Oxygen radical-induced mitochondrial DNA damage and repair in
pulmonary vascular endothelial cell phenotypes
Valentina
Grishko1,
Marie
Solomon1,
Glenn L.
Wilson2,
Susan P.
LeDoux2, and
Mark N.
Gillespie1
Departments of 1 Pharmacology and 2 Cell Biology and
Neuroscience, College of Medicine, University of South Alabama,
Mobile, Alabama 36688
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ABSTRACT |
Mitochondrial (mt) DNA is damaged by free radicals. Recent
data also show that there are cell type-dependent differences in mtDNA
repair capacity. In this study, we explored the effects of xanthine
oxidase (XO), which generates superoxide anion directly, and menadione,
which enhances superoxide production within mitochondria, on mtDNA in
pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV)
endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC
phenotypes, with a rank order of sensitivity of (from most to least)
PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the
iron-catalyzed formation of hydroxyl radical. Damage to the nuclear
vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA
damage quickly. Menadione-induced mtDNA damage was avidly repaired in
MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA
lesions at nucleotide resolution showed that damage patterns were
similar between EC phenotypes, but there were disparities between XO
and menadione in terms of the specific nucleotides damaged. These
findings indicate that mtDNA in lung vascular ECs is damaged by XO- and
menadione-derived free radicals and suggest that mtDNA damage and
repair capacities differ between EC phenotypes.
mitochondrial dexoyribonucleic acid; oxidant sensitivity; cytotoxicity
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INTRODUCTION |
IT IS WIDELY
APPRECIATED that the mitochondrial genome is prone to
oxidant-mediated damage, being 10- to 100-fold more sensitive than
nuclear DNA (36). Moreover, mutations and deletions in the
mitochondrial genome in cells of the central nervous system and
elsewhere have been linked to neurodegenerative disorders and other
age-related diseases (11, 27, 30, 32, 33). Depletion of
mitochondrial (mt) DNA with ethidium bromide is also known to suppress
ATP synthesis and cause defects in cell function (8).
These findings indicate that mtDNA damage could play a causal role in
disorders linked to excessive generation of reactive oxygen species.
Pulmonary vascular endothelial cells (ECs) are among the most important
targets of reactive oxygen species elaborated in acute lung injury
(6, 7, 23). Emerging data suggest that there may be
important differences between phenotypically distinct EC populations
residing at different segments of the pulmonary circulation (1,
9, 24, 25). Although the effects of oxygen radicals on mtDNA in
ECs from the lung have yet to be studied, a recent report by Ballinger
and colleagues (3) demonstrated that the mitochondrial
genome in human umbilical vein ECs was damaged by hydrogen peroxide and
peroxynitrite. Accordingly, a proximate goal of the present study was
to determine if mtDNA in pulmonary arterial (PA), microvascular (MV),
and pulmonary venous (PV) ECs was sensitive to reactive species-induced
damage. In addition, because cytotoxic reactive species can be
generated from both exogenous and endogenous sources, mtDNA damage in
lung ECs exposed to xanthine oxidase (XO), which produces reactive
species directly, was compared with that induced by menadione, which
promotes endogenous oxidant generation by redox cycling within the
mitochondria. Finally, we used pharmacological tools to confirm that
the mtDNA damage evoked by these stimuli in PAECs involved the
iron-catalyzed production of hydroxyl radicals as it does in other
systems (19, 22).
Accumulating data show that mitochondria are well endowed with
enzymatic pathways that repair oxidative DNA damage (4, 14). Defective mtDNA repair has also been associated with
certain human pathologies (13, 15). Interestingly, there
may be cell type-specific differences in mtDNA repair capabilities as
evidenced by recent findings (20) that primary cultures of
selected glial cell populations exhibit disparities in the rates at
which oxidative mtDNA damage is repaired. Even more provocative is the
observation that sensitivity to oxidant-induced mtDNA damage and death
in these central nervous system cell populations do not seem to be related to the mitochondrial antioxidant burden and, instead, are
closely linked to the capacity for mtDNA repair (20).
