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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Medicine and Neonatology,
University of Debrecen, Hungary; Department of Laboratory Sciences,
Kanazawa University, Japan; Department of Medicine, University of
Minnesota, Minneapolis; and the James Graham Brown Cancer Center,
University of Louisville, KY.
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 Aerobic organisms are well endowed with enzymatic
oxidant defense systems, which provide protection against activated
oxygen species. Damage caused by reactive oxygen can be greatly
amplified by "free" redox active iron.1-2 For example,
iron-rich Staphylococcus aureus is 3 orders of magnitude
more susceptible to killing by hydrogen peroxide than are iron-poor
staphylococci.3 Conversely, depletion of cellular iron
powerfully protects eukaryotic and prokaryotic cells against oxidant
challenge.4-5 One abundant source of potentially toxic
iron is heme, and both exogenous and endogenous heme can
synergistically enhance oxidant-mediated cellular damage.6-10
Heme, a ubiquitous iron-containing compound, is quite hydrophobic,
readily enters cell membranes, and greatly increases cellular susceptibility to oxidant-mediated killing.8 Heme also
acts as a catalyst for the oxidation of low-density lipoprotein (LDL), generating products toxic to endothelium.9,11-12 The toxic
effects of heme may be important in a number of pathologies. These
include not only acute conditions such as intravascular hemolysis
(which can lead to renal failure) but also more insidious processes
such as atherogenesis in which intralesional deposits of iron (perhaps derived from erythrocytes, which are known to intrude into
atherosclerotic lesions) have been observed.13
As a defense against such toxicity, normal cells upregulate heme
oxygenase-1 (HO-1) and ferritin. HO is a heme-degrading enzyme that
opens the porphyrin ring, producing biliverdin and carbon monoxide and
releasing iron.14-15 Three genes encode for 3 isoenzymes for HO.16 HO-1, identified as a 32.8-kd stress
protein,17 is transcriptionally inducible by a variety of
agents, such as heme, oxidants, and cytokines.17-21
Ferritin, a multimeric protein with a very high capacity for storing
iron, consists of 24 subunits of 2 types (heavy chain and light
chain).22 The central importance of HO-1 was recently
highlighted by the discovery of a child with HO-1 deficiency who
exhibited extensive endothelial damage.23 Similar damage
to endothelium, as well as hepatic and renal cytotoxicity, has been
observed in transgenic knockout mice deficient in HO-1.24 In both the human patient and mice, very high concentrations of circulating heme were present.
Free hemoglobin (Hb) in plasma, when oxidized, can provide heme to
endothelium, which greatly enhances cellular susceptibility to
oxidant-mediated cell injury.25,26 We hypothesized that oxidation of free Hb in plasma could threaten vascular endothelial cell
integrity via oxidative modification of LDL and that oxidized LDL might
also induce cytoprotectants such as HO-1 and ferritin. The present
investigations represent an attempt to determine whether the cytotoxic
effects of circulating Hb might, in fact, derive from oxidized species
of LDL. The results indicate that components of LDL, primarily lipid
hydroperoxides, are distinctly cytotoxic and probably account for a
substantial portion of the observed toxic effects of free Hb in plasma.
Intravascular hemolysis associated with persistent endothelial
damage was shown to be the main characteristic in a child diagnosed
with HO-1 deficiency.23 We suggest that the endothelial
damage observed in this case was mediated in part by oxidation of LDL
catalyzed by metHb-derived heme.
