MITOCHONDRIAL DNA DAMAGE TRIGGERS

MITOCHONDRIAL DYSFUNCTION AND APOPTOSIS IN

OXIDANT-CHALLENGED LUNG ENDOTHELIAL CELLS

Mykhaylo Ruchko1,3, Olena Gorodnya1,3, Susan P. LeDoux2,

Mikhail F. Alexeyev2,3, Abu-Bakr Al-Mehdi1,3, and Mark N. Gillespie1,3

University of South Alabama College of Medicine, Departments of1Pharmacology and of 2Cell Biology and Neuroscience, and the

3Center for Lung Biology, Mobile, AL 36688

Correspondence: Mark N. Gillespie, Ph.D.

Department of Pharmacology

University of South Alabama

College of Medicine

Mobile, AL 36688

Telephone: 251-460-64697

Fax: 251-460-6798

e-mail: mgillesp@jaguar1.usouthal.edu

Running Head: Mitochondrial DNA Damage and Endothelial

Cell Apoptosis

Key words: xanthine oxidase; endothelial cells;

apoptosis

Articles in PresS. Am J Physiol Lung Cell Mol Physiol (November 24, 2004). doi:10.1152/ajplung.00255.2004

Copyright © 2004 by the American Physiological Society.

2

ABSTRACT

Oxidant-induced death and dysfunction of pulmonary vascular cells play

important roles in the evolution of acute lung injury. In pulmonary artery

endothelial cells (PAECs), oxidant-mediated damage to mitochondrial (mt)

DNA seems to be critical in initiating cytotoxicity inasmuch as over-

expression of the mitochondrially-targeted, human DNA repair enzyme,

hOgg1, prevents both mtDNA damage and cell death (Amer. J. Physiol: Lung

Cell Molec. Physiol. 283: L205-10, 2002). The mechanism by which mtDNA

damage leads to PAEC death is unknown, and the present study tested the

specific hypothesis that enhanced mtDNA repair suppresses PAEC

mitochondrial dysfunction and apoptosis evoked by xanthine oxidase (XO).

PAECs transfected with either an adenoviral vector encoding hOgg1 linked to

a mitochondrial targeting sequence or with empty vector were challenged

with ascending doses of XO plus hypoxanthine. Quantitative Southern blot

analyses revealed that, as expected, hOgg1 over-expression suppressed XO-

induced mtDNA damage. Mitochondrial over-expression of hOgg1 also

suppressed the XO-mediated loss of mitochondrial membrane potential.

Importantly, hOgg1 over-expression attenuated XO-induced apoptosis as

detected by suppression of caspase 3 activation, by reduced DNA

fragmentation, and by a blunted appearance of condensed, fragmented

nuclei. These observations suggest that mtDNA damage serves as a trigger

for mitochondrial dysfunction and apoptosis in XO-treated PAECs.

3

INTRODUCTION

Oxidant-induced death and dysfunction of pulmonary vascular

endothelial cells (ECs) play important roles in the evolution of acute lung

injury. A number of molecular targets of reactive oxygen species (ROS) have

been identified, including membrane phospholipids, transporters, enzymes,

transcription factors, DNA, etc (16). The mitochondrial genome is far more

vulnerable to oxidative stress in comparison to nuclear DNA (2, 3, 11, 17)

because of its open, circular structure and relatively limited repair capacity.

Oxidative damage to the mitochondrial genome leads to altered

mitochondrial gene expression and an imbalance in the availability of

proteins involved in electron transport chain (2). Respiratory chain

dysfunction could lead to increased ROS production and formation of a

vicious cycle thereby killing cells either necrotically, apoptotically or by some

combination thereof (12).

We recently advanced the concept that mtDNA in lung vascular ECs

serves as a sentinel molecule in which persistent or severe damage promotes

oxidant-mediated cell death (5). In support of this idea we showed, first,

that sensitivity to xanthine oxidase (XO)-induced cytotoxicity among

pulmonary artery (PA), microvascular, and pulmonary vein EC phenotypes

was inversely related to repair of XO-mediated mtDNA damage (10). Second

and more recently, we found that over-expression of a mitochondrially-

targeted human 8-oxoguanine-DNA glycosylase (hOgg1) repair enzyme

4

attenuated mtDNA damage and enhanced cell survival in oxidant-challenged

PAECs and other cell types (5, 6). However, the mechanism by which

oxidant-mediated damage to mtDNA causes cell death remains unknown. In

the present study, we tested the specific hypothesis that mtDNA damage is a

proximate trigger for mitochondrial dysfunction and linked to activation of an

apoptotic death pathway in XO-challenged PAECs.

