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Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3442-3450
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Ferriporphyrins and endothelium: a 2-edged sword promotion of
oxidation and induction of cytoprotectants
József Balla,
György Balla,
Viktoria Jeney,
György Kakuk,
Harry S. Jacob, and
Gregory M. Vercellotti
From the Department of Medicine and Department of Pediatrics,
University Medical School of Debrecen, Debrecen, Hungary, and the
Division of Hematology, University of Minnesota, Minneapolis, MN.
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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% 51Cr 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. 2000;95:3442-3450)
© 2000 by The American Society of Hematology.
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Introduction |
Acute hepatic porphyrias, a failure of
iron-protoporphyrin IX(heme) biosynthesis and
accumulation of porphyrin precursors,1-3 cause a myriad of
disabling symptoms and may even be fatal.4 Therapeutically,
infusion of hematin has proven efficacious by correcting heme
metabolism5-9 but has been accompanied by considerable toxicities, including vasculitis and coagulopathy.8,10-17
Finnish investigators recently successfully introduced heme arginate in the treatment of acute hepatic porphyrias without evidence of vasculopathy or thrombotic side effects.18-23
Iron is a marked potentiator of oxidant damage in diverse systems and
plays a substantial role in phagocyte-mediated vascular injury.24-26 For example, prolonged preexposure of
endothelium to the iron chelator deferoxamine prevented activated
polymorphonuclear cell (PMN)- or hydrogen peroxide
(H2O2)-mediated endothelial
cell damage.24 We have shown that one critical feature of
highly damaging iron to endothelium is permeation of the metal into
cells.27 Chelation of iron by lipophilic
chelators, such as 8-hydroxyquinoline, results in the accumulation of
catalytically active lipophilic iron chelates in endothelial lipid
compartments; endothelium pretreated with 8-hydroxyquinoline-iron
chelate was exquisitely sensitive to both endogenous and exogenous
oxidant stress. A physiologically ubiquitous hydrophobic iron chelate,
heme, readily concentrated within the hydrophobic milieu of intact
endothelium and greatly amplified reactive oxygen species-mediated
cytotoxicity.28 The plasma heme-binding protein, hemopexin,
attenuated sensitization of endothelium by affecting heme-iron uptake
and catalytic activity.28-30 Purified hemopexin, when
preincubated with heme for 15 minutes in stoichiometric amounts,
completely abrogated heme-augmented endothelial oxidant
injury.28 Furthermore, heme served as a catalyst for the
oxidation of low-density lipoprotein (LDL) from native LDL; the toxic
moieties of oxidized LDL could, in turn, mediate endothelial activation
and damage.31
Our previous studies have demonstrated that endothelial cells respond
to chronic heme exposure by up-regulating heme oxygenase and ferritin
synthesis.32 The latter was critically important in
providing cytoprotection against further iron-driven oxidant damage.
Heme oxygenase is a heme-degrading enzyme that opens the porphyrin ring
producing biliverdin and carbon monoxide and releasing iron.33,34 Three genes encode for 3 isoenzymes for heme
oxygenase.35 Heme oxygenase-1, identified as the 32.8-kd
stress protein,36 is the form transcriptionally inducible
by a variety of agents, such as heme, oxidants, and
cytokines.34,36-41 Heme oxygenase-2 is constitutively
active as a metabolizer of heme. The function of heme oxygenase-3 is
still under investigation. Ferritin is the major intracellular depot of
nonmetabolic iron. This multimeric protein consists of 24 subunits of 2 types (heavy chain and light chain) and has a very high capacity for
storing iron (up to 4500 mol of iron per mol of
ferritin).42,43 The heavy chain of ferritin manifests
ferroxidase activity.43,44 The proportion of heavy and
light subunits depends on the iron status of the cell or tissue and
varies among organs and species.42,43
We hypothesize that heme arginate's lack of vascular side
effects in vivo may be due to its inefficient catalyses of
oxidant-mediatedendothelial cell injury. In order to test whether
various heme compounds used clinically in the treatment of porphyria
are vasculotoxic in vitro, we examined the effects of
ferriporphyrins with different hydrophobic properties on oxidant
susceptibility of endothelial cells or LDL. We also tested
ferriporphyrin's ability to induce the synthesis of heme oxygenase and
ferritin in endothelium.
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Materials and methods |
Reagents
Medium 199, fetal calf serum (FCS) and Hank's balanced salt
solution (HBSS) were purchased from Life Technology (Rockville, MD);
dispase II was from Gibco (Grand Island, NY); and hydroxyethyl starch
was from Du Pont (Wilmington, DE). 51CrO4 (as
the sodium salt) was purchased from Amersham Corp (Piscataway, NJ).
Iron deuteroporphyrin IX, iron deuteroporphyrin IX,2,4-bis-glycol, iron
deuteroporphyrin IX,2,4-bis-sulfonate, and iron coproporphyrin III were
synthesized by Porphyrin Products (Logan, UT). Heme arginate was
obtained from Dr Falk Pharma, GmbH (Freiburg, Germany), and Huhtamäki Oy Pharmaceuticals (Helsinki, Finland). For the
preparation of hematin stock solutions (1 and 5 mmol/L), hemin was dissolved in the dark in NaOH 20 mmol/L and used within 1 hour after preparation. Subsequent centrifugation of hematin solutions (1 mmol/L and 5 mmol/L) at
12 000g for 10 minutes resulted in losses of less than 0.5%
and 5% of the heme content, respectively. All other reagents used were
purchased from Sigma (St Louis, MO) unless otherwise specified.