Materials
Endothelial cell isolation and culture
Preparation of human neutrophils Polymorphonuclear leukocytes (PMNs) were isolated from human volunteers after informed consent (following guidelines of the Committee on the Use of Human Subjects in Research of the University of Debrecen, Hungary).9Hb preparation Purified Hb was prepared from fresh blood drawn from volunteers by using ion-exchange chromatography on diethylaminoethanol-sepharose CL-6B column.25,26 Hb was assessed for purity by means of isoelectric focusing. MetHb was formed by incubation of Hb with 1.5-fold molar excess K3Fe(CN)6 over heme followed by dialysis. CyanometHb was prepared by the addition of 2-fold excess NaCN to metHb followed by gel filtration. For spectral analysis of Hb in plasma, samples were diluted 6-fold with saline and scanned with the use of diluted Hb-free plasma as blank. Oxyhemoglobin Hb, metHb, and hemichrome concentrations were determined as described by Winterbourn.27Preparation of human LDL LDL was isolated from plasma derived from EDTA-anticoagulated venous blood of healthy volunteers28 who had fasted overnight; 1 mg/mL EDTA was used. Before LDL separation, plasma was incubated with 80 µM heme, 20 µM ferrohemoglobin (ferroHb), 20 µM metHb, 20 µM metHb liganded to haptoglobin, 20 µM cyanometHb, 80 µM metmyoglobin, or 80 µM cytochrome c for 2 hours at 37°C. In separate experiments, plasma anticoagulated with 5 U/mL heparin was incubated with PMNs (107 cells per milliliter), either resting or activated by 500 ng/mL phorbol myristate acetate (PMA), in the presence or absence of ferroHb (80 µM in heme) at 37°C for 90 minutes; PMNs were then removed by centrifugation at 400g for 5 minutes at room temperature, followed by a 2-hour incubation at 37°C in the presence of 1 mg/mL EDTA before the isolation of LDL. Density of plasma was adjusted to 1.3 g/mL with potassium bromide (KBr), and a 2-layer gradient was made in a Quick-Seal polyallomer ultracentrifuge tube (Beckman Instruments, Palo Alto, CA) by layering 0.9% NaCl on 10 mL density-adjusted plasma, which was then centrifuged at 302 000g for 3 hours at 4°C (VTi 50.2 rotor) (Beckman Instruments). For the small plasma samples from the HO-1-deficient child (1.5 mL), the density was adjusted to 1210 g/L with KBr, and after the 2-layer gradient was made in a 5.1-mL Quick-Seal tube, a single spin-gradient ultracentrifugation was performed at 228 000g for 90 minutes at 4°C (VTi 65.2 rotor; Beckman Instruments) to isolate LDL.29 Purity of the LDL fraction was checked by agarose gel electrophoresis. The LDL samples were kept at 4°C and protected from light, and the protein content was determined by the BCA protein assay (Pierce, Rockford, IL).Measurement of oxidative resistance and detection of oxidative modification of LDL We used a microassay, based on the kinetics of heme-catalyzed lipid peroxidation of LDL, to assess the resistance of lipoprotein to oxidative modification.29 Briefly, in heme-catalyzed oxidation of LDL, heme degradation occurs inversely with formation of lipid oxidation products, including conjugated dienes and lipid hydroperoxide; thus, heme degradation functions as a probe for the lipid peroxidation process. The kinetics of heme disappearance is monitored spectrophotometrically at 405 nm in an automated microplate reader (Bio-Tek Instruments, Winooski, VT). The oxidative resistance of LDL was characterized by change in time ( T) at maximum
velocity (Vmax) in seconds (the time period until the
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 the decrease in
oxidative resistance of LDL. Importantly, because the HO-1-deficient
child's plasma was stored for 10 to 11 months, we established that
storage of plasma samples of healthy individuals (n = 54) for 12 months at  70°C does not alter the oxidative resistance of LDL
(T = 3017 ± 1194 versus 3131 ± 1381 seconds before and after
storage, respectively). The conjugated diene formation and generation
of thiobarbituric acid-reactive substances (TBARSs) in LDL (50 µg/mL) were measured as in our previous studies.9 Lipid
hydroperoxide (LOOH) was detected by means of the ferrous oxidation in xylenol orange assay.30 The
 -tocopherol content of LDL was determined as
described.28 Reactive amino groups in LDL were estimated
with fluorescamine with the use of lysine as a standard.9
The electrophoretic mobility of lipoprotein was determined by agarose
gel electrophoresis by means of the Hydragel LIPO plus Lp(a) kit
(Sebia, Issy-les-Moulineaux, France).