5

METHODS

Rat pulmonary artery endothelial cell culture and treatment: Rat

PAECs were isolated and cultured as described previously (1). In brief, main

pulmonary arteries were isolated from 250-300g Sprague-Dawley rats killed

with an overdose of Nembutal. Isolated arteries were opened and the intimal

lining was carefully scraped with a scalpel. The harvested cells were then

placed into flasks (Corning Inc., Corning, NY) containing F12 Nutrient Mixture

and Dulbecco’s modified Eagle’s medium (DMEM) mixture (1:1)

supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and

0.1 mg/ml streptomycin (Gibco BRL Products, Grand Island, NY). Culture

medium was changed once per week and, after reaching confluence, the cells

were harvested using a 0.05% solution of trypsin (Gibco BRL). The

endothelial cell phenotype, confirmed by acetylated LDL uptake, Factor VIII-

RAg immunostaining, and the lack of immunostaining with smooth muscle

cell α-actin antibodies (Sigma, St. Louis, MO), persisted for at least 15

passages.

Enzymatic oxidation of hypoxanthine by XO was used as source of

reactive oxygen species for cell treatment. Confluent PAECs transfected with

adenovirus were challenged for 1 hour with the indicated concentrations of

XO and 0.5 mM hypoxanthine in Hanks’ Balanced Salt Solution (HBSS). After

treatment, cells were either processed immediately or placed into cell culture

media for recovery.

6

Adenoviral vector construct and cell transfection: Recombinant

adenoviruses were generated according to established protocols (8) using

commercially available plasmids (AdMax, Microbix, Toronto, Canada). Briefly,

the shuttle plasmid pDC512 was modified to incorporate a CMV promoter, a

polylinker and an internal ribosome entry site of the encephalomyocarditis

virus. Both viruses contained an enhanced green fluorescent protein as their

second cistron. The Ad.889 contained no first cistron and was used as a

control for non-specific viral effects, whereas Ad.979 contained hOgg1 gene

used in our previous studies (5, 6). In Ad.979, the hOgg1 gene is modified

to incorporate a stop codon and is preceded by a synthetic sequence

consisting of the Kozak sequence and codon-optimized sequences encoding

the mitochondrial targeting sequence (MTS) for human MnSod and a

hemagglutinin (HA) tag.

Nearly confluent PAECs were transfected with Ad.889 as control or

Ad.979 with concentrations corresponding to multiplicity of infections (moi)

of one (1x) or two (2x) viral particles per cell. Cells were studied 48 hours

after transfection.

Detection of mtDNA damage with quantitative Southern blot analysis:

Vector-transfected and hOgg1-transfected PAECs cultured on 35-mm plates

were challenged for 1 h with the indicated concentrations of XO and 0.5 mM

hypoxanthine in HBSS and immediately lysed with 3 ml of TE buffer

7

containing 0.5% SDS and 0.3 mg/ml proteinase K overnight at 37 oC. DNA

was isolated using extraction with 1 M NaCl, 10 min at room temperature

and purified twice with chloroform/isoamyl alcohol, 24:1. After precipitation

with ethanol and reconstitution in water purified DNA was treated with

DNase-free RNase and digested with Bam HI (Roche Diagnostics,

Indianapolis, IN) - 10 U/mg DNA, overnight at 37 oC. Following restriction,

samples were precipitated, dissolved in TE buffer and precise DNA

concentrations were measured on the Hoefer DyNA Quant 200 fluorometer

(Pharmacia Biotech, San Francisco, CA) using Hoechst H33258 dye. Samples

containing 2 mg DNA were treated with NaOH (final concentration 0.1 N)