Endothelial cell isolation and culture
As in our previous studies,45 human umbilical vein
endothelial cells (HUVECs) were isolated from human umbilical veins. Human aortic endothelial cells and human dermal microvascular endothelial cells were grown as described.32 Endothelial
cells were identified by their morphology, the presence of von
Willebrand factor, and the ability to take up acetylated LDL. The cell
cultures were studied within 48 hours of reaching confluence.
Preparation of human neutrophils
As described previously,28 PMNs were isolated from blood
of human volunteers after informed consent (following the guidelines of
the Commitee on the Use of Human Subjects in Research of the University
of Minnesota and Medical University of Debrecen).
Endothelial cell cytotoxicity assays
Confluent endothelial cell monolayers grown in 2-cm2
wells were radiolabeled with 2 µCi of
[51Cr]Na2CrO4 in cell culture
medium overnight. The monolayers were washed 3 times with HBSS and
exposed to H2O2 (100 µmol/L)
or phorbol myristate acetate (PMA) (100 ng/mL)-activated PMNs (2:1 PMN:endothelial cell ratio)
in HBSS for 2 hours. Spontaneous 51Cr release was less than
10%. Endothelial cell cytotoxicity mediated by oxidation of LDL (200 µg/mL) was measured at 4 hours, and the spontaneous 51Cr
release was below 15%. Samples and cells were processed in the dark.
Specific cytotoxicity values were calculated as described previously.28
Preparation of human LDL
Plasma LDL was prepared from EDTA (1 mg/mL)-anticoagulated venous
blood of healthy volunteers who had fasted overnight. LDL was isolated
from plasma by rate zonal density gradient ultracentrifugation after a
2000g centrifugation of blood at 4°C for 20 minutes as previously described.31 Blood, plasma, and LDL samples were processed in subdued light on ice. LDL fraction was assessed for purity
by means of agarose electrophoresis and used in the experiments within
24 hours of isolation. The LDL cholesterol was measured enzymatically
on a Synchron CX5 autoanalyzer (Beckman Instruments, Brea, CA), and LDL
protein concentration was determined by BCA protein assay (Pierce,
Rockford, IL).
Oxidation of LDL
Oxidation of LDL (200 µg/mL protein) was catalyzed by hematin (5 µmol/L) or heme arginate (5 µmol/L), and the lipid peroxidation was monitored by
measuring the formation of conjugated dienes, lipid hydroperoxides and
thiobarbituric acid-reactive substances. LDL oxidation was promoted by
the presence of H2O2 (50 µmol/L). Lipid hydroperoxide in LDL was detected by
means of the ferrous oxidation in xylenol orange (FOX)
assay.46 Briefly, 50 µL of LDL samples or
cumene hydroperoxide as standard peroxide were added to 450 µL of
reaction mixture containing ferrous sulfate (250 µmol/L), xylenol orange (100 µmol/L), sorbitol (100 mmol/L), and
sulfuric acid (25 mmol/L). The formation of
Fe3+-xylenol orange complex was followed
spectrophotometrically at 560 nm for 30 minutes at room temperature.
The thiobarbituric-acid colorimetric assay and the measurement of
conjugated dienes were performed as described previously.31
The kinetics of lipid peroxidation of LDL was characterized by the
length of initiation phase (lag time) in minutes, the maximal velocity
of propagation phase (Vmax) in milliabsorbance units/minute, and the
time period passing until the maximal velocity of propagation phase
( T at Vmax) in minutes.47 Samples were processed in the
dark. For cytotoxicity assays and induction of heme oxygenase gene, the
LDL was incubated with either hematin or heme arginate in the presence
of H2O2 for 45 minutes at 37°C prior to the
endothelial cell experiments.
Heme determinations
At the end of incubations of HUVECs with heme solutions, the cell
monolayers grown in 24-well tissue-culture plates were washed 3 times
with 1 mL of HBSS, and the endothelial cells removed with 250 µL of
concentrated formic acid. The heme content of the formic acid-solubilized HUVECs was determined spectrophotometrically at 398 nm with the use of an extinction coefficient of
1.5 × 105
mol/L 1 · Cm1.28
LDL-associated heme was measured spectrophotometrically as a
heme-pyridine complex. Pyridine (0.35 mL) and 0.15 mL of 1.0 N NaOH were added to 1.25 mL of 200 µg/mL LDL
samples. After vortexing, the samples were divided into 2 equal parts.
The first was oxidized by 25 µL of 3 mmol/L
K3Fe(CN)6, and the second was reduced by 1 mg
of dithionite. The absorbances were measured at 541 and 557 nm,
respectively, with the use of the oxidized samples as blanks. For
calculation of the results, the differences between optical densities
at 541 and 557 nm were used, with the extinction coefficient of
2.07 × 104
mol/L1 · Cm1.31
Apolipoprotein B-100 was measured by the Unimate 3 ApoB immunoturbidometric assay (F. Hoffmann-La Roche AG, Basel, Switzerland) to calculate the number of molecules of heme per LDL particle.
