presence of human plasma although at 2 orders of magnitude greater
concentration for both heme arginate and heme.
The hydrophobicity of various heme analogues (ferriporphyrins) is critical for entry into cells and
required for the synergistic oxidative toxicity. Substitution of vinyl side chains of heme with
hydrogen does not alter the hydrophobicity of the resultant ferriporphyrin (iron deuteroporphyrin
IX) and cytotoxicity remains high. In contrast, if water solubility of heme is increased with the
arginate counterion or the vinyl side chains of heme are substituted by sulfonate, propionate, or
glycol leading to hydrophilic ferriporphyrins (iron deuteroporphyrin IX,2,4-bis-sulfonate, iron
coproporphyrin III, and iron deuteroporphyrin IX,2,4-bis-glycol), these ferriporphyrins fail to
sensitize cells to oxidants or activated polymorphonuclear leukocytes (8).
Although free heme is rapidly incorporated into hydrophobic domains of cells and serves as a
source of highly damaging iron, the question remains as to whether intact heme liganded to
proteins, as in hemoglobin, might also transfer heme to vascular endothelium. Whereas reduced
(ferro- or oxy-) hemoglobin is relatively innocuous to endothelial cells, oxidized (ferri- or met-)
hemoglobin greatly amplifies oxidant mediated endothelial injury (Fig. 1B) (15, 16). This is
because ferrihemoglobin readily releases its heme moieties as first demonstrated by Bunn and
Jandl (17). Released heme from ferrihemoglobin is rapidly incorporated into hydrophobic
domains of cultured endothelium and serves as a source of highly damaging iron. Although
ferrohemoglobin itself is not capable of sensitizing vascular endothelial cells to oxidant injury,
we and others have shown it can readily be oxidized to heme-releasing ferrihemoglobin in the
presence of inflammatory cell-derived oxidants (15, 18, 19). For instance, polymorphonuclear
leukocytes, when activated with phorbol ester, rapidly oxidize ferrohemoglobin to
ferrihemoglobin (15). Accordingly, ferrohemoglobin oxidized to ferrihemoglobin by activated
PMNs can provide heme to endothelium which greatly enhances cellular susceptibility to
oxidant-mediated cell-injury (15, 16). Another candidate for generating ferrihemoglobin is nitric
oxide. Reaction of nitric oxide with free hemoglobin produces ferrihemoglobin, and leads to
decreased nitric oxide bioavailability, causing pulmonary hypertension, vascular damage, and
end-organ injury as reviewed by Gladwin et al (20).
The initial release of heme from ferrihemoglobin can be inhibited by complexing with the
hemoglobin-binding protein, haptoglobin (17). If metheme binding to globin is strengthened by
haptoglobin, cyanide, or the released heme is religanded to hemopexin, ferrihemoglobin loses
much of its capacity to sensitize endothelium to reactive oxygen (Fig. 1C) (15). The
hemoglobin:haptoglobin complex is eliminated from the circulation at least in part through the
recently characterized CD163 receptor (21), which is expressed exclusively by cells of the
The importance of heme release from ferrihemoglobin in such toxicity is emphasized by the fact
that ferrohemoglobin or other heme proteins, such as metmyoglobin and cytochrome c, none of
which readily release heme (22), do not alter endothelial integrity (Fig. 1B). At higher
concentrations of free ferrihemoglobin in plasma (such as might occur in certain hemolytic
diseases, atherosclerosis and malaria infections) the normal mechanisms for control of
hemoglobin (haptoglobin/hemopexin) can be overwhelmed and released heme will enter the
These previous studies and others indicating that hemoglobin might behave as a biologic Fenton
reagent(23, 24) made us wonder whether hemoglobin in plasma could provide heme-iron to
endothelium in vivo. As was the case in vitro, we found in vivo that oxyhemoglobin does not
serve as a source of damaging heme-iron to endothelium. In contrast, oxidation of hemoglobin to
ferrihemoglobin by phagocyte-mediated oxidation fosters transfer of heme to the vessel wall and
aggravates endothelial cell damage in the short term. Ferrihemoglobin present in plasma also
increases the level of endothelial cell associated heme in lung (25) indicating that protective
effects of haptoglobin (26), hemopexin (6, 13, 14), and albumin can be overwhelmed and the
delivery of heme-iron to the endothelium can occur in vivo (25).
Hemoglobin-, heme- and iron-mediated oxidation of low-density lipoprotein (LDL)
It appears that endothelium may not be the only target of heme-mediated oxidation reactions.
