SPECIAL COMMUNICATION
Peroxynitrite causes endothelial cell monolayer barrier dysfunction

James L. Knepler Jr., Loui N. Taher, Mahesh P. Gupta, Carolyn Patterson, Fred Pavalko, Michael D. Ober, and C. Michael Hart

Departments of Medicine and Physiology, Richard L. Roudebush Veterans Affairs and Indiana University Medical Centers, Indianapolis, Indiana 46202


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (·NO) attenuates hydrogen peroxide (H2O2)-mediated barrier dysfunction in cultured porcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD, Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am J Physiol Lung Cell Mol Physiol 280: L116-L126, 2001). However, ·NO rapidly combines with superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>) to form the powerful oxidant peroxynitrite (ONOO-), which we hypothesized would cause PAEC monolayer barrier dysfunction. To test this hypothesis, we treated PAEC with ONOO- (500 µM) or 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM). SIN-1-mediated ONOO- formation was confirmed by monitoring the oxidation of dihydrorhodamine 123 to rhodamine. Both ONOO- and SIN-1 increased albumin clearance (P < 0.05) in the absence of cytotoxicity and altered the architecture of the cytoskeletal proteins actin and beta -catenin as detected by immunofluorescent confocal imaging. ONOO--induced barrier dysfunction was partially reversible and was attenuated by cysteine. Both ONOO- and SIN-1 nitrated tyrosine residues, including those on beta -catenin and actin, and oxidized proteins in PAEC. The introduction of actin treated with ONOO- into PAEC monolayers via liposomes also resulted in barrier dysfunction. These results indicate that ONOO- directly alters endothelial cytoskeletal proteins, leading to barrier dysfunction.

nitrotyrosine; actin; catenin


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASCULAR ENDOTHELIUM provides a barrier that prevents the unregulated passage of fluids and macromolecules across the vessel wall. Reactive oxygen species (ROS), as well as nonoxidant inflammatory mediators, disrupt the endothelial barrier by activating intracellular signaling pathways that stimulate altered endothelial cell (EC) cytoskeletal architecture and the formation of paracellular gaps in the monolayer (14). Endothelial barrier dysfunction in the lung is generally believed to contribute to noncardiogenic edema and to abnormalities in gas exchange and lung compliance in acute respiratory distress syndrome (ARDS). Inhaled nitric oxide (·NO) has been investigated as a therapeutic modality in ARDS because of its ability to promote the selective vasodilatation of pulmonary vessels in ventilated regions of the lung. To date, however, clinical studies have demonstrated that ·NO-induced improvements in gas exchange are short-lived (10, 38). Additional studies, however, indicate that ·NO attenuates oxidant-induced endothelial barrier dysfunction in diverse experimental models (13, 18, 20, 27, 36, 37, 41) and in patients with ARDS (4). Together, these studies suggest that ·NO, delivered at the appropriate time and concentration, exerts a barrier protective effect on vascular endothelium during oxidative stress.

On the other hand, inflammatory mediators that disrupt the endothelial barrier stimulate the production of superoxide (O<UP><SUB>2</SUB><SUP>−</SUP></UP>) (24) as well as ·NO (7). O<UP><SUB>2</SUB><SUP>−</SUP></UP> combines with ·NO to form the potent oxidant peroxynitrite (ONOO-) (2). For example, both bradykinin and the calcium ionophore A-23187 stimulated ONOO- production via O<UP><SUB>2</SUB><SUP>−</SUP></UP> and ·NO production in cultured bovine aortic endothelial cells (28). Additional evidence supports a role for ONOO- in the pathogenesis of acute lung injury and ARDS. Nitrotyrosine residues were detected in lung tissue from patients with ARDS (22, 29). Furthermore, bronchoalveolar lavage fluid and plasma from ARDS patients contain elevated levels of nitrated and oxidized proteins (15, 31). ONOO- infusion into a rabbit lung increased hydrostatic pressure, capillary permeability, and lung weight (1) consistent with a pathogenetic role for ONOO- in lung injury.