Heme oxygenase enzyme activity assay
Heme oxygenase enzyme activity was measured by bilirubin and carbon
monoxide generation.32 HUVECs grown in 10-cm-diameter tissue-culture dishes were treated with control media, hematin (10 µmol/L), iron deuteroporphyrin IX (10 µmol/L), iron deuteroporphyrin IX,2,4-bis-glycol (10 µmol/L), iron deuteroporphyrin IX,2,4-bis-sulfonate (10 µmol/L), and iron coproporphyrin III (10 µmol/L) (all the ferriporphyrins were dissolved in
NaOH [20 mmol/L] and were added to media or buffer
with the final pH 7.4), or heme arginate (10 µmol/L)
(dissolved in media or buffer with final pH 7.4) for 60 minutes. In
separate experiments, endothelial cells were exposed to LDL (50 µg/mL
protein) alone, LDL conditioned by hematin (1.25 µmol/L), and H2O2 (6.25 µmol/L), or LDL conditioned by heme arginate (1.25 µmol/L) and H2O2 (6.25 µmol/L) for 60 minutes followed by replacement of the
test solutions with complete media for 8 hours. Samples and cells were
processed in the dark. The endothelial cell monolayers were washed,
scraped with a rubber policeman, and centrifuged at 1000g for
10 minutes at 4°C. The pellet was suspended in MgCl2 (2 mmol/L) phosphate (100 mmol/L) buffer
(pH 7.4), frozen and thawed 3 times, and sonicated on ice prior to centrifugation at 18 800g for 15 minutes at 4°C. The
supernatant was added to the reaction mixture (400 µL) containing
glucose 6-phosphate (2 mmol/L), glucose 6-phosphate
dehydrogenase (0.2 units), hemin (20 µmol/L), and rat
liver cytosol (2 mg protein) for bilirubin generation. In some
experiments, heme arginate (20 µmol/L) was used as a
substrate for heme oxygenase. The assays were started by the addition
of NADPH (0.8 mmol/L). After incubation for 60 minutes
at 37°C in the dark, the formed bilirubin was extracted with
chloroform and a optical density of 464 to 530 nm
was measured (extinction coefficient, 40 mmol/L1 · Cm1 for
bilirubin), or the carbon monoxide was determined with
the use of gas chromatography. Heme oxygenase enzyme activity is
expressed as picomole of bilirubin formed per milligram of cell protein per 60 minutes or as microliter of carbon monoxide formed per milligram
of cell protein per 60 minutes.
Ferritin assay
Endothelial cell ferritin content was measured in cells grown in
6-well tissue-culture plates treated with control media or various
ferriporphyrins (10 µmol/L), as indicated, for 60 minutes; the culture media were then replaced with ferriporphyrin-free medium for 15 hours. Samples and cells were processed in the dark. HUVECs' ferritin content was measured after the cells were washed 3 times with HBSS (Ca and Mg free), solubilized with 1% Triton X-100,
0.5% Nonidet P-40 in Tris-HCl (10 mmol/L) buffer (pH
7.2) containing EDTA (5 mmol/L) and
phenylmethylsulfonyl fluoride, and centrifuged at 10 000g for
10 minutes at 4°C. The supernatant was analyzed for ferritin with
the use of the Stratus fluorometric enzyme immunoassay
system.32 The results are expressed as nanogram of ferritin
per milligram of cell protein. The protein content of the endothelial
cell monolayers was determined by means of BCA protein assay.
Heme oxygenase and ferritin messenger RNA analysis
Heme oxygenase messenger RNA (mRNA) and H- and L-chain ferritin
content were analyzed in HUVEC cultured in 10-cm-diameter tissue-culture dishes after endothelial cells were exposed to test
solutions, as described for the measurement of heme oxygenase enzyme
activity above, for 1 hour and then replaced with complete media for an
additional 4 hours. Samples and cells were processed in the dark.
Endothelial cell RNA was isolated by the RNAzol method (TEL-TEST, Inc,
Friendswood, TX). The RNA transferred to nylon membranes was then
hybridized at 42°C with 32P-labeled complementary DNA
(cDNA) probes synthesized by random primed DNA labeling for human heme
oxygenase32,36 and H ferritin and L
ferritin.32,48 Autoradiograms were quantified by
computer-assisted videodensitometry and expressed as arbitrary
OD units.
Statistical analysis
Data are expressed as means ± SE The Student t test was
employed for comparisons.
| |
Results |
As shown in Figure 1 (and reported
previously in another context28), 1-hour exposure of
cultured endothelial cells to hematin (5 µmol/L)
markedly aggravated H2O2 (Figure 1A, second
bar) or phorbol-stimulated polymorphonuclear
oxidant-mediated cytotoxicity (Figure 1B, second
bar). Substitution of vinyl side chains of heme with
hydrogen does not alter the hydrophobicity of the resultant ferriporphyrin, iron deuteroporphyrin IX; accordingly,
hypersusceptibility was similarly provoked (seventh
bars). In contrast, if water
solubility of heme is conferred associatively with the arginate
counterion (heme arginate) (5 µmol/L) (third
bars)49 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
[5 µmol/L] [fourth bars], iron
coproporphyrin III [5 µmol/L] [fifth
bars], and iron deuteroporphyrin IX,2,4-bis-glycol [5 µmol/L] [sixth bars]), these
ferriporphyrins failed to sensitize cells to H2O2
or activated polymorphonuclear leukocytes. This was not
HUVEC-specific since the same results were observed when the target
cells were human aortic and human dermal microvascular endothelial
cells instead of HUVECs (data not shown). We asked: could hematin
sensitize endothelial cells to oxidative challenge in the presence of
plasma? After all, plasma is enriched with binding proteins, such as
albumin and hemopexin, known to inhibit heme-mediated cell
damage.28-30 Exposure of HUVECs to hematin (600 µmol/L in whole human plasma) (plasma concentration
attained in heme arginate- or hematin-treated porphyria patients)
synergized cellular oxidant damage with added
H2O2 (100 µmol/L)
(36.8 ± 5.1% [hematin plus H2O2]
versus 4.3 ± 3.8% [H2O2 alone]
51Cr release; P = .001), with an optimal
hematin-exposure duration of 60 minutes. In contrast, cytotoxicity
studies showed little added toxicity to HUVECs incubated with heme
arginate (600 µmol/L) (heme arginate plus
H2O2) in plasma for 60 minutes and then
challenged with H2O2 (100 µmol/L) (14.8 ± 5.9% versus 4.3 ± 4.1%
[H2O2 alone] 51Cr release; not
significant).