Glial cell types that exhibit avid mtDNA repair are less sensitive to oxidant-mediated cytotoxicity than are closely related cell populations that are relatively deficient in eliminating mtDNA lesions. To determine whether lung EC phenotypes are different in terms of their
capacities for mtDNA repair, we examined the rates at which XO- and
menadione-induced mtDNA lesions were removed from rat main PAECs,
MVECs, and PVECs. In addition, to probe the association between mtDNA
repair capacity and EC sensitivity to oxidant stress, we evaluated the
relationship between repair kinetics and cell death in the three lung
EC phenotypes.
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METHODS |
Rat main PAEC, MVEC, and PVEC cultures.
Main PAs and PVs were isolated from 250- to 300-g Sprague-Dawley rats
killed with an overdose of Nembutal. Isolated arteries were opened, and
the intimal lining was carefully scraped with a scalpel. Pulmonary
MVECs were harvested from tryptic digests of peripheral lung tissue
(24). The harvested cells were then placed into flasks
(Corning, Corning, NY) containing Ham's F-12 nutrient mixture and DMEM
mixture (1:1) supplemented with 10% fetal bovine serum, 100 U/ml of
penicillin, and 0.1 mg/ml of streptomycin (GIBCO BRL, Grand Island,
NY). The culture medium was changed once a week, and after reaching
confluence, the cells were harvested with a 0.05% solution of trypsin
(GIBCO BRL) and passsaged up to 15 times. The EC phenotype was
confirmed by acetylated low-density lipoprotein uptake, factor
VIII-related antigen immunostaining, and the lack of
immunostaining with smooth muscle cell
-actin antibodies
(Sigma, St. Louis, MO).
Detection of mtDNA damage with quantitative Southern and
ligation-mediated PCR analyses.
PAECs cultured on 100-mm petri plates were challenged with ascending
doses of either XO plus hypoxanthine (0.5 mM) or menadione. Quantitative Southern analysis was used to examine changes in the
equilibrium density of the lesions in the entire mitochondrial genome
(14). DNA isolated from the ECs was precipitated,
resuspended in Tris-EDTA buffer (pH 8.0), and treated with DNase-free
RNase. Purified DNA was then digested with BamHI, and
complete digestion was verified on minigels. After restriction, the
samples were resuspended in a small volume of Tris-EDTA buffer and
precisely quantified with a Hoefer TKO minifluorometer and standards
kit. Samples containing 5-10 µg of DNA were heated, cooled at
room temperature, and then incubated for 15 min with sodium hydroxide (0.1 N) to cleave DNA at the sites of oxidative injury to the deoxyribose backbone. Samples were then combined with 5 µl of loading
dye, loaded onto a 0.6% alkaline gel, and electrophoresed in an
alkaline buffer. After the gel was washed, DNA was transferred by
vacublotting (Millipore, Bedford, MA) onto a Zeta-Probe GT nylon
membrane (Bio-Rad Laboratories, Hercules, CA) and cross-linked to the
membrane with a GS Gene Linker (Bio-Rad). After a 10-min preincubation,
the membrane was hybridized with a PCR-generated genomic DNA probe for
the mitochondrial genome. The probe used to hybridize to mtDNA was
generated via PCR from a mouse mtDNA sequence with the use of the
following primers: 5'-GCAGGAACAGGATGAACAGTCT-3' from the sense strand
and 5'-GTATCGTGAAGCACGATGTCAAGGGATGTAT-3' from the antisense strand.
The 725-bp product recognized a 10.8-kb restriction fragment when
hybridized to rat mtDNA digested with BamHI. Using a similar
strategy, we also probed a 3.4-kb sequence in the 5' promoter of the
nuclear vascular endothelial growth factor (VEGF)
gene. The membranes were washed according to the manufacturer's instructions, and hybridization bands were detected with a Bio-Rad GS-250 molecular imager. Changes in the equilibrium "lesion" density were calculated as
ln of the quotient of
hybridization intensities in the treated and control bands
(5).