Oxidative modification of LDL is implicated in the pathogenesis of atherosclerosis (27-33). LDL
particles entering subendothelial "sanctuaries" within the artery wall can become trapped and
exposed to oxidative stresses. LDL oxidation has been shown to foster recruitment of
macrophages and, by binding to scavenger receptors on the surface of macrophages, oxidized
LDL can ultimately generate foam cells. Oxidized LDL (hereafter, LDLox ) is also directly
cytotoxic, particularly to vascular endothelial cells. Such damage would presumably exacerbate
atheroma formation both by allowing LDL to penetrate the endothelial barrier, promoting platelet
adherence and smooth muscle growth factor production.
We earlier proposed that heme might be a possible physiological mediator of LDL oxidation and
subsequent endothelial cell injury (27). The process of heme-mediated LDL oxidation involves
coupled interactions between LDL, heme, oxidants, and antioxidants. The initial step in these
complex reactions is the spontaneous insertion of heme into LDL particles. The inserted heme
directly promotes extensive oxidative modification of LDLsuch modification can be amplified
by trace amounts of hydrogen peroxide (Fig. 2), PMN-derived oxidants or preformed lipid
hydroperoxides within the LDL. Depletion of -tocopherol in LDL is followed by the formation
of conjugated dienes, lipid hydroperoxides, and thiobarbituric acid-reactive substances.
Heme will oxidatively modify both the lipid moiety of LDL as well as the apoprotein, which
latter can be detected through increased anodal electrophoretic mobility. This increased mobility
reflects a loss of net positive charge, arising from oxidative destruction of amine groups, which
can also be assayed independently by measurement of free amino groups on the LDL particles.
Fluorescamine-titratable free amino groups of apolipoprotein B-100 progressively fall during
exposure of LDL to heme. During these oxidative reactions between heme, LDL, and peroxides,
the heme ring (protoporphyrin IX) is degraded, with resultant release of free iron. Both the
destruction of porphyrin ring and the release of ferrozine-trappable free iron are evidently
involved in LDL oxidation. The oxidative scission of the porphyrin ring, presumably via reaction
with lipid hydroperoxides, can be detected spectrophotometrically by the decrease in heme
absorption at 412 nm (Fig. 2). The subsequent release of free iron results in iron catalysis of
oxidation of further heme, fatty acids, cholesterol, and apolipoprotein B-100 in LDL particles.
The importance of this degradation is emphasized by the fact that conjugated diene formation
occurs in parallel with the release of iron. The released iron-driven component in heme-mediated
LDL oxidation is critical since desferrioxamine attenuates both the oxidative modification of
LDL and the degradation of heme (27).
The requirement for intimate association between LDL and heme in LDL oxidation is supported
by experiments employing hemopexin. This serum protein, present in remarkably high plasma
concentration ( 1g/L), binds heme with extraordinary avidity (34) and will prevent insertion of
heme into LDL (27). Not surprisingly, hemopexin, in stoichiometric amounts with heme, inhibits
oxidative modification of LDL and subsequent endothelial cell damage (Fig. 3). As would also be
expected, given the exceptional affinity of hemopexin for heme, other proteins such as
haptoglobin and albumin at equimolar concentration do not protect LDL from heme-catalyzed
oxidation. Potentially relevant to in vivo vascular damages are studies demonstrating that
activated PMNs potentiate oxidation of LDL catalyzed by heme-iron. That such heme-induced
LDL oxidation may be involved in vascular damage is supported by the finding that LDL
oxidized by heme is extremely cytotoxic to endothelial cells (Fig. 3A). Lipid soluble butylated
hydroxytoluene, probucol and the 21-amino steroid lazaroid, U74500A, are all potent inhibitors
of heme-catalized LDL oxidation and subsequent cytotoxicity (Fig. 3B). Although the reaction of
LDL with heme produces a markedly toxic oxidized LDL in less then 2 hours, more prolonged
incubation actually reduces the LDL toxicity (Fig. 4A). Measurement of lipid peroxidation
products led us to the conclusion that there is a strong connection between the lipid
hydroperoxide content and the toxicity of the LDL oxidized by heme (Fig. 4B). This is in
agreement with other experiments wherein specific enzymatic reduction of LOOH to LOH yields
LDL with minimal toxic effect (vide infra).