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| Fig 1.
Exposure of endothelial cells to iron deuteroporphyrin
IX, hematin, heme arginate, and other substances.
Iron deuteroporphyrin IX, like hematin, but unlike
heme arginate, iron deuteroporphyrin IX,2,4-bis-sulfonate, iron
coproporphyrin III, or iron deuteroporphyrin IX,2,4-bis-glycol,
sensitized endothelial cells to H2O2 and
activated PMN-mediated cytolysis. Confluent 51Cr-labeled
human umbilical vein endothelial cells grown in 24-well (2 cm2/well) tissue-culture plates were incubated with medium
199 alone (first bars), 5 µmol/L
hematin (second bars), 5 µmol/L heme
arginate (third bars), 5 µmol/L iron
deuteroporphyrin IX,2,4-bis-sulfonate (fourth bars), 5 µmol/L iron coproporphyrin III (fifth
bars), 5 µmol/L iron deuteroporphyrin
IX,2,4-bis-glycol (sixth bars), or 5 µmol/l iron deuteroporphyrin IX (seventh
bars) in 500 µL of media 199 for 60 minutes. After
removal of solutions, the cells were washed with HBSS and exposed for 2 hours to (A) 100 µmol/L H2O2
or (B) PMA (100 ng/mL-activated neutrophils (2:1
PMN:endothelial cell ratio). Results represent the
percentage of specific cytotoxicity (mean ± SE) of at least 3 experiments performed in duplicate.
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Since incubation of endothelial cells with heme increased the
expression of heme oxygenase,32,45 we tested whether other ferriporphyrins could act as inducers of heme oxygenase mRNA and enzyme
activity. As demonstrated in Figure 2, heme
arginate (third lane) and iron deuteroporphyrin IX (seventh lane)
induced heme oxygenase mRNA to a similar degree as hematin (second
lane). Accompanying this mRNA induction, expression of heme oxygenase
enzyme activity was markedly enhanced in endothelial cells exposed to
heme arginate (Figure 2D, third bar) or iron deuteroporphyrin IX
(Figure 2D, seventh bar). Iron deuteroporphyrin IX,2,4-bis-sulfonate
(fourth lanes and bars), iron coproporphyrin III (fifth lanes and
bars), and iron deuteroporphyrin IX,2,4-bis-glycol (sixth lanes and
bars) did not alter endothelial heme oxygenase mRNA level and enzyme activity. Heme arginate did not provoke oxidant-mediated endothelial damage but entered cells similarly to hematin and iron deuteroporphyrin IX, since mRNA level and enzyme activity for heme oxygenase were markedly induced in HUVECs. In support, 1-hour exposure of HUVECs to
heme arginate (5 µmol/L) in FCS free media increased
endothelial cell heme content to an extent similar to what was observed
after hematin treatment (5 µmol/L) (from
68 ± 18 pmol heme per milligram endothelial cell protein to
5.04 ± 0.6 and 3.94 ± 0.91 nmol heme per milligram
endothelial cell protein, respectively). Comparable heme uptake can be
obtained in the presence of human plasma although at 2 orders of
magnitude greater concentration for both heme arginate and hematin.
One-hour exposure of HUVECs to heme arginate (600 µmol/L) or hematin (600 µmol/L) in
human plasma enhanced endothelial cell heme content to
1.24 ± 0.12 nmol heme per milligram protein and 1.16 ± 0.21
nmol heme per milligram endothelial cell protein, respectively. The
heme content of cytosol was below levels of detection in these
experiments. Thus, accurate cytosolic penetration of the
ferriporphyrins could not be determined. Similarly to endothelium, lipoproteins also compete for heme added to plasma. When plasma LDL was
incubated with equimolar concentrations of heme arginate and hematin
for 2 hours at 37°C, 1.06% and 1.54% of the added heme became
associated with LDL particles, respectively.
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| Fig 2.
Heme oxygenase induction by heme analogues.
(A) For heme oxygenase mRNA Northern analysis, confluent human
umbilical vein endothelial cells cultured in 10-cm tissue-culture
dishes were incubated with medium 199 alone (first lane), 10 µmol/L hematin (second lane), 10 µmol/L heme arginate (third lane), 10 µmol/L iron deuteroporphyrin IX,2,4-bis-sulfonate
(fourth lane), 10 µmol/L iron coproporphyrin III
(fifth lane), 10 µmol/L iron deuteroporphyrin
IX,2,4-bis-glycol (sixth lane), or 10 µmol/L iron
deuteroporphyrin IX (seventh lane) in media 199 for 60 minutes followed
by an additional 4 hours' incubation with hematin and heme
analogue-free medium. RNA was isolated, electrophoresed, blotted, and
hybridized with a 32P-labeled heme oxygenase cDNA probe.
(B) Densitometry tracings of heme oxygenase mRNA bands expressed as
arbitrary OD units. (C) The corresponding 28S and 18S
ribosomal RNA of the Northern blot in (A). (D) Heme
oxygenase enzyme activity (picomolar of bilirubin formed per milligram
of cell protein per 60 minutes) measured at 8 hours after exposure of
endothelial cell monolayers to the same compounds as above. Results
represent the enzyme activity (mean ± SE) of at least 3 experiments
done in duplicate.