Ligation-mediated PCR was used to evaluate oxidative lesions at
single-nucleotide resolution in the "common deletion sequence" of
the mitochondrial genome, which is already known to be prone to
mutation and deletions underlying aging-related disease (11, 28). DNA from control and oxidant-treated cells was
isolated and denatured to produce single-strand DNA with terminal 5'
phosphate groups. After annealing a primer to the known 3' sequence,
extension was performed to yield double-strand DNA fragments, the
lengths of which were determined by the site of cleavage; i.e., the
site of the lesion. A linker was then ligated to the blunt-end duplex DNA, and the DNA was amplified by multiple cycles of PCR with a nested
primer and another primer complementary to the linker sequence.
Subsequently, the reaction mixture was run on a sequencing gel and
electroblotted to a nylon membrane. The membrane was then hybridized
with a single-strand probe, analyzed with a phosphorimager, and
compared with a Maxim-Gilbert sequence ladder. A spatial map of lesion
density was then constructed by expressing normalized hybridization
intensities at each specific nucleotide in the 50-bp sequence of interest.
Cytotoxicity assay.
The cytotoxic effects of ascending doses of either XO or menadione were
determined 24 h after removal of the oxidant generators with the
commercially available MTT assay (Promega, Madison, WI).
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RESULTS |
Initial studies evaluated dose-response relationships for XO- and
menadione-induced mtDNA damage in the three EC phenotypes. Cells were
exposed to the free radical-generating systems for 1 h, a duration
shown by preliminary experiments to be sufficient for mtDNA damage to
plateau at any given XO concentration. Representative Southern blot
analyses of the concentration-dependent effects of XO are shown in Fig.
1. It is evident that XO decreased
hybridization intensity, which is indicative of mtDNA damage, in all
three phenotypes, but PVECs tended to be the most sensitive compared
with the other two EC populations. Changes in steady-state mtDNA lesion
density were calculated as a function of the XO concentration and
pooled for four experiments and are displayed for each EC phenotype in Fig. 1. The apparent rank order of sensitivity to XO-induced mtDNA damage was (from most to least) PVECs > PAECs > MVECs.

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Fig. 1.
Rat pulmonary venous (PV), pulmonary arterial (PA), and
pulmonary microvascular (MV) endothelial cells (ECs) were cultured
under control (Con) conditions or challenged with indicated
concentrations of xanthine oxidase (XO) plus 0.5 mM hypoxanthine for
1 h, after which the cells were lysed. High molecular weight DNA
was isolated and digested to completion with BamHI. DNA
samples were exposed to 1 N NaOH before Southern blot analysis and
hybridization with a mitochondrial (mt) DNA-specific probe.
Top: representative autoradiogram of quantitative Southern
analysis of alkali-labile mtDNA damage. There are 2 lanes each for
control and XO-treated samples. Note diminished hybridization
intensity, indicative of mtDNA damage. Bottom: calculated
increases in equilibrium lesion density normalized to 10 kb. Values are
means ± SE for 4 experiments.
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Representative Southern blot analyses depicting the effects of
menadione are shown in Fig. 2. Like XO,
the reactive species generated endogenously in response to
menadione-damaged mtDNA, but the relative sensitivities of the EC
phenotypes appeared to be different. Calculated increases in
steady-state lesion density, also displayed in Fig. 2, indicate that
MVECs and PVECs were roughly equal in terms of the ability of menadione
to damage mtDNA, whereas PAECs were somewhat less sensitive, especially
at the lowest menadione dose tested.

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Fig. 2.
Rat MVECs, PVECs, and PAECs were cultured under control
conditions or challenged with menadione for 1 h, after which the
cells were lysed. High molecular weight DNA was isolated and digested
to completion with BamHI. DNA samples were exposed to 1 N
NaOH before Southern blot analysis and hybridization with a
mtDNA-specific probe. Top: representative autoradiogram of
quantitative Southern analysis of alkali-labile mtDNA damage. Note
diminished hybridization intensity indicative of mtDNA damage.