Determination of fatty acids Lipids extracted from LDL samples (500 µg/mL) were hydrolyzed, and fatty acids methylated. After extraction of fatty acid methylesters with n-hexane, the fatty acid analysis was performed with the use of a gas chromatograph (Hewlett Packard, Palo Alto, CA) coupled to a mass selective detector.31Iron and heme determinations Iron was measured spectrophotometrically as ferrozine-iron complex in reducing environment.9 LDL-associated heme was determined spectrophotometrically at 398 nm after the addition of 300 µL formic acid to LDL samples (100 µL) at a concentration of 1.5 mg/mL with the use of an extinction coefficient of 1.5 × 105 M 1 × cm1.32
Apolipoprotein B-100 was measured by the Unimate 3 ApoB immunoturbidometric assay (F Hoffmann-La Roche, Basel, Switzerland) to
calculate the molar ratios of iron and heme to apolipoprotein B.
Endothelial cell cytotoxicity assay Confluent endothelial cell monolayers grown in 24-well tissue-culture plates were washed 3 times with HBSS and then exposed to LDL samples (200 µg/mL) treated with 5 µM heme in medium 199. In some experiments, LDL was pretreated with 4 mM glutathione (GSH) and/or 2 U/mL GSH peroxidase. In the experiments with organic lipid-hydroperoxides, endothelial cells were treated with cumene hydroperoxide at a concentration of 5 to 100 µM. After a 4-hour incubation, the test solutions were replaced with 500 µL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) solution (0.5 mg/mL) in HBSS, and monolayers were incubated for an additional 6 hours. The reduced MTT was measured spectrophotometrically at 570 nm after the formazan was dissolved in 100 µL 10% sodium dodecyl sulfate and 500 µL hot isopropanol containing 20 mM HCl.Measurement of intracellular GSH HUVEC monolayers were treated with LDL or organic lipid-hydroperoxide test solutions, and intracellular GSH content was determined by using a kinetic assay.33HO enzyme activity assay HO activity in endothelial cell microsomes was measured by bilirubin generation34 after exposure of the cells to LDL isolated from variously treated plasma in medium 199 for 60 minutes followed by an 8-hour incubation with complete medium alone. HO activity is expressed as picomoles of bilirubin formed per milligram of cell protein per 60 minutes.Ferritin assay Endothelial cell ferritin content was measured after treatment of cells with control medium or LDL test solutions in medium 199 for 1 hour. After a further 16 hours of incubation in complete medium, the cells were solubilized34 and analyzed for ferritin content with the use of the IMx ferritin enzyme immunoassay (Abbott Laboratories, Abbott Park, IL). The results are expressed as nanograms of ferritin per milligram of cell protein.HO messenger RNA analysis HO-1 messenger RNA (mRNA) content was analyzed in confluent endothelial cells incubated with control medium or LDL test solutions as described for the measurement of HO enzyme activity. After 4 hours of exposure, total cellular RNA was isolated, electrophoresed, and transferred to nylon membrane. The position of 28S and 18S ribosomal RNA and the equal loading of samples were checked by ethidium bromide staining. After hybridization with biotin-labeled (Bioprime DNA Labeling System) (Life Technologies) complementary DNA (cDNA) for HO-1,25,34 the probes were detected by a chemiluminescent detection system (Photogene System 2.0) (Life Technologies). Autoradiographs were quantified by computer-assisted videodensitometry.Statistical analysis We should note that, in the healthy subjects group, SD stands for the between-subjects SD, whereas in the HO-1-deficient child it represents the within-subject SD. The distributions of the LDL-tocopherol and lipid peroxidation parameters were approximated by
normal distributions, with parameter estimates taken from the empirical
frequency distributions. The probabilities of observing values as
extreme as those observed in the HO-1-deficient child in healthy
subjects were calculated from these distributions.
LDL isolated from plasma preincubated with either metHb or heme
was found to be markedly cytotoxic (Figure
1A). In contrast, LDL isolated from
plasma preincubated with ferroHb or other heme proteins such as
metmyoglobin or cytochrome c, all of which avidly bind
heme,35-37 failed to harm endothelial cell monolayers.