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The hematin induction of the heme oxygenase gene was accompanied by
increased ferritin synthesis in endothelial cells.32,45 Therefore, we examined whether endothelial cell ferritin content was
affected by other ferriporphyrins. Like hematin (Figure
3B, second bar), iron deuteroporphyrin IX
strikingly increased the ferritin level in endothelial cells (Figure
3B, seventh bar). In contrast, heme arginate enhanced endothelial cell
ferritin content to much less an extent (Figure 3B, third bar). Heavy- and light-chain ferritin mRNA levels were not affected
by heme arginate and iron deuteroporphyrin IX (Figure 3A, third and
seventh lanes), concordant with previous studies42,48,50,51
and demonstrating that iron-mediated regulation of ferritin synthesis
occurs at the posttranscriptional/translational level. Treatment of
cells with iron deuteroporphyrin IX,2,4-bis-sulfonate (fourth lanes and
bar), iron coproporphyrin III (fifth lanes and bar), and iron deuteroporphyrin IX,2,4-bis-glycol (sixth lanes and bar) did not affect
mRNA levels for H and L ferritin chains and ferritin content in
endothelium.
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| Fig 3.
Effect of ferriporphyrins on ferritin expression.
Human umbilical vein endothelial cell monolayers were treated with
medium 199 alone (first lanes and bar) or equimolar (10 µmol/L), various ferriporphyrins, including hematin
(second lanes and bar), heme arginate (third lanes and bar), iron
deuteroporphyrin IX,2,4-bis-sulfonate (fourth lanes and bar), iron
coproporphyrin III (fifth lanes and bar), iron deuteroporphyrin
IX,2,4-bis-glycol (sixth lanes and bar), or iron
deuteroporphyrin IX (seventh lanes and bar), in media 199 for 60 minutes followed by additional incubations with ferriporphyrin free
medium. (A) Northern blot analysis of mRNA for heavy-chain (H) and
light-chain (L) ferritin at 4 hours after treatment of endothelial
cells. Upper Panel: After transfer and hybridization, the H-ferritin
and L-ferritin mRNAs are recognized by 32P-labeled cDNA
probes. Lower Panel: Equal quantities of 28S rRNA were shown in the
ethidium bromide-stained agarose gel. (B) Ferritin content at 16 hours
after treatment of endothelium was measured for the same groups as in
(A). Results are expressed as nanograms of ferritin per milligram of
endothelial cell protein and represent mean ± SE of at least 3 experiments performed in duplicate. P < .004 heme arginate
(third bar) versus control (first bar).
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Conversion of heme by heme oxygenase to carbon monoxide, biliverdin,
and bilirubin is accompanied by the release of iron from the porphyrin
ring, which can drive the synthesis of ferritin.48 To
assess the degradation of the porphyrin ring in endothelial cells, we
measured the generation of bilirubin and carbon monoxide. Since human
endothelial cells have very low levels of biliverdin reductase activity
(data not shown), we used rat liver cytosol (a rich source of
biliverdin reductase) to reduce biliverdin to bilirubin for the heme
oxygenase enzyme activity assay.32,45,52 When heme arginate
(20 µmol/L) was used as a substrate for heme oxygenase, less bilirubin (79.7 ± 12.8 versus 259.1 ± 22.1
pmol bilirubin formed per milligram of cell protein per 60 minutes) and
carbon monoxide (7.6 ± 1.2 versus 22.0 ± 4.2
µL × 103 carbon monoxide formed per
milligram of cell protein per 60 minutes) were generated in isolated
endothelial cell microsomes compared with what happened when hematin
(20 µmol/L) was used as a substrate.
We have previously demonstrated that intracellular ferritin protects
endothelium from oxidant-mediated cytolysis and does so in a
dose-responsive manner.32 The induction of ferritin synthesis by pretreatment of endothelial cells with hematin (10 µmol/L) for 1 hour followed by a further incubation
for 15 hours provided resistance to the very damaging combination of
newly added hematin (5 µmol/L) plus
H2O2 (100 µmol/L) (Figure
4, second bar). Prolonged
preincubation of cells with iron deuteroporphyrin IX (Figure 4, seventh
bar) attenuated cytotoxicity derived from hematin plus
H2O2 (Figure 4, first bar). In contrast,
endothelial cells challenged by hematin plus
H2O2 were not protected from cytolysis after
pretreatment for 16 hours with heme arginate (Figure 4, third bar),
iron deuteroporphyrin IX, 2,4-bis-sulfonate (fourth bar), iron
coproporphyrin III (fifth bar), or iron deuteroporphyrin IX,2,4-bis-glycol (sixth bar).
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| Fig 4.
Pretreatment of endothelium.
Hematin and iron deuteroporphyrin IX pretreatment of endothelium
provided cell protection against
hematin/H2O2-mediated lysis. Human umbilical
vein endothelial cells were pretreated with control medium (first bar)
or 10 µmol/L of ferriporphyrins, consisting
of hematin (second bar), heme arginate (third bar),
iron deuteroporphyrin IX,2,4-bis-sulfonate (fourth bar), iron
coproporphyrin III (fifth bar), iron deuteroporphyrin IX,2,4-bis-glycol
(sixth bar), or iron deuteroporphyrin IX (seventh bar), for 60 minutes, and the culture media were then replaced with
ferriporphyrin-free medium for 15 hours. Endothelial cell oxidant
stress was then provided by exposure of the cells to hematin (5 µmol/L) for 60 minutes followed by
H2O2 (100 µmol/L) for 2 hours. Results are the specific cytotoxicity (mean ± SE) of 3 experiments performed in duplicate.