during 15 min at 37 oC, mixed with loading dye and resolved in 0.6%

agarose alkaline gel at 1.5 V/cm during 16 h. After electrophoresis gel was

washed and DNA was vacuum transferred to Zeta-Probe GT nylon membrane

(Bio-Rad Laboratories, Hercules, CA). Membrane with cross-linked DNA was

pre-hybridized during 30 min and then hybridized with 32P-labeled probe

overnight at 55 Co. The probe to mtDNA was generated by PCR using rat

mtDNA sequence as template and the following primers: 5’-

GCAGGAACAGGATGAACAGTCT-3’ for the sense strand and 5’-

GTATCGTGAAGCACGATGTCAAGGGATGAG-3’ for the anti-sense strand. The

725-bp product was hybridized with a 10.8-kb restriction fragment of rat

mtDNA digested with Bam HI. After hybridization the membrane was washed

and exposed to X-ray film (Kodak XAR-5) to obtain an autoradiographic

image or placed on phosphorimaging screen and scanned with Bio-Rad GS-

8

250 Molecular Imaging System. Changes in the equilibrium lesion density

were calculated as negative ln of the quotient of hybridization intensities in

treated and control bands (4).

Measurement of mitochondrial membrane potential: The membrane

fluorescent dye, JC-1 (Molecular Probes, Eugene, OR), was used for the

measurement of mitochondrial membrane potential. The green-fluorescent

JC-1 selectively enters mitochondria and exists as a monomer at low

membrane potential. However, at higher potentials, JC-1 forms red-

fluorescent “J-aggregates”, that exhibit a broad excitation spectrum and

large shift in emission with maximum at 590 nm. For dye loading, cells were

incubated with 1 µM JC-1 in HBSS for 30 min at 37 oC prior to cell treatment

with XO. Changes in mitochondrial membrane potential were observed with

Olympus IX70 fluorescence microscope.

Isolation of mitochondrial fraction: Crude mitochondrial fraction was

prepared by the differential centrifugation method. In brief, PAECs were

rinsed three times with phosphate buffered saline (PBS) and two times with

0.25M sucrose, 10 mM triethanolamine-acetic acid, pH 7.8 at room

temperature and harvested in ice-cold 0.25M sucrose, 1 mM EDTA, 10 mM

triethanolamine-acetic acid, pH 7.8. All the following steps were carried out

at 0-4 oC. The cell suspension was transferred to a Dounce grinder and

homogenized with 10 strokes. Cell debris and nuclei were removed by

9

centrifugation at 1000 g for 10 min. The pellet was then washed with

homogenizing buffer twice following centrifugation. Post-nuclear

supernatants were spun at 15000 g for 15 min to pellet the mitochondrial

fraction. For Western blot analysis the pellet was resuspended in 2% SDS

electrophoresis loading buffer.

Immunoblot analyses of hOgg1 and caspase 3: Human Ogg1 was

detected in crude mitochondrial extract, prepared as described above, and

caspase 3 was detected in total cell lysate using Western immunoblot

analyses. Control and treated cells were harvested 4 hours after XO

exposure. For preparation of total cell lysate, cells were washed twice with

PBS and lysed in 2% SDS electrophoresis loading buffer after which 15 - 25

µg protein was applied to an SDS/12% polyacrylamide gel. Following

separation, samples were transferred to nitrocellulose filters (BioRad

Laboratories, Hercules, CA). Membranes blocked in 5% nonfat dry milk in

PBS with 0.1% Tween 20 (PBS-T) were incubated overnight at 4 oC with

primary, rabbit polyclonal antibodies recognizing either the activated form of

caspase 3 (Cell Signaling Technology, Beverly, MA) or hOgg1 (Novus

Biologicals, Littleton, CO), both of which were diluted 1:600 in blocking

solution. After washing, the membranes were incubated with 1:7,500 diluted

HRP-conjugated goat anti-rabbit IgG (Calbiochem-Novabiochem Corp., San

Diego, CA) for 1 h at room temperature and then revealed by

chemiluminescence with the ECL detection kit (Amersham, England).

10

Immunocytochemcial analysis of activated caspase 3: For

immunocytochemical analysis of caspase 3, control and XO-treated PAECs

were fixed with 2% paraformaldehyde in PBS (1 h at 4 oC) 4 hours after

treatment. Fixed cells were washed 3 times with 50 mM Tris-HCl (pH 7.4),

150mM NaCl, 0.1% Triton X-100 (TBS-T), blocked with 5% normal goat

serum in TBS-T and incubated with primary antibody against activated form

of caspase 3 (Cell Signaling Technology, Beverly, MA), diluted 1:120 in TBS-

T with 3% BSA, at 4 oC overnight. After this, cells were incubated with Texas

Red-labeled anti-rabbit IgG (Vector Laboratories, Burlingame, CA) diluted

1:100 in TBS-T with 3% BSA for 1 hour at room temperature. Slides were

then mounted with fluorescent mounting media (DAKO, Carpinteria, CA) and

observed with Olympus IX70 fluorescence microscope. Photomicrographs

were taken with a SPOT digital camera using SPOT 2 software (Diagnostic

Instruments, Sterling Heights, MI)

Morphological assessment of nuclear condensation: PAECs were

prepared for microscopy as described for caspase 3 immunocytochemistry,

except they were stained with Hoechst 33258 dye (Molecular Probes,

Eugene, OR) to demarcate nuclear morphology.