Bottom: calculated increases in equilibrium lesion density
normalized to 10 kb. Values are means ± SE for 4 experiments.
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In the nonvascular cell systems examined thus far, the iron-catalyzed
production of hydroxyl radicals seems to play an important role in
oxidant-mediated DNA damage (19, 22). To determine whether
XO- and menadione-induced mtDNA damage in lung ECs involved a similar
mechanism, PAECs were treated with XO (5 mU/ml) and menadione (100 µM) in the absence and presence of the putative hydroxyl radical
scavenger dimethylthiourea (100 µM) (17, 34) or the iron
chelator deferoxamine (150 µM). Cells were harvested 60 min after the
simultaneous addition of the free radical generators and inhibitory
agents, and quantitative Southern analysis was used to quantify changes
in the steady-state density of mtDNA lesions. As shown in Fig.
3, both dimethylthiourea and
deferoxamine suppressed XO- and menadione-induced mtDNA damage to
an approximately equal extent, although deferoxamine tended to be
less effective in attenuating XO-mediated damage than damage induced by
menadione.

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Fig. 3.
Rat PAECs were challenged with either XO (5 mU/ml) or
menadione (MEN; 100 µM) in the presence and absence of 100 µM
dimethylthiourea (DMTU) or 150 µM deferoxamine (DFX) for 60 min,
after which cells were harvested and the extent of mtDNA damage was
determined by quantitative Southern analysis. Representative
autoradiogram (top) indicates that although neither DMTU nor
DFX altered the baseline hybridization intensity, both agents
suppressed the profound decrease in hybridization caused by either XO
or menadione. Increases in the steady-state lesion density as
determined in 4 separate experiments were pooled, and the effects of
DMTU and DFX are expressed as percent inhibition of the responses to XO
and menadione alone (bottom). All treatment groups were
significantly depressed relative to control groups (P < 0.05) by 1-way ANOVA and Dunnett's test).
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The deleterious effects of XO and menadione on the nuclear
VEGF gene also were explored. No damage to the
VEGF gene was detected at any XO or menadione dose examined
(data not shown).
To examine mtDNA repair capacity, the three EC phenotypes were treated
with 10 mU/ml of XO to produce an increase of ~1.5 strand breaks/10
kb in equilibrium lesion density. The cells were harvested immediately
after a 1-h treatment with XO or were allowed to repair the damage for
4 or 24 h in XO-free medium. The representative Southern blot
analyses shown in Fig. 4 indicate that
PVECs exhibited a rather low capacity for mtDNA repair compared with
the other two EC phenotypes. As also displayed in Fig. 4, when mtDNA
integrity is expressed as the percentage repaired, i.e., as a
percentage of the lesion burden present immediately after XO treatment,
it was evident that PVECs were virtually incapable of repairing
XO-induced mtDNA damage, at least over a 24-h period. Conversely,
time-dependent repair in PAECs and MVECs resulted in almost complete
repair of the lesions within 24 h. The outcome of a companion
experiment that used 200 µM menadione to promote reactive species
generation is shown in Fig. 5. In this
instance, repair in PVECs and MVECs was almost complete within 4 h
and approached 100% removal of the lesions. Repair in PAECs was
somewhat slower but was still nearly complete by 24 h.

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Fig. 4.
Rat PVECs, PAECs, and MVECs were cultured under control
conditions or treated for 1 h with 10 mU/ml of XO plus 0.5 mM
hypoxanthine. After XO treatment, cells were harvested immediately (0 h) or placed in XO-free medium and allowed to recover (repair) for 4 or
24 h. At the indicated times, high molecular weight DNA was
isolated, digested, and subjected to quantitative Southern blot
analysis. Top: representative autoradiogram of quantitative
Southern blot analysis of alkali-labile mtDNA damage in control and
XO-treated EC phenotypes as a function of time after the removal of XO.