These results suggest that the release of heme from metHb is an
important precedent event in generating toxic (presumably oxidized)
LDL. Therefore, we conducted similar experiments using various
strategies to stabilize the heme moiety.35,38-39
Haptoglobin or cyanide was shown by Bunn and
Jandl35 to strengthen heme-globin liganding, preventing heme release from metHb. Preincubation of metHb with sodium
cyanide or stoichiometric amounts of haptoglobin prevented the
generation of toxic species of LDL.
Although ferroHb in plasma does not itself provoke oxidation of LDL, we25 and others40-41 have demonstrated that it can readily be oxidized to heme-releasing metHb in the presence of inflammatory cell-derived oxidants. Under the conditions shown in Figure 1B, PMNs, when activated with PMA, markedly oxidize Hb (approximately 70%) in plasma within 30 minutes. Concordantly, as shown in Figure 1B, if endothelial cells were exposed to LDL isolated from plasma containing ferroHb and PMA-activated PMNs, endothelial cell toxicity was observed. Importantly, neither PMA-activated PMNs alone nor ferroHb alone caused the generation of cytotoxic LDL. Oxidation of ferroHb by PMA-activated PMNs in plasma was significantly inhibited by catalase; concomitantly, LDL isolated from plasma containing ferroHb, PMA-activated PMNs, and catalase caused reduced endothelial cell cytotoxicity (data not shown). Evidence that the same toxic species of LDL might accumulate in vivo
derived from additional experiments involving LDL isolated from the
plasma of the HO-1-deficient child reported earlier.23 As
shown in Figure 2A, LDL isolated from the
plasma of this child showed the same cytotoxic effects as were obtained
with LDL isolated following preincubation of normal plasma with metHb.
In this child, chronic intravascular hemolysis and severe endothelial
cell damage were present.23 Spectral analysis of plasma
from this child (Figure 2B) revealed that plasma Hb was predominantly
metHb. The proportion of total Hb present as metHb was around 80%
(approximately 60 µM). Intriguingly, the child's LDL had increased
electrophoretic mobility (Figure 2C), suggesting the loss of net
positive charge, which was confirmed by measurement of
fluorescamine-titratable free amino groups on the LDL particle;
fluorescamine-reactive amino group content fell to 732 mol/molapolipoprotein B-100 as compared with control (978 mol/mol
apolipoprotein B-100). Similar alterations in the anodal mobility of
LDL occurred when normal plasma was exposed to metHb in vitro, whereas
ferroHb had no effect (Figure 2C).
As might be expected, the oxidative resistance of the HO-1-deficient
boy's LDL was virtually zero (Table 1),
and there was only 0.2 mol/mol apolipoprotein B-100
Further evidence for ongoing in vivo heme-catalyzed LDL oxidation derives from measurements of the fatty acid composition of the HO-1-deficient child's LDL. It contained 1.7 times more saturated and monounsaturated fatty acids and a lower percentage (20.18% versus 54.86%) of polyunsaturated fatty acids as compared with control LDL. There was an accumulation of palmitic acid (28.71% versus 11.49%) and a relative lack of linoleic acid (3.43% versus 41.04%) and arachidonic acid (1.65% versus 6.88%). Interestingly, and for unexplained reasons, the percentages of the highly oxidizable polyunsatured fatty acids and of eicosatrienoic and docosahexaenoic acids were higher in his LDL (5.25% versus 0% and 9.85% versus 2.84%, respectively). These results raised the question of what kinds of cytotoxic materials might be present in oxidized LDL. LDL is a complex mixture that includes triglycerides, cholesterol esters, phospholipids, unesterified cholesterols, lysophosphatidylcholine, phosphatidylethanolamine, diacylglycerol, ceramide, and some phosphatidylinositol. Oxidation leads to formation of a wide range of biologically active products, and some of these, such as 7-oxysterols, have been reported to be highly cytotoxic.42 However, we suspected that the majority of the toxicity of oxidized LDL might derive from the high concentrations of LOOH (Table 2), which is chemically very similar to organic hydroperoxides, such as cumene hydroperoxide. In support, preincubation of oxidized LDL with reduced GSH or GSH peroxidase (which will relatively specifically reduce the LOOH to the alcohol) abolished almost 100% of the cytotoxic effects of the LDL. In further support of the toxicity of the LOOH per se, when endothelial cells were exposed to a concentration of cumene hydroperoxide approximately equal to the LOOH content of the toxic LDL, almost identical cytotoxicity was observed. Finally, if endothelial cells were exposed to either LOOH-containing LDL or cumene hydroperoxide, we observed a precipitous decline in intracellular GSH content as previously described.33 Exposure of endothelial cells to sublethal amounts of oxidized LDL