DNA fragmentation assay: Quantitative ELISA detecting mono- and

oligonucleosomes in the cytoplasmic fraction of cell lysates was used to

determine fragmentation of nuclear DNA during apoptosis caused by XO

11

treatment. Assay was performed with Cell Death Detection ELISA kit (Roche

Diagnostics, Germany) according to supplied protocol. Pulmonary artery

endothelial cells grown and treated in 24-well culture plates were washed

twice with PBS and 500 µ l of lysis buffer per well were added. After

incubation for 30 min at room temperature lysates were transferred to

microcentrifuge tubes and centrifuged at 15000 g for 10 min at 4 oC.

Supernatant was diluted 1:3 with lysis buffer and 20 µl were used for ELISA.

Reaction product abundance was determined by measuring the absorbance

at 405 nm (reference wavelength 490 nm) with a 7520 Microplate Reader

(Cambridge Technology, Cambridge, MA).

Statistical analysis: Data are expressed as the mean ± the standard

error of the mean. Depending on the experimental design, differences in

mean values were assessed using un-paired t-tests or one way analyses of

variance combined with Neumann-Kuels tests. P values less than 0.05 were

taken as evidence of statistical significance.

12

RESULTS

To test the hypothesis that mtDNA damage is a proximate cause of

ROS-induced apoptosis, a construct encompassing the hOgg1 gene fused to

MTS from human MnSOD was inserted into an adenoviral vector (Figure 1,

top panel) and transfected into PAECs. The efficiency of transfection and

mitochondrial localization of the recombinant protein was determined by

Western analysis of mitochondrial fractions. Transient transfection of PAECs

with adenoviral vector encoding hOgg1-MTS construct at 1 and 2 moi was

associated with substantial increases in mitochondrial hOgg1 content (Figure

1, bottom panel) in comparison to cells transfected with empty vector. The

increase in mitochondrial hOgg1 was slightly greater at an moi of 2x relative

to 1x.

Initial experiments verified that XO + hypoxanthine damaged mtDNA

and that adenoviral-mediated over-expression of mitochondrially-targeted

hOgg1 led to a reduction in the XO-induced increase in mtDNA equilibrium

lesion density. The representative quantitative Southern blot displayed in

Figure 2 (upper panel) shows that XO, as expected, decreased hybridization

intensity of the mtDNA probe in a dose-related manner, thus indicating an

increase in equilibrium lesion density. Densitometric analysis of hybridization

intensity followed by calculation of the increase in lesions per 10 kb indicated

that the 1 h challenge with XO at doses of 2 and 5 mU/ml was accompanied

by increases of 0.4 and 1.3 lesions/10 kb, respectively (Figure 2, bottom

13

panel). Also as expected on the basis of previous observations (9, 10),

lesions in the nuclear VEGF gene could not be detected at these XO doses

using the quantitative Southern blot approach (data not shown). Finally, and

also as predicted on the basis of earlier work, mitochondrial over-expression

of hOgg1 was accompanied by a dose-related suppression of XO-induced

mtDNA damage at both XO concentrations tested (Figure 2).

Having verified that XO-induced mtDNA damage is suppressed by

over-expression of mitochondrially-targeted hOgg1, we next addressed the

question of whether protection of mtDNA was linked to preservation of

mitochondrial function. To address mitochondrial functional activity, we used

fluorescence of the mitochondrial membrane potential-dependent dye, JC-1.

After loading with JC-1, PAECs were treated with XO as described in Materials

and Methods and analyzed 1 h thereafter and again 4 h after treatment.

There were no differences in mitochondrial membrane potential compared to

control in any samples analyzed at the 1 h time-point (data not shown).