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Oxidative damage to endothelial cells can also be mediated by
oxidized LDL.31 Heme-catalyzed lipid peroxidation of LDL
was monitored by the formation of conjugated dienes, lipid
hydroperoxides, and thiobarbituric acid-reactive substances (Figure
5; Table 1). The kinetics of lipid peroxidation of LDL mediated by heme
arginate/H2O2 (Figure 5, closed circles)
differed from that mediated by hematin/H2O2 (closed squares). Measurement of LDL-conjugated diene formation demonstrated that the lag time and T at Vmax were longer (74 versus
48 minutes and 82 versus 51 minutes) and Vmax was slower (112.7 versus
66.6 milliabsorbance units per minute) for LDL exposed to heme
arginate/H2O2 (closed circles) compared with
LDL exposed to hematin/H2O2 (closed squares)
(Figure 5). In the absence of H2O2, the
kinetics of LDL oxidation by heme arginate alone (open circles) or
hematin alone (open squares) was characterized by a lag phase lasting
more than 3 hours. During long-term incubations, hematin alone enhanced
the lipid peroxidation of LDL more than heme arginate alone. As shown
in Table 1, addition of hematin to LDL led to
increased formation of conjugated dienes, lipid hydroperoxides, and
thiobarbituric acid-reactive substances by 18 hours of incubation
while with heme arginate similar oxidation required 36 hours. In these
respects, hematin-catalyzed oxidation of LDL was approximately twice as
active as heme arginate-mediated oxidation of LDL. Since the number of
heme molecules associated with LDL particles was the same in LDL
exposed to hematin in serum free solution as in LDL exposed to heme
arginate (12.7 ± 0.4 versus 12.1 ± 0.9 heme molecules per
LDL particle), the more efficient free-radical catalysis in LDL by
hematin could not be attributed to quantitative characteristics in
these experiments (Figure 5; Table 1). Potentially
relevant to in vivo vascular damage was the fact that oxidative
modification of LDL mediated by heme was cytotoxic to endothelium. The
cytotoxicity of heme arginate/H2O2-conditioned LDL to endothelial cells was significantly less than endothelial cell
cytotoxicity evoked by LDL conditioned with
hematin/H2O2 (Figure
6) (P < .004).
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| Fig 5.
Kinetics of lipid peroxidation of LDL catalyzed by
hematin or heme arginate.
The LDL (200 µg/mL protein) was exposed to hematin (5 µmol/L) (closed squares) or heme arginate (5 µmol/L) (closed circles) in the presence of
H2O2 (75 µmol/L), hematin
alone (5 µmol/L) (open squares), heme arginate alone
(5 µmol/L) (open circles), and
H2O2 alone (75 µmol/L) (open
triangles). Closed triangles represent native LDL alone. The lipid
peroxidation was monitored spectrophotometrically at 234 nm at 37 °C
for 120 minutes to assess the formation of conjugated dienes.
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View this table:
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Table 1.
TBARS, total lipid hydroperoxide and conjugated diene
formation in a low density lipoprotein exposed to hematin or heme
arginate
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| Fig 6.
Heme arginate-conditioned and hematin-conditioned LDL.
Heme arginate-conditioned LDL induced less endothelial cell lysis than
hematin-conditioned LDL. Human umbilical vein endothelial cells were
incubated with LDL (200 µg/mL protein) treated with hematin (5 µmol/L) plus H2O2 (75 µmol/L), LDL treated with heme arginate (HA) (5 µmol/L) plus H2O2, and LDL
treated with H2O2 alone in HBSS for 4 hours.
Endothelial cells were also exposed to native LDL alone, and for
control cells, HBSS was free of lipoprotein. Results were the
percentage of specific cytotoxicity (mean ± SE) of 3 experiments
performed in duplicate. P < .004 versus LDL treated with
hematin plus H2O2.
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The up-regulation of heme oxygenase mRNA and enzyme activity in
endothelium was shown to occur after sublethal stress of oxidatively modified LDL.41 We probed for expression of heme oxygenase
as a marker for oxidative stress in endothelial cells mediated by hematin- or heme arginate-modified LDL. Exposure of endothelium to LDL
conditioned with hematin/H2O2 for 1 hour
resulted in a marked cellular expression of heme oxygenase mRNA and
enzyme activity (Figure 7, fifth lanes and
bars). In contrast, native LDL (second lanes and bars) or LDL exposed
to H2O2 (third lanes and bars) did not affect
mRNA level and enzyme activity for heme oxygenase in endothelial cells.
Induction of heme oxygenase was also observed when endothelial
monolayers were exposed to LDL conditioned with heme
arginate/H2O2 (sixth lanes and bars), albeit
heme arginate/H2O2-conditioned LDL was a
significantly weaker inducer (P < .001) compared with hematin/H2O2-conditioned LDL. In these
studies, the induction of heme oxygenase mRNA and enzyme activity by
hematin (10 µmol/L) alone served as a positive
control (fourth lanes and bars). In experiments in which oxidation of
LDL was catalyzed by hematin or heme arginate, heme per se could not be
ascribed as the cause of the induction of heme oxygenase mRNA and
enzyme activity, since in the course of catalyzing the oxidation of
LDL, heme itself undergoes degradation.31,47 Treatment of
LDL with antioxidants prior to its exposure to
hematin/H2O2 prevented the oxidation of
lipoproteins and the induction of both mRNA and enzyme activity for
heme oxygenase, in spite of the fact that heme content of LDL remained
high.41 In support, 1.25 µmol/L of
butylated hydroxytoluene or 12.5 µmol/L of alpha
tocopherol added to LDL (50 µg/mL) prior to its exposure to heme
arginate/H2O2 also abrogated the induction of
heme oxygenase enzyme activity (36.8 ± 8.2 and 32.5 ± 8.4
versus 298.3 ± 17.7 pmol of bilirubin formed per milligram of
cell protein per 60 minutes, respectively), which was accompanied by
prevention of lipid peroxidation of LDL; 93% of the added hemes
remained LDL associated. Heme arginate alone (87.5 nmol/L) (7% of the added heme) did not enhance heme
oxygenase enzyme activity in HUVECs (32.8 ± 8.4 versus
39.5 ± 5.4 pmol of bilirubin formed per milligram of cell protein
per 60 minutes). Thus, in these studies, oxidation of LDL per se could
be ascribed as the cause for the induction of heme oxygenase in
endothelial cells.