There are 2 lanes each for control and XO-treated samples. Note
recovery of hybridization intensity at 4 and 24 h, indicative of
repair of mtDNA damage evident immediately after XO treatment.
Bottom: calculated repair of mtDNA lesions expressed as
percent reversal of the initial lesion density. Values are means ± SE for 4 experiments.
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Fig. 5.
Rat PVECs, PAECs, and MVECs were cultured under control
conditions or treated for 1 h with 200 µM menadione. After
menadione treatment, cells were harvested immediately (0 h) or placed
in menadione-free medium and allowed to recover for 4 or 24 h. At
the indicated times, high molecular weight DNA was isolated, digested,
and subjected to quantitative Southern blot analysis. Top:
representative autoradiogram of quantitative Southern blot analysis
of alkali-labile mtDNA damage in control and menadione-treated EC
phenotypes as a function of time after removal of menadione. Note
recovery of hybridization intensity at 4 and 24 h, indicative of
repair of mtDNA damage Bottom: calculated repair of mtDNA
lesions expressed as percent reversal of the initial lesion density.
Values are means ± SE for 4 experiments.
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We next assessed whether the phenotypic differences in the XO- and
menadione-induced mtDNA damage and repair described above were
accompanied by differences in oxidant-mediated cytotoxicity determined
24 h after removal of the oxidant-generating stimuli. As shown in
Fig. 6, PVECs were the most sensitive to
XO-mediated cell death followed by PAECs and MVECs. In the case of
menadione-induced cytotoxicity, MVECs and PAECs were of approximately
equal sensitivity, followed by PVECs.

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Fig. 6.
Concentration-dependent cytotoxicity evoked by XO
(top) and menadione (bottom) as assessed by MTT
assay in PAECs, MVECs, and PVECs. Note different rank order of
sensitivities of the different EC phenotypes to the free radical
generators. Each point is the mean ± SE of at least 4 determinations.
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Ligation-mediated PCR was used to determine whether the nucleotide
specificity of XO-mediated mtDNA damage differed among the EC
phenotypes or from damage induced by menadione. These analyses focused
on a 200-bp sequence containing one of the break sites for the
"common" 5-kb deletion known to occur in aging (11, 28). Representative autoradiograms depicting ligation-mediated PCR determinations of XO- and menadione-induced damage in the three EC
phenotypes are shown in Figs. 7 and
8, respectively. A qualitative analysis
of these data reveals two significant features. First, as would be
predicted from the Southern blot analyses described previously, the
increased hybridization intensities associated with increasing XO and
menadione concentrations indicate that both agents caused dose-related
damage to specific nucleotides within the 200-bp segment of the
mitochondrial genome. Second, and more important, the pattern of damage
for each agent did not differ markedly between the EC phenotypes.

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Fig. 7.
Ligation-mediated PCR analysis of nucleotide-specific damage within
the common deletion sequence of the mtDNA heavy strand in control
PAECs, PVECs, and MVECs and in cells treated with either 10 or 5 mU/ml
of XO plus 0.5 mM hypoxanthine (nos. at top). DNA samples
were treated with (+) and without ( ) 0.1 N NaOH to cleave to the
deoxyribose backbone at sites of oxidative damage. A Maxim-Gilbert
sequence ladder shows the nucleotide bases guanine (G), adenine (A),
thymine (T), and cytosine (C). Letters and numbers at far
right, base and position, respectively. Damage at a given
nucleotide is indicated by an increase in hybridization intensity. Note
1) increasing hybridization intensities at specific
nucleotides with increasing XO concentration and, most importantly,
2) the similar patterns of damage between the 3 EC
phenotypes.
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Fig. 8.
Ligation-mediated PCR analysis of nucleotide-specific damage within
the common deletion sequence of the mtDNA heavy strand in control
PAECs, PVECs, and MVECs and in cells treated with either 200 µM or
300 µM menadione (nos. at top). Letters and nos. at far
right, base and position, respectively. Damage at a given
nucleotide is indicated by an increase in hybridization intensity. Note
1) increasing hybridization intensities at specific
nucleotides with increasing menadione concentration and, most
importantly, 2) the similar patterns of damage between the 3 EC phenotypes.