If water soluble heme (heme arginate) is added to LDL, lipid peroxidation is characterized by a
longer lag phase and T at Vmax as well as a slower propagation phase compared to heme-
mediated lipid peroxidation of LDL as judged by conjugated diene formation (8). The results of
several independent assays for LDL oxidation stimulated by heme or heme arginate all support
the conclusion that heme arginate promotes LDL oxidation less efficiently. Accordingly, the
cytotoxicity of heme arginate-conditioned LDL to endothelial cells was significantly less than
endothelial cell cytotoxicity evoked by LDL conditioned with heme. However, the number of
heme molecules associated with LDL particles was the same in LDL exposed to heme versus
LDL exposed to heme arginate (both in serum), suggesting that the more efficient oxidation of
LDL by heme versus heme arginate is partially a function of increased hydrophobicity. In diluted
serum (20%), Camejo also observed heme binding to LDL leading as well as its oxidation in the
presence of hydrogen peroxide (35).
Continuous monitoring of the process of oxidative modification of LDL is possible by measuring
the decreasing absorbance of heme at 405-412 nm, since in heme-catalyzed oxidation of
lipoprotein heme degradation occurs concurrently with formation of lipid oxidation products
including conjugated dienes and lipid hydroperoxide. Thus, heme degradation functions as a
probe for lipid peroxidation processes (27). Based on the kinetics of heme-catalyzed lipid
peroxidation we developed an assay for the clinical laboratory to judge the susceptibility of LDL
to oxidative modification (36), a risk factor of atherosclerosis. The oxidative resistance of LDL
was characterized by T at maximum velocity (Vmax) in seconds, the time point of maximal
velocity of heme degradation as defined by the maximum change in absorbance of heme in the
propagation phase of lipid peroxidation. The shortening of T at Vmax indicates a decrease in
oxidative resistance of LDL. This novel assay is suitable for testing large numbers of LDL
samples on an automated microplate reader. The advantages of our method over existing
measurements (37-39) are the ability to follow the kinetics of LDL lipid peroxidation at a visible
wavelength and to use Na2EDTA during isolation and analysis of LDL.
In addition to free heme (27, 40), a number of heme proteins such as hemoglobin (41),
myoglobin (42), horseradish peroxidase (43), myeloperoxidase (44), and lipoxygenase (45, 46)
have been reported to act as oxidants of LDL. However, the mechanisms involved are by no
means clear. In a plasma-free model, hemoglobin reacting with hydrogen peroxide was shown to
induce lipid peroxidation of LDL accompanied by oxidative cross-linking of apolipoprotein B-
100 via the formation of ferryl hemoglobin and the subsequent generation of radicals on the
globin surface (47). The authors of that study concluded that negligible heme transfer from
hemoglobin to LDL, or none at all, occurred under the oxidative conditions they employed.
Oxidation of hemoglobin to the ferryl state by peroxides has been reported to be accompanied by
tyrosyl radical formation (48, 49). In end-stage renal failure patients on chronic hemodialysis
therapy, a high degree of apolipoprotein B-100 modification resulting from covalent association
of hemoglobin with LDL was observed (50). The authors postulated that a tyrosyl radical species
of hemoglobin that forms by oxidation of ferrihemoglobin to ferrylhemoglobin with hydrogen
peroxide induces cross-linking of LDL accompanied by an increase in dityrosine formation, and
the modification of lipoprotein occurs through a mechanism independent of lipid peroxidation.
Our studies offer an alternative pathway for modification of LDL by hemoglobin in plasma
involving heme release from ferrihemoglobin. The results reported (51) generally support such a
mechanism insofar as maneuvers which restrict heme transfer to LDL uniformly diminish or
block LDL oxidation. We hypothesized that oxidation of free hemoglobin in plasma could
threaten vascular endothelial cell integrity via oxidative modification of LDL by heme. Indeed,
LDL isolated from plasma incubated with either ferrihemoglobin or heme was found to be
markedly cytotoxic. In contrast, LDL isolated from plasma incubated with ferrohemoglobin or
other heme proteins such as metmyoglobin or cytochrome c, all of which avidly bind heme,
failed to harm endothelial cell monolayers. These results suggest that the release of heme from
ferrihemoglobin is an important precedent event in generating toxic (presumably oxidized) LDL.
Therefore, we conducted similar experiments using various strategies to stabilize the heme
moiety. Preincubation of ferrihemoglobin with sodium cyanide or stoichiometric amounts of
haptoglobin stabilized the heme:globin complex and prevented the generation of LDLox.
The above findings might explain why haptoglobin polymorphisms were found in clinical studies
to be a risk factor in the pathogenesis of atherosclerosis (52).