ONOO- could contribute to endothelial barrier dysfunction during lung injury by several potential mechanisms. The diffusion of ONOO- across cell membranes (33) indicates that ONOO- generated outside the EC compartment as well as that generated within ECs could directly interact with intracellular targets. ONOO- reacts with numerous targets including protein tyrosine residues to form nitrotyrosine (25). Nitration of target proteins could thereby modulate the structure or function of molecules, leading to cell dysfunction. ONOO--induced oxidant stress could also disrupt actin polymerization (8) or activate upstream signaling cascades, e.g., tyrosine kinases (45), that result in the modification of cytoskeletal proteins, leading to barrier dysfunction. ONOO--induced accumulation of cGMP in EC could regulate vascular EC shape and function (35), whereas activation of poly(ADP-ribose) synthetase (PARS) by ONOO- can suppress mitochondrial respiration and cause endothelial dysfunction (44). These reports emphasize that ONOO- can potentially alter endothelial function through complex mechanisms. The current study extends these previous reports by directly examining the ability of ONOO- to modulate cytoskeletal architecture and monolayer barrier function in cultured pulmonary artery endothelial cells (PAEC).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PAEC isolation and culture. A well-characterized model using PAEC monolayers was employed to examine how ONOO- alters endothelial barrier function independent of its effects on vascular perfusion. EC were isolated from the main pulmonary artery of pigs as previously reported (23). Once confluent, PAEC were passaged by treatment with trypsin to polycarbonate filters or 24-well tissue culture plates. After cells reached secondary confluence, the concentration of fetal bovine serum was decreased from 10% to 4% (maintenance medium). In all experiments, PAEC were studied 3-6 days after confluence, with control and experimental dishes matched according to cell line, number of passages, and number of days postconfluence. Monolayers were identified as ECs by phase-contrast microscopy and periodically by immunofluorescent staining for factor VIII antigen (5).

ONOO- treatment protocols. PAEC were exposed to either ONOO- (Cayman Chemicals, Ann Arbor, MI) or 3-morpholinosydnonimine hydrochloride (SIN-1; Molecular Probes, Eugene, OR), a compound that simultaneously generates ·NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP>, which combine at near-diffusion limited rates to form ONOO- (3). Stock solutions of ONOO- (20 mM in 0.3 M NaOH) and SIN-1 [10 mM in Hanks' balanced salt solution (HBSS; GIBCO)] were prepared immediately before addition to the culture medium. PAEC monolayers were treated for 2 h with RPMI 1640 medium containing 4% fatty acid-free albumin (Sigma), 20 mM HEPES (Sigma), and ONOO- or SIN-1 (1-500 µM) at 37°C. In selected experiments, because of the short half-time of ONOO- in neutral solutions (30), PAEC were also treated with repeated doses of ONOO- (150 µM) every minute for 4-8 min. In other experiments, cysteine (5 mM for 1 h before treatment with SIN-1), a scavenger of ONOO- (6), was included during treatment with SIN-1. In all experiments, control monolayers were treated with culture medium to which an equivalent volume of NaOH or HBSS vehicle had been added.

Measurement of PAEC cytotoxicity. Each PAEC monolayer was treated with ONOO- or SIN-1 for 2 h. To determine the effect of ONOO- or SIN-1 on PAEC viability, we collected the medium from each monolayer. PAEC were then washed thoroughly with HBSS and collected by scraping with a rubber policeman into 10 mM Tris · HCl buffer, pH 7.4, containing 0.2% Triton X-100. PAEC injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH) as described previously (23). Duplicate aliquots (40 µl) of culture medium and cell lysates were placed in 96-well microtiter plates, and LDH activity was measured by monitoring the consumption of nicotinamide adenine dinucleotide (Sigma) at 340 nm with a spectrophotometric plate reader (17). Results are expressed as the percentage of total LDH activity released to the culture medium: %LDH release = [LDH activity in medium/(LDH activity in cells + medium)] × 100.

Measurement of SIN-1-generated reactive species. ONOO- generation by SIN-1 was confirmed by spectrophotometrically monitoring the oxidation of dihydrorhodamine 123 (DHR; Molecular Probes) to rhodamine at 500 nm (molar extinction coefficient = 78,000 M-1 · cm-1) (21). ONOO-, but neither O<UP><SUB>2</SUB><SUP>−</SUP></UP> nor ·NO, oxidizes DHR. DHR was prepared in dimethyl sulfoxide (DMSO; 10 mM) as a stock solution that was added to culture medium to generate a final DHR concentration of 50 µM. The oxidation of DHR to rhodamine by SIN-1 (500 µM) was measured for 2 h at 37°C in a medium identical to that used in studies of barrier function consisting of RPMI 1640 plus 4% albumin and 20 mM HEPES, pH 7.4. The ·NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO; 100 µM), superoxide dismutase (SOD; 15 U/ml), or catalase (50 U/ml) was added to DHR- and SIN-1-containing samples as indicated.

The amount of ·NO generated by SIN-1 in the presence and absence of SOD was determined with a nitric oxide-specific sensor (ISO-NOP200; World Precision Instruments, Sarasota, FL). Calibration of the sensor was performed according to the specifications of the manufacturer. A calibration curve was generated daily by adding known S-nitroso-N-acetylpenicillamine (SNAP; Biomol, Plymouth Meeting, PA) concentrations in 0.5 mM EDTA, pH 9.0, to 0.1 M copper sulfate, pH 4.0. SNAP concentrations were converted to ·NO concentrations with the formula [SNAP] × 0.539 = [·NO] per the ISO-NOP200 manufacturer's instructions. The amount of ·NO generated by SIN-1 was measured at 37°C for 20 min by the addition of 500 µM SIN-1 to RPMI 1640 medium containing 4% BSA in the presence or absence of 