isolated from plasma containing metHb markedly induced HO-1 mRNA
(Figure 3A), a sensitive marker for
oxidative stress. Accompanying this mRNA induction, HO activity was
also significantly enhanced (Figure 3D). A similar increase in the
expression of HO-1 mRNA and enzyme activity was observed in endothelial
cells exposed to LDL preincubated with heme instead of metHb (Figure 3A,D). In contrast, LDL isolated from ferroHb-treated plasma did not
alter the HO-1 mRNA level and enzyme activity in endothelial cells
(Figure 3A,D). Similar effects were observed in the case of ferritin;
LDL from plasma exposed to metHb or heme caused a doubling of
endothelial ferritin content, whereas ferroHb failed to induce ferritin
synthesis (Figure 3E). In keeping with a posttranscriptional control of
ferritin synthesis, ferritin light- and heavy-chain mRNA levels were
not affected in these experiments (data not shown).
These changes in HO and ferritin synthesis cannot be ascribed to heme
per se because, in the course of catalyzing the oxidation of LDL, heme
itself undergoes degradation.9 Furthermore, the addition
of antioxidants to LDL prior to its exposure to heme prevents the
oxidation of lipoprotein and the induction of both mRNA and enzyme
activity for HO in endothelium, in spite of the fact that the heme
content of LDL remains high.32,43 In support, 200 µM
Further experiments suggest that conditions in circulating plasma may
permit the oxidation of Hb to metHb. Thus, if ferroHb-containing plasma
is exposed to PMA-activated PMNs for 90 minutes and the LDL is then
isolated and added to endothelial monolayers, induction of HO-1 mRNA
and enzyme activity occur (Figure 4A,D).
Under the same conditions, endothelial ferritin likewise accumulates
(Figure 4E). In contrast, LDL from ferroHb-free plasma exposed
to PMA-activated PMNs does not increase either HO-1 mRNA or enzyme
activity (Figure 4A,D) and has no effect on ferritin content (Figure
4E). In addition, LDL derived from ferroHb-containing plasma incubated
with resting (nonstimulated) PMNs also has no effect on either HO-1 or
ferritin expression (Figure 4).
Since endothelial cytolysis was induced by the HO-1-deficient child's
LDL, we wondered whether it was also capable of enhancing the
expression of HO-1 and ferritin in endothelial cells of healthy subjects. Exposure of endothelial cells derived from healthy subjects to sublethal stress of the child's LDL led to marked increase in
enzyme activity for HO (35 ± 3 versus 163 ± 18 pmol bilirubin formed per milligram of cell protein per 60 minutes) and doubled ferritin content (15.4 ± 2.3 versus 26 ± 7.5 ng/mg cell protein). The increase in ferritin synthesis after exposure of endothelium to the
HO-1-deficient child's LDL was significantly blunted (
The pronounced vascular pathologies described for both an HO-1-deficient human and mice in which this enzyme has been knocked out suggest that defective heme catabolism (and, by implication, heme itself) can have serious pathologic effects. Whereas heme may be directly cytotoxic, the present investigations were an attempt to determine whether the observed in vivo effects of HO-1 deficiency might, at least in part, represent an indirect process. Specifically, we hypothesized that extensive, heme- or heme iron-mediated oxidation of LDL might produce oxidized forms of LDL with appreciable cytotoxicity. In support of this concept, LDL isolated from plasma preincubated with either heme or metHb is markedly cytotoxic to cultured endothelial cells. Furthermore, similarly toxic LDL was present in the plasma of the HO-1-deficient child. Conversion of ferroHb to metHb appears to be essential for the ensuing oxidation of LDL, presumably because metHb readily releases heme, whereas ferroHb does not. That metHb and not ferroHb readily releases free heme was