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| Fig 7.
LDL conditioned with heme arginate or
hematin.
Heme arginate-conditioned LDL was a weaker inducer for heme oxygenase
than hematin-conditioned LDL. (A) For heme oxygenase mRNA analysis,
human umbilical vein endothelial cell monolayers were incubated for 1 hour with LDL (50 µg/mL protein) alone (second lane), LDL treated
with H2O2 (6.25 µmol/L)
(third lane), LDL treated with hematin (1.25 µmol/L)
plus H2O2 (6.25 µmol/L)
(fifth lane), and LDL treated with heme arginate (1.25 µmol/L) plus H2O2 (sixth
lane) in HBSS, and then replaced with medium for 4 hours. For negative
control (first lane), HBSS was free of lipoprotein, and for positive
control (fourth lane), cells were exposed to hematin alone (10 µmol/L) for 1 hour followed by a 4-hour incubation
with culture medium. RNA was isolated, electrophoresed, blotted, and
hybridized with a 32P-labeled heme oxygenase cDNA probe.
(B) Autoradiograph was quantified by videodensitometry and expressed as
arbitrary OD units. (C) Ethidium bromide-stained
nylon membrane with the corresponding 18S and 28S rRNA for the
above autoradiogram. (D) Heme oxygenase enzyme activity (picomole of
bilirubin formed per milligram of cell protein per 60 minutes) was
measured at 8 hours after exposure of endothelium to the same test
solutions as for Northern blot in (A). Results represent enzyme
activity (mean ± SE) of at least 3 experiments done in duplicate.
P < .001 versus bar 5.
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Discussion |
In acute life-threatening porphyric attacks, hematin has been
successfully used 5-9 although there may develop harmful
effects on the vasculature, such as vasculitis, thrombosis, and
disseminated intravascular coagulation.8,10-17 Heme
arginate treatment for acute porphyric attacks has been very effective
without evidence of vascular side effects.18-23 In our
studies, heme arginate, unlike hematin, did not amplify hydrogen
peroxide or activated polymorphonuclear leukocyte-mediated endothelial
cell cytotoxicity. Nonpermeant heme analogues, iron deuteroporphyrin
IX,2,4-bis-sulfonate, iron coproporphyrin III, and iron
deuteroporphyrin IX,2,4-bis-glycol, also failed to sensitize
endothelial cells to oxidants. In contrast, brief exposure of
endothelium to the lipid-soluble ferriporphyrin iron
deuteroporphyrin IX sensitized cells to oxidant injury mediated by
hydrogen peroxide or activated neutrophils.
Heme can also threaten vascular endothelial cell integrity by its
ability to catalyze the oxidation of LDL.31 LDL oxidized by
heme is extremely cytotoxic to endothelial cells. The kinetics of LDL
lipid peroxidation mediated by heme arginate in the presence of
H2O2 was characterized by a longer lag phase
and T at Vmax as well as a slower propagation phase compared with
hematin-mediated lipid peroxidation of LDL as judged by conjugated
diene formation. The results of several independent assays for LDL
oxidation production of thiobarbituric acid-reactive substances,
conjugated diene formation, and lipid hydroperoxide
generationstimulated by hematin alone or heme arginate alone all
support the conclusion that heme arginate promoted LDL oxidation less
efficiently. Accordingly, heme
arginate/H2O2-conditioned LDL was less
cytotoxic to cultured human endothelial cells than hematin/H2O2-conditioned LDL.
Heme arginate did not provoke oxidant-mediated endothelial damage but
nevertheless entered endothelial cells similarly to hematin and iron
deuteroporphyrin IX; heme oxygenase mRNA level and enzyme activity were
markedly increased in cells exposed to heme arginate as in hematin- or
iron deuteroporphyrin IX-treated cells (Figure 2). This substantial
induction of heme oxygenase gene was accompanied by increased
endothelial ferritin synthesis. In spite of the similar enhancement of
heme oxygenase mRNA and enzyme activity, heme arginate only doubled
endothelial cell ferritin content, while in endothelium exposed to
hematin and iron deuteroporphyrin IX, ferritin increased 20-fold and
13-fold, respectively. To assess differences in degradation of the
porphyrin ring, heme arginate or hematin was used as a substrate for
heme oxygenase. We measured the generation of bilirubin and carbon
monoxide by using isolated microsomes from endothelium as a source of
heme oxygenase. Less bilirubin and carbon monoxide were generated when
heme arginate was used as a substrate for heme oxygenase than when
hematin was used. The lower level of iron release from the heme
arginate porphyrin ring may explain the blunted ferritin response.
Heavy- and light-chain ferritin mRNA levels were not
altered by heme arginate, hematin, or iron deuteroporphyrin IX,
concordant with previous studies42,43,48,50,51 and
demonstrating that in these cell systems, iron-mediated regulation of
ferritin synthesis occurred primarily at the
posttranscriptional/translational level.
Prolonged incubation of endothelial cells with heme rendered them
remarkably resistant to oxidant challenge via the induction of heme
oxygenase and ferritin.32 The ultimate cytoprotectant against iron-driven oxidant injury was identified as
ferritin32 in a wide range of experimental conditions. Such
protection arises from the ability of ferritin to sequester
intracellular iron and/or to exhibit ferroxidase activity. Endothelial
cells, when incubated with iron deuteroporphyrin IX, were also markedly
induced to increase heme oxygenase mRNA and enzyme activity (Figure 2)
as well as their ferritin content (Figure 3). The remarkably increased
synthesis of heme oxygenase and ferritin proteins were associated with
resistance to oxidative stress imposed by the highly damaging
combination of hematin and H2O2 (Figure 4).