However, as seen in the photomicrographs and in the pooled data presented

in Figure 3, a marked diminution in JC-1 fluorescence was evident 4 h after

XO challenge of vector-transfected PAECs in comparison to non-treated cells,

thus indicating XO-mediated dissipation of mitochondrial membrane potential

(Figure 3A, C, and E). While transfection with mitochondrially-targeted

hOgg1 failed to alter JC-1 fluorescence in control cells, hOgg1 over-

14

expression prevented the dissipation in JC-1 fluorescence caused by XO

(Figure 3B, D and E).

We next determined if the observed protective actions of hOgg1 over-

expression on mtDNA integrity and mitochondrial function were associated

with suppression of XO-induced apoptosis in PAECs. To assess apoptosis, we

evaluated the extent of DNA fragmentation, the presence of activated

caspase 3, and the appearance of condensed, fragmented, Hoescht-stained

nuclei. Initial experiments to delineate the time-course of DNA

fragmentation indicated the appearance of oligonucleosomes beginning at 3

hours with a peak at 4-6 hours after the XO treatment (data not shown). All

the following apoptosis-detection assays were therefore performed 4h after

the cells were challenge with XO 1h. The Western blot analyses shown in the

top panel of Figure 4 indicate that while XO treatment at doses of 2 and 5

mU/ml increased the extent of active caspase 3, this effect was blunted in

hOgg1 over-expressers relative to cells transfected with empty vector. These

results were confirmed by immunocytochemistry with antibody specific to

activated caspase 3 (Figure 4, bottom panel). We wondered whether the

residual caspase 3 activation occurring in the presence of hOgg1 over-

expression at the higher dose of XO (5 mU/ml) was related to oxidant-

mediated reduction of mitochondrial hOgg1 or to the possibility that the ROS

produced in response to this high XO dose overwhelmed the protection

afforded by hOgg1. Mitochondrial hOgg1 was therefore measured by Western

15

blot analyses at the two levels of transfection in control and XO-treated

PAECs and found not to differe between the two treatment groups (Data not

shown). Thus, the residual caspase 3 activation associated with the 5 mU/ml

XO can most likely be attributed to hOgg1 activity that is insufficient to afford

complete protection against the high ROS production.

Another index of apoptosis, nuclear condensation, was detected by

staining with Hoechst 33258 dye. As seen in the photomicrographs and

pooled data presented in Figure 5, while XO-treated cells displayed numerous

condensed, fragmented nuclei (Figure 5C and E), over-expression of

mitochondrially-targeted hOgg1 protein almost completely eliminated this

morphological change (Figure 5D and E). Finally, the outcome of

experiments using the ELISA for DNA fragmentation were consistent with the

effects of hOgg1 over-expression on XO-induced caspase 3 activation and

formation of apoptotic nuclei. As shown in Figure 6, PAECs transfected with

adenoviral vector encoding hOgg1-MTS construct exhibited a significant

reduction of DNA fragmentation (~40%) after treatment with XO at a dose of

5 mU/ml in comparison to cells transfected with empty vector. The ELISA

assay for oligonucleosome formation failed to detect apoptotic cell death at

an XO dose of 2 mU/ml nor an effect of vector- or hOgg1-transfection alone.

Although the disparity between the presence of oligonucleosome formation

and caspase 3 activation at the 2 mU/ml XO dose is difficult to explain, it

seems reasonable to suspect that it is related to differing time courses of

16

DNA fragmentation and caspase 3 activation and/or different sensitivities of

the assays employed.

17

DISCUSSION

Apoptosis in endothelial cells derived from both systemic and

pulmonary vascular beds as well as other, non-endothelial cell types is an

important mechanism of cell death induced by oxidative stress. It is also

widely appreciated that mitochondria play a central role in the apoptotic

process. A key feature within the apoptosis cascades is dissipation of

mitochondrial membrane potential, increased mitochondrial oxidant

production and apoptogenic protein release, caused by opening of the

permeability transition pore (13). The processes downstream from the

release of proteins from mitochondria, such as apoptosis-inducing factor,

cytochrome c, Smac/DIABLO etc., are also reasonably well established.

However, the intra-mitochondrial events initiated by oxidant challenge that

activate the apoptotic cascade are not yet clarified.