first demonstrated by Bunn and Jandl.35 This observation prompted us to hypothesize that, following dissociation of heme from metHb, the free heme may readily enter lipoprotein particles. Indeed, LDL particles successfully compete for heme released from metHb in plasma despite the presence of haptoglobin, hemopexin, and albumin. In fact, it has been estimated that, when heme is added to whole plasma, approximately 80% binds immediately to LDL and high-density lipoprotein.44 Once heme is lodged within the LDL, spontaneous oxidative reactions involving small amounts of preformed LOOH (or other oxidizing equivalent) will lead to oxidative lysis of the heme group and release of heme iron45 within the LDL particle. Most likely, it is this heme iron that catalyzes the further oxidative breakdown of heme as well as the accelerated oxidation of polyunsaturated fatty acids and other components of the LDL. This is supported by the observation that inclusion of the iron chelator, desferrioxamine, in incubations containing heme and LDL prevents the loss of intact heme and also stops the appearance of oxidation products such as LOOH and conjugated dienes.9 In plasma-free solutions,9,11 heme was earlier reported to
catalyze LDL oxidation, generating products toxic to endothelial cells.
In diluted serum (20%) as well, heme was shown to bind to LDL leading
to its oxidation in the presence of hydrogen peroxide.12 However, in the latter case, the possible formation of cytotoxic products was not investigated. Although a number of heme proteins Our studies offer an alternative pathway for modification of LDL by Hb in plasma involving heme release from metHb. The results reported here generally support such a mechanism insofar as maneuvers that restrict heme transfer to LDL uniformly diminish or block LDL oxidation. These observations raised the question of the nature of the toxic substances that might arise from heme- or heme iron-mediated LDL oxidation. LDL contains a number of components, and oxidized forms of several of these have been proposed as responsible for the toxicity of oxidized LDL. However, our results suggest that an accumulation of LOOH is the predominant toxic species within oxidized LDL because specific enzymatic reduction of LOOH to LOH yields LDL with minimal toxic effects. Furthermore, we find that, on an equimolar basis, LOOH within oxidized LDL and an organic hydroperoxide, cumene hydroperoxide, have very similar toxic effects on endothelial cells. We have shown earlier that endothelial cells exposed to oxidized LDL increase the expression of HO-1 as well as ferritin.43 Here, we present evidence that this induction probably involves LDL-associated hydroperoxides (or secondary oxidation events caused by these peroxides). LDL derived from metHb-containing plasma induces endothelial cells to increase HO-1 and ferritin synthesis. In contrast, LDL from plasma containing ferroHb fails to alter the expression of HO-1 and ferritin in endothelial cells. Since oxidation of Hb to metHb is essential for endothelial perturbation by LDL, we sought to model oxidant conditions that might be relevant to vascular pathophysiology. Previous studies have demonstrated that activated PMNs efficiently oxidize Hb contained in erythrocytes to metHb.40-41 In the present studies, we demonstrate that ferroHb in plasma is rapidly oxidized when exposed to activated, but not resting, PMNs. Moreover, LDL derived from plasma containing ferroHb and activated PMNs enhances the expression of both HO-1 and ferritin in endothelium. Such oxidized LDL can also threaten vascular endothelial cell integrity depending on its concentration. In addition to being a possible marker of exposure to oxidizing events, HO-1 and ferritin have been demonstrated to be cytoprotective in various models.25,32,34,55-67 Iron-driven oxidative damage of endothelial cells, such as cytotoxicity provoked by oxidized LDL or inflammatory cells, can be prevented by induction of ferritin synthesis.32,34,55 The protective role of ferritin against iron-driven oxidative stress is attributable to high sequestering capacity for inorganic iron and ferroxidase activity of its H chain.34 Intracellular ferritin was shown to abolish iron-catalyzed oxidation of LDL.61 Increased expression of HO-1 in endothelial cells was associated with inhibition of monocyte