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On the other hand, the pattern of damage to specific nucleotides did
differ between menadione and XO. Figure 9
shows a comparison of damage maps for XO- and menadione-induced mtDNA
damage in PAECs. In this analysis, hybridization intensities for each
nucleotide, as determined from four ligation-mediated PCR analyses,
were scored from 1 to 6, with 1 reflecting the least prevalent and 6 indicative of the most prevalent lesions, and were plotted as a
function of the specific nucleotide. The resulting maps revealed that
there were a number of nucleotides frequently damaged by XO but not by
menadione and vice versa. In further support of such differences in
nucleotide specificity, the proportion of four nucleotides with severe
damage, defined as a damage level
3 from the ligation-mediated PCR analyses, was determined and, as shown in Table
1, was guanine = adenine > thymine > cytosine in the case of XO-mediated damage and
guanine > adenine > thymine > cytosine for damage
evoked by menadione. The rate of damage to guanine in menadione-treated cells exceeded that in XO-treated ECs.

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Fig. 9.
Ligation-mediated PCR-generated map of nucleotide-specific damage
evoked by XO (A) and menadione (B) in cultured
PAECs. The analysis focused on a 54-nucleotide span within the common
deletion sequence of the mtDNA heavy chain. Position of bars indicates
damage to a specific nucleotide; bar height indicates relative
prevalence of damage as determined by hybridization intensities
normalized to a scale of 1 (least lesions) to 6 (most prevalent
lesions). Note differences in patterns of damage between XO and
menadione.
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DISCUSSION |
Multiple studies (11, 27, 30, 32, 33) show that
mutations and deletions in the mitochondrial genome contribute to pathogenesis in a spectrum of diseases. The first report examining the
sensitivity of systemic vascular EC mtDNA to oxidant stress appeared
recently (3), and it showed that mtDNA damage evoked by
reactive oxygen and nitrogen species was accompanied by diminished ATP
synthesis, impaired mitochondrial RNA expression, and altered mitochondrial membrane potential. It is unknown whether mtDNA in lung
vascular ECs is sensitive to oxidant-mediated damage; resolution of
this issue was the proximate goal of the present study. In a related
context, in light of the increasing appreciation that there are
segmental differences among EC phenotypes in the pulmonary circulation
(1, 9, 24, 25), we determined if there were disparities
between ECs harvested from PA, MV, and PV segments of the rat lung in
terms of their sensitivities to oxidant stress. Finally, because the
pulmonary vascular endothelium can be damaged by oxidants elaborated
from both exogenous and endogenous sources, we compared the effects of
XO and menadione, respectively, for their abilities to erode mtDNA integrity.
The present results show that XO- and menadione-generated oxidants
cause prominent damage to the mitochondrial genome in three lung EC
phenotypes, with no detectable effects on nuclear DNA. The relative
sensitivity of mtDNA compared with that in the nucleus was also noted
by Ballinger et al. (3) for systemic vascular smooth cells
and ECs as it has been for other nonvascular cells (36).
The reason that mtDNA is more sensitive than nuclear DNA has
traditionally been ascribed to the lack of protective histone muscle
proteins and the relative underdevelopment of DNA repair mechanisms,
although more recent work shows that mitochondria do exhibit effective
DNA repair for some types of damage (4).