However, no associated cytoprotection against iron-driven oxidant
injury was afforded if only a high level of heme oxygenase enzyme
activity was present without a substantial increase in ferritin
content, as seen after endothelial cells are exposed to heme arginate
(Figure 4, third bar). Nonpermeant ferriporphyrins, iron
deuteroporphyrin IX,2,4-bis-sulfonate, iron coproporphyrin III, and
iron deuteroporphyrin IX,2,4-bis-glycol, were noninducers for heme
oxygenase and ferritin in endothelium and did not provide
cytoprotection against oxidant damage.
Catabolism of heme by heme oxygenase may rid the cells of a
membrane-permeant form of iron, but the resultant nonheme iron would
represent a potential hazard unless sequestered by
ferritin.32 The cytoprotective nature of ferritin against
iron-driven oxidative stress is attributable to high sequestering
capacity for inorganic iron and ferroxidase activity of its H
chain.32 The cytoprotectant role of heme oxygenase and
ferritin has been confirmed in various models.59-63 A study
by Vile et al64 demonstrated the adaptive role of ferritin in oxidative stress in human skin fibroblast; Van
Lenten et al26 showed that intracellular ferritin abolishes iron-induced LDL modification; and Lin et al53 described
the protective effects of the H ferritin chain in human leukemia cells, confirming the antioxidant role of ferritin. Increased coexpression of
heme oxygenase and ferritin can be observed in humans and in animal
models.52,54-65 A recent study from our laboratory has revealed that ferritin is highly expressed in endothelial cells of
human atherosclerotic lesions54 while Wang et
al55 have shown that there is increased expression of heme
oxygenase-1 in atherosclerotic endothelium. In vivo, induction of heme
oxygenase and ferritin protects rats against rhabdomyolysis-induced
renal failure.56 Heme oxygenase can be important for
cardiac xenograft survival in rats, as hearts from heme
oxygenase-1 knockout mice were rapidly rejected.57 Heme
oxygenase- 2-deficient mice were more sensitive to hyperoxia-induced
oxidative lung injury with the absence of ferritin
induction.58 Heme oxygenase-1 deficiency in a human was
accompanied by fragmentation hemolysis and increased oxidant
sensitivity of lymphoblastoid cell line derived from the patient.59 Conversion of heme by heme oxygenase to
biliverdin and bilirubin was demonstrated to protect neurons against
oxidative stress.60,61 Induction of heme oxygenase was
associated with inhibition of monocyte chemotaxis induced by oxidized
LDL.62 In fact, it has been shown that bilirubin is an
important antioxidant.63 In addition to bilirubin, the
carbon monoxide produced during the degradation of heme has attracted a
great deal of interest on its potential function in regulating vascular
tone via guanylate cyclase.66 Despite the fact that heme
oxygenase and ferritin can provide cytoprotection against
heme-catalyzed injury in many ways, our previous studies and those
presented here emphasize the paramount role of iron sequestration by
ferritin in these cell systems.
The induction of heme oxygenase can be a response to oxidative
modification of LDL in endothelial cells.41 Heme oxygenase mRNA and enzyme activity were increased after cells were exposed to
either hematin/H2O2-conditioned LDL or heme
arginate/H2O2-conditioned LDL (Figure 7). That
heme arginate promoted LDL oxidation less efficiently than heme was
confirmed by the finding that heme
arginate/H2O2-conditioned LDL was a
significantly weaker inducer for heme oxygenase mRNA and enzyme
activity than hematin/H2O2-conditioned LDL.
We conclude that heme arginate, in contrast to hematin, was an
inefficient free-radical catalyst in endothelial cells and a less
potent catalyst for the oxidative modification of LDL particles, features possibly explaining the safety of heme arginate treatment for
porphyria. The beneficial effect of exogenous heme for patients with
acute hepatic porphyria may be due to repletion of the intrahepatic heme pool, which restores heme proteins, and to
negative feedback reducing the overall porphyrin synthesis through
inhibition of hepatic delta-aminolevulinic acid
synthase.1-9,67 The theoretical concern with the
administration of heme is that induction of heme oxygenase increases
the catabolism of heme, depriving the cell of essential heme. Indeed,
coadministration of tin protoporphyrin, a competitive inhibitor for
heme oxygenase,68 prolonged the biochemical remission
produced by heme arginate in acute hepatic porphyria.69 In
our study, heme arginate acted as a gene regulator for heme oxygenase
in endothelial cells as effectively as hematin, but endothelial cell
heme oxygenase degraded porphyrin rings of heme arginate less than the
use of hematin did. Possibly the arginine moieties
protect and stabilize the porphyrin ring.
Hematin-induced coagulopathy and thrombocytopenia have been
reported11,12,17; hematin inhibits coagulation and enhances platelet aggregation. In contrast, heme arginate treatment does not
alter hemostasis.70,71 Our findings may help to explain why
heme arginate can replenish cellular heme deficiency in a nonvasculopathic form.
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Acknowledgment |
We thank Dr Claus A. Pierach (Abbott Northwestern Hospital,
Minneapolis, MN) for helpful discussions.
| |
Footnotes |
Submitted April 14, 1999; accepted January 31, 2000.
Supported in part by National Institutes of Health grant HL-55552;
Hungarian government grants OTKA-T029558/021023, ETT-1161361996, and
FKFP-06171999; and the Cecil J. Watson Research Laboratory (J.B.).
Reprints: Gregory M. Vercellotti, Box 293, 420 Delaware St SE,
Minneapolis, MN 55455; e-mail: verce001{at}maroon.tc.umn.edu.
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.
| |
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Pro |
Top
Abstract
Introduction