transmigration induced by oxidized LDL65 and may also protect neurons from oxidative injury.66-67 To what extent the products (bilirubin and carbon monoxide) might explain such protection is a matter of current debate.68-71 These results may have further relevance to other pathologic conditions in which extracellular metHb, heme, and iron are present. These include renal failure, which can occur in instances of acute intravascular hemolysis, as well as the progression of atherosclerosis. Oxidation of LDL is recognized as one of the early events in atherogenesis.72 In fact, in an animal model, intravascular hemolysis increases the atherogenicity of diet-induced hypercholesterolemia.73 Interestingly, up-regulation of HO-1 and ferritin genes in endothelium may also occur in the early phase of progression of atherosclerotic lesions55,74 possibly reflecting cellular responses to heme- or iron-generated lipid peroxidation products. We think it is likely that the interactions between Hb/heme and LDL in
plasma may explain some of the pathologies observed in the human
patient and in mice deficient in HO-1. In the HO-1-deficient child,
both intravascular hemolysis and endothelial cell injury were prominent
features,23 and at least the latter pathology is also
quite evident in HO-1 knockout mice.24 In the present studies, spectral analysis revealed that oxidation of Hb to metHb occurred in the child's plasma and demonstrated that iron was accumulating in his LDL. Several independent assays for LDL
oxidation MetHb represents a hazard to vascular endothelial cells by not only catalyzing the oxidation of LDL but sensitizing endothelium to oxidant damage.25 MetHb releases free heme to endothelial cells; this heme initially sensitizes endothelium to oxidant stress but later induces the cytoprotectants HO-1 and ferritin.25 In the present studies, we also observed an up-regulation of HO-1 and ferritin after endothelial cells derived from healthy humans were exposed to the LDL of the HO-1-deficient child. Inhibition of HO enzyme activity in endothelium blunted the rapid ferritin response to the child's LDL, suggesting that the induction of ferritin synthesis was in part due to iron liberated from endogenous heme. It is tempting to speculate that endothelial cells of the HO-1-deficient child were prone to oxidative damage arising from both heme-mediated oxidation of LDL and, perhaps, an associated lack of adaptive response (ie, induction of HO-1 and ferritin synthesis). Overall, our results suggest that heme derived from free Hb in plasma may threaten vascular endothelial cell integrity via oxidative modification of LDL; this lipoprotein, in turn, induces the cytoprotectants heme oxygenase and ferritin.
We thank Zoltán Vokó for his contribution to the statistical analysis and Alice Dobolyi for technical assistance.
Submitted November 2, 2001; accepted March 15, 2002.
Supported in part by Hungarian government grants OTKA-029558/037883, ETT-041/037, FKFP-0617, and NKFP-1/007 and by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (A.Y.). J.W.E. is supported by The Commonwealth of Kentucky Research Challenge Trust Fund and by National Institutes of Health grant DK 58882.
V.J. and J.B. contributed equally to these studies.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: József Balla, Pf 19, Nagyerdei krt 98, 4012 Debrecen, Hungary; e-mail: balla{at}ibel.dote.hu.
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F. A. D. T. G. Wagener, H. E. van Beurden, J. W. von den Hoff, G. J. Adema, and C. G. Figdor The heme-heme oxygenase system: a molecular switch in wound healing Blood, July 15, 2003; 102(2): 521 - 528. [Abstract] [Full Text] [PDF]
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J. Balla, G. M. Vercellotti, K. Nath, A. Yachie, E. Nagy, J. W. Eaton, and G. Balla Haem, haem oxygenase and ferritin in vascular endothelial cell injury Nephrol. Dial. Transplant., July 1, 2003; 18(90005): v8 - 12. [Abstract] [Full Text] [PDF]
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C. J. Mingone, S. A. Gupte, S. Quan, N. G. Abraham, and M. S. Wolin Influence of Heme and Heme Oxygenase-1 Transfection of Pulmonary Microvascular Endothelium on Oxidant Generation and cGMP Experimental Biology and Medicine, May 1, 2003; 228(5): 535 - 539. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
HO-1 and ferritin