The objective of present study was to test the hypothesis that XO-

induced damage to mtDNA was a proximate trigger for mitochondrial

dysfunction and apoptosis in cultured pulmonary artery endothelial cells. To

test this hypothesis, PAECs were transfected either with empty adenoviral

vector or with vector encoding hOgg1 linked to an MTS. 8-Oxoguanine-DNA

glycosylase is the principle enzyme for repair of 7,8-dihydro-8-oxoguanine

(8-oxoG), one of the most important mutagenic lesion in DNA induced by

reactive oxygen species (7). In the present study, mitochondrial targeting of

hOgg1 was verified by Western analysis. Our previous report showed that

18

transfection with an identical construct using a similar strategy was

accompanied by a prominent increase in Ogg-like activity in the

mitochondrial fraction in comparison to nucleus or cytosol (5). Cells were

challenged with ascending doses of XO plus hypoxanthine and the association

between mtDNA damage and oxidatively-induced apoptosis was examined.

As expected on the basis of our previous studies (5, 6), hOgg1 over-

expression was accompanied by protection of mtDNA against XO-mediated

damage. It should be emphasized that at the doses of XO employed, we

have never detected oxidative damage to the nuclear genome using either

quantitative Southern analyses (10) or ligation-mediated polymerase chain

reaction methods (9, 10), findings consistent with the high sensitivity of

mtDNA to oxidative damage in comparison to nuclear gene (2, 11, 14, 17).

Thus, the impact of hOgg1 on mitochondrial function and cell survival can be

ascribed to protection of mtDNA and not to an unsuspected effect on oxidant-

mediated effects elsewhere in the cell. Protection of mtDNA from XO-

mediated damage was associated with preservation of mitochondrial function

assessed in terms of mitochondrial membrane potential. This preservation of

mitochondrial membrane potential could further enhance the protective

actions of hOgg1 over-expression against mtDNA damage since, as pointed

out by Santos and co-workers (14), mitochondrial membrane potential is

critical driving force for mitochondrial protein import, including DNA repair

enzymes. Most importantly, mitochondrial over-expression of hOgg1

19

protected against XO-induced apoptosis, as evidenced by blunted activation

of caspase 3, accumulation of oligonucleosomes, and formation of

condensed, fragmented nuclei. Because of the specificity of hOgg1 as a DNA

repair enzyme, its targeting to mitochondria, and the attendant protection of

mtDNA from oxidant-mediated damage, these findings support the

conclusion that mtDNA damage is a proximate cause of mitochondrial

dysfunction and that mtDNA damage is linked specifically to induction of

apoptosis in XO-challenged PAECs. These findings, in addition, corroborate

observations by Shukla et al. in asbestos-treated, hOgg1 over-expressing

HeLa cells (15).

Results of this study indicate that the extent of mtDNA damage in

response to XO dictates whether PAECs survive or activate an apoptotic

death pathway. One of questions associated with this conclusion pertains to

the mechanism by which mtDNA damage initiates the apoptotic cascade.

Previous reports have suggested that mtDNA damage activates a “feed

forward” process wherein mtDNA damage initiates persistent oxidant

production that, in cells surviving the oxidant insult, is terminated by mtDNA

repair (14). Non-surviving cells exhibit decreased mitochondrial membrane

potential and persistent mtDNA damage. Additional studies will be required

to determine whether such a feed forward system, terminated by mtDNA

repair, is involved with the PAEC response to oxidant stress.

20

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FIGURE LEGENDS

FIGURE 1: Integration of the hOgg1-MTS construct into pulmonary artery

endothelial cells. TOP PANEL - hOgg1-MTS construct transfected into PAECs

using an adenoviral vector Ad.979. The construct was prepared with human

SOD mitochondrial targeting sequence and human Ogg-1c gene. BOTTOM

PANEL - Representative Western blot analysis of hOgg1 protein in

mitochondrial fractions from non-transfected PAECs (CON), PAECs

transfected with empty vector at a multiplicity of infection (moi) of 2x (V)-

and with the hOgg1-MTS construct (Ogg) at moi’s of 1x and 2x.

FIGURE 2: Quantitative Southern blot analysis of alkali-detectable mtDNA

damage in pulmonary artery endothelial cells. TOP PANEL - Representative

autoradiograms of Southern blot of mtDNA from vector (V)- and hOgg1-MTS

(Ogg)-transfected cells without treatment or after 1 hour exposure to XO (2

or 5 mU/ml). BOTTOM PANEL - Calculated changes in equilibrium lesion

density, normalized to 10 kb. Bars reflect the mean ± standard error of 4

determinations. Note that transfection with hOgg1 significantly (*P<0.05)

suppressed mtDNA damage caused by XO in comparison to vector-

transfected cells.