We found that the EC phenotypes exhibited different sensitivities to
mtDNA damage, with MVECs being less sensitive to XO than PAECs and
PVECs. On the other hand, MVECs were most sensitive to
menadione-induced mtDNA damage, with PAECs somewhat more resistant. The
factors underlying these divergent sensitivities are unknown. Certainly, different abundances of cellular antioxidants and
scavenging enzymes could play a role, although to what extent these
pathways protect mtDNA from oxidant-mediated damage has not been
systematically addressed. It is also possible that the EC phenotypes
are intrinsically different in terms of the ability of XO and menadione
to promote oxidant generation. For example, ECs are known to
internalize XO by endocytosis, after which the free radicals generated
intracellularly are capable of disrupting nitric oxide-dependent
signaling (21). It is thus possible that the different
sensitivities of mtDNA in the EC phenotypes studied in the present
report are related to different capacities for XO binding and
endocytosis. Counter to this idea, a recent preliminary finding from
our laboratory that XO-induced mtDNA damage in cultured PAECs is
unaffected by suppression of XO uptake argues that free radicals
generated by extracellular XO are sufficient to erode mtDNA integrity
(31). In the case of menadione, it should be considered
that mitochondrial metabolism is a determinant of its ability to
produce superoxide anion (16). As such, EC
phenotype-dependent metabolic differences could account for their
differing sensitivities to menadione-induced mtDNA damage.
It is widely appreciated that the iron-catalyzed formation of hydroxyl
radicals plays an important role in oxidant-mediated DNA damage
(19, 22). The oxidizing species are probably not diffusible hydroxyl radicals but rather hydroxyl radicals generated in
the immediate vicinity of iron bound to the DNA target (18, 22). In this regard, it appears uncertain whether in the intact cell there is sufficient mtDNA-bound iron for the Fenton reaction to
proceed. To address this issue, we examined the effects of the putative
hydroxyl radical scavenger dimethylthiourea (17, 34) and
found that it suppressed both XO- and menadione-induced mtDNA damage.
More interestingly, deferoxamine, an iron chelator, also blocked mtDNA
damage evoked by both stimuli in PAECs. These observations suggest that
mtDNA, like nuclear DNA, appears to bind sufficient iron to support
hydroxyl radical formation via the Fenton reaction and that the
ultimate mtDNA-damaging reactive species is hydroxyl radical. It should
be noted that these experiments were performed only in PAECs, but it
seems likely that the involvement of iron and the hydroxyl radical can
be extended to the other EC phenotypes insofar as the nucleotide
pattern of damage in the mitochondrial genome failed to display any
phenotype-dependent differences (see below).
Another mechanism suspected of governing the sensitivity of mtDNA to
oxidant-mediated damage is the mtDNA repair pathways. In contrast to
the traditional concept that mitochondria are deficient in DNA repair
(10), experiments in nonvascular cells indicate that they
are, at a minimum, equipped to perform base excision repair of the
mitochondrial genome (4). Because mtDNA repair has not
been documented in vascular cell types, the present study determined if
lung ECs were able to remove oxidant lesions evoked by XO and
menadione. As might be expected, pulmonary vascular ECs were generally
able to repair lesions evoked by both stimuli, but there were rather
prominent phenotype-dependent differences. XO-induced damage was
repaired well by PAECs and MVECs but not in cells derived from PVs.
Menadione-induced mtDNA lesions were repaired proficiently by all three
phenotypes, but PAECs exhibited a somewhat slower rate than the other
two. The concept that different phenotypes can exhibit divergent mtDNA
repair capacity is not without precedent. In a study of closely related
glial cell types, Hollensworth and coworkers (20) showed
that astrocytes, which are highly resistant to oxidant-induced mtDNA
damage, exhibit more efficient mtDNA repair than oligodendrocytes or
microglia, which are relatively prone to oxidant-induced mtDNA damage.
The abundance of various antioxidant pathways or scavengers did not correlate with mtDNA sensitivity to oxidants in these glial cell lines.