In studies, Shaklai s group recently revealed that haptoglobin phenotypes differ in their ability to
inhibit heme transfer from hemoglobin to LDL (53). Heme transfer from ferrihemoglobin to LDL
was demonstrated to be almost completely blocked by haptoglobin 1-1 BUT only partially by
haptoglobin 2-2. Accordingly, haptoglobin 1-1 was shown to inhibit hemoglobin induced
oxidation of lipoprotein more vigorously compared to haptoglobin 2-2. This difference in
'antioxidant' capacity of different haptoglobin types may help explain why individuals with
haptoglobin 2-2 have more severe atherosclerotic disease compared to those with haptoglobin 1-1
Although ferrohemoglobin in plasma does not itself provoke oxidation of LDL, as mentioned
above hemoglobin can readily be oxidized to heme-releasing ferrihemoglobin in the presence of
inflammatory cell-derived oxidants (8, 15, 18). Accordingly, if endothelial cells are exposed to
LDL isolated from plasma containing ferrohemoglobin and activated PMNs, oxidative
endothelial damage develops (51). Importantly, neither activated PMNs alone nor
ferrohemoglobin alone causes the generation of cytotoxic LDL. Oxidation of ferrohemoglobin by
activated PMNs in plasma can be inhibited by catalase and LDL isolated from plasma containing
ferrohemoglobin, activated PMNs and catalase has reduced endothelial cell cytotoxicity.
In a recent study we have shown that LDL-associated lipid hydroperoxides convert
ferrohemoglobin to ferrihemoglobin in a dose-dependent manner as well (Fig. 5) (54). Reduction
of the lipid hydroperoxide content of LDL with GSH/glutathione peroxidase prevents the
formation of ferrihemoglobin. Interestingly, haptoglobin does not inhibit this oxidation but does
prevent heme release from the resultant ferrihemoglobin.
The results of several independent assays for LDL lipid peroxidation support the conclusion that
ferrihemoglobin-derived heme promotes LDL oxidation (Table 1) (51). Shortening of T at Vmax
by ferrihemoglobin is paralleled by a rapid decrease in the -tocopherol content of LDL, which is
followed by the formation of conjugated dienes, lipid hydroperoxides (LOOHs) and
thiobarbituric acid-reactive substances (TBARS). In contrast, ferrihemoglobin complexed with
haptoglobin or cyanomethemoglobin does not alter either T at Vmax or the -tocopherol content
of LDL. This also prevents the generation of conjugated dienes, lipid hydroperoxides and
thiobarbituric acid-reactive substances in LDL.
These observations raised the question of the nature of the toxic substance(s) which might arise
from hemoglobin/heme iron-mediated LDL oxidation. Oxidation of LDL leads to formation of a
wide range of biologically active products, and some of these, such as 7 -hydroperoxycholesterol
(55) and 7-oxysterols (56), have been reported to be highly cytotoxic. In this regard, ebselen, a
seleno organic compound which has hydroperoxide reducing activity, protects against LDLox
induced cell death in human fibroblast cells (57). It appears that accumulated LOOH is the
predominant toxic species within LDLox catalyzed by heme because specific enzymatic
reduction of LOOH to LOH yields LDL with minimal toxic effects (51). Furthermore, we find
that, on an equimolar basis, LOOH within LDLox and an organic hydroperoxide, cumene
hydroperoxide, have very similar toxic effects on endothelial cells.
How endothelium adapts and survives in an iron-rich environment: heme oxygenase-1 and
Early support for the notion of heme oxygenase and ferritin being an antioxidant cytoprotective
stratagem of endothelium derived from the strange time-dependent effects of heme exposure on
the oxidant sensitivity of endothelial cells (Fig. 6) (58). Following a brief incubation with
exogenous heme (as little as 1 hour), endothelial cells become extraordinarily sensitive to oxidant
challenge (e.g., reagent hydrogen peroxide or activated PMN). However, endothelial cells briefly
exposed to heme in the same way and then allowed to incubate in the absence of heme for more
prolonged periods (12-72 hours) become highly resistant to oxidant mediated injury (Fig. 6A)
(58). Exposure of cells to ferrihemoglobin yielded similar time dependent effects on oxidant
susceptibility (15, 16).
Because heme and various other stimuli were known to cause the induction of both heme
oxygenase-1 (59-63) and ferritin (64-66) in other cell types, we wondered whether one or both of
these proteins might be expressed in endothelial cells previously exposed to heme and then
incubated for longer periods in response to heme. Indeed, we found that both heme oxygenase-1
mRNA level and enzyme activity as well as both H and L ferritin synthesis were upregulated in
endothelial cells a few hours after initial exposure to heme (Fig. 7) (58). Heme oxygenase,
through cleavage of the heme ring, will release intracellular iron which, in turn, can promote the
synthesis of ferritin. Alternatively, heme itself might enhance ferritin synthesis directly by
increasing RNA translation (66).