24

FIGURE 3: Mitochondrial membrane potential, assessed in terms of JC-

flouescence, in pulmonary artery endothelial cells 4 hours after a 1h XO

treatment. Photomicrographs of vector- (A, C) and hOgg1-MTS- (B, D)

transfected PAECs (moi: 2x) taken 4h after culture for 1h in the absence (A,

B) or presence of 5 mU/ml of XO (C, D). Cells were loaded with JC-1 (1 µM)

during 30 min prior to XO treatment. Note the mitochondria depolarization in

vector-transfected cells after treatment. E. Pooled data displaying mean ±

S.E. JC-1 flourescence intensity for vector-transfected, control cells (CON),

cells treated with 5 mu/ml XO, transfected with hOgg1-MTS (Ogg), or

challenged with XO in the presence of hOgg1 over-expression (XO + Ogg).

*Significantly decreased in comparison to all other groups (N=4).

FIGURE 4: TOP PANEL – Representative Western blot analysis of vector (V)-

and hOgg1-MTS (Ogg)-transfected cells without treatment or treated with XO

(2 or 5 mU/ml) probed with antibody to activated caspase 3. Cells were

harvested 4 hours after 1h treatment with XO-treatment. BOTTOM PANEL -

Immunocytochemistry of vector- (Vector) and hOgg1-MTS- (Ogg) transfected

PAECs (moi: 2x) cultured for 1 hour in the absence or presence of 5 mU/ml

of XO. Antibodies against the active form of caspase 3 were applied 4h after

XO teatment. Note increased abundance of activated caspase 3 in pulmonary

artery endothelial cells 4 hours after XO treatment but not in cells over-

expressing mitochondrially-targeted hOgg1.

25

FIGURE 5: Nuclear condensation in pulmonary artery endothelial cells after

XO treatment. Photomicrographs of vector- (A, C) and hOgg1-MTS- (B, D)

transfected PAECs (moi: 2x) cultured for 1 hour in the absence (A, B) or

presence of 5 mU/ml of XO (C, D) stained with Hoechst 33258. Cells were

harvested 4 hours after beginning of XO-treatment. Note the appearance of

apoptotic nuclei in vector-transfected cells after treatment (arrows). E.

Pooled data displaying mean ± S.E. of the percentage of condensed,

fragmented nuclei for vector-transfected, control cells (CON), cells treated

with 5 mU/ml XO, transfected with hOgg1-MTS (Ogg), or challenged with XO

in the presence of hOgg1 over-expression (XO + Ogg). *Significantly

increased in comparison to CON. **Significantly increased in comparison to

CON but decreased in comparison to XO (N=4-6).

FIGURE 6: DNA fragmentation in pulmonary artery endothelial cells 4 hours

after XO treatment. Reduced results of quantitative ELISA assay detecting

mono- and oligonucleosomes in the cytoplasmic fractions of vector (V)- and

hOgg1-MTS (Ogg)-transfected cells without treatment or treated with XO (2

or 5 mU/ml). Bars reflect the mean ± S.E. *Significantly increased in

comparison to cells not exposed to XO. **Significantly reduced in comparison

to vector-transfected cells treated with 5 mU/ml XO (N=6).

26

FIGURE 1

pCMV MTS hOGG 1c eGFPIRES

CON V Ogg-1x Ogg-2x

27

*

*

*

*

FIGURE 2

No treatment XO - 2mU XO - 5mU

mtDNA

V Ogg-1x Ogg-2x V Ogg-1x Ogg-2x V Ogg-1x Ogg-2x

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

2.5Vector

Ogg-1x

Ogg-2x

No treatment XO-2mU XO-5mU

28

FIGURE 3

E.

*

Con XO Ogg XO + Ogg0

25

50

75

100

125

Control

XO(5 mU/ml)

Vector Ogg

29

FIGURE 4

Ogg + XOOgg Vector + XO

Vector

Vector

Control XO (2 mU/ml) XO (5 mU/ml)

V Ogg-1x 2xV Ogg-1x 2xV Ogg-1x 2x

30

FIGURE 5

E.

*

**

Con XO Ogg XO + Ogg0

10

20

30

Control

XO(5 mU/ml)

Vector Ogg

31

FIGURE 6

*

**

**

0.0 2.0 5.00

1

2Vector - 2xOgg - 1xOgg - 2x

XO (mU/ml)