As expected on the basis of numerous previous reports (e.g., Refs. 6,
7), the oxidant stresses examined in the present study were
lethal to lung ECs. A provocative pattern emerged when the sensitivity
to XO-mediated cell death was compared with phenotype-dependent differences in mtDNA repair. The EC phenotype that repaired XO-induced mtDNA damage most avidly (MVECs) was resistant to its cytotoxic actions, whereas PVECs, which were deficient in repair of XO damage, were most sensitive to its cytotoxicity. In this regard, the
aforementioned study (20) on glial cell phenotypes found
that the propensity for menadione-induced apoptosis, which is
associated with activation of caspase-9, was inversely related to the
rate of repair of menadione-induced damage to mtDNA. Moreover,
enhancement of mtDNA repair in either HeLa cells (12) or
PAECs (unpublished observations) through stable or transient
transfection, respectively, with the gene for the DNA repair enzyme,
Ogg1, linked to a mitochondrial localization sequence
confers protection against oxidant-mediated mtDNA damage and
cytotoxicity. Although the available data are far from conclusive, it
is tempting to speculate that the capacity to repair mtDNA damage or,
stated differently, the persistence of mtDNA damage, could be a
determinant of sensitivity to oxidant-mediated EC death.
Using ligation-mediated PCR, we found that the nucleotide specificity
by which XO and menadione damaged an ~200-bp sequence located within
the common deletion fragment of the mitochondrial heavy chain was
internally consistent; with only minor exceptions, the same nucleotides
were damaged regardless of the EC phenotype examined. On the other
hand, there were differences between XO and menadione in terms of the
nucleotide specificity of the damage, with the rate of damage to
guanine and adenine similar for XO, whereas guanines seemed to be
preferentially damaged by menadione. Neither the reasons for
these differences nor their biological significance can be explained at
present. Nevertheless, it is likely that although the proximate radical
species generated by both XO and menadione is superoxide anion, the
different anatomic locations of superoxide anion generation,
extracellular or cytoplasmic for XO versus intramitochondrial for
menadione, could afford different opportunities for interactions with
other reactive species. As a consequence, the rates and/or nature of
the radical species interacting with mtDNA could differ, thus
accounting for the divergent nucleotide specificities. In a related
context, an emerging concept relative to the biology of DNA damage is
that certain nucleotides within a given sequence may be prone to damage
or deficiently repaired and could thus play more important roles in
determining cellular outcomes (29). Against this
background, perhaps phenotype-dependent differences in the cytotoxic
actions of XO and menadione are somehow linked to the different
nucleotide specificities of the damage they induce. Clearly, much
additional study will be required to address this possibility.
In summary, the present study shows that XO- and menadione-generated
oxidants preferentially erode mtDNA integrity relative to nuclear DNA
in lung EC phenotypes. There appear to be phenotype-dependent differences in the propensity for mtDNA damage, in the capacity for
mtDNA repair, and in the cytotoxicity associated with these oxidant
stresses that warrant additional investigation. An outstanding question prompted by our study as well as by the preceding report on oxidant mtDNA damage in systemic vascular ECs by Ballinger et al.
(3) pertains to its biological significance. An editorial (35) accompanying the Ballinger paper
appropriately emphasized the vagaries in causally linking EC
mtDNA mutations and deletions to progressive vasculopathies like
atherosclerosis because it is believed that 80-90% of the total
mtDNA pool must be affected before disease progression takes place
(26). On the other hand, our observation that mtDNA repair
kinetics may be predictive of XO-induced lung EC death, coupled with
the finding of Ballinger et al. (3) that mtDNA damage was
associated with impaired ATP synthesis and reduced mitochondrial gene
expression, raises the interesting possibility that acute damage to
mtDNA could be a proximate cause of EC dysfunction and death. Thus even
in the absence of mtDNA mutation or deletion, damage to mtDNA could be significant in the various forms of acute lung EC injury in which oxidants are suspected of playing a pathogenic role.
 |
ACKNOWLEDGEMENTS |
This investigation was supported by National Heart, Lung, and Blood
Institute Grants HL-38495 and HL-58243.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: M. N. Gillespie, Dept. of Pharmacology, Univ. of South Alabama College of
Medicine, Mobile, AL 36688 (E-mail:
mgillesp{at}jaguar1.usouthal.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 September 2000; accepted in final form 5 January 2001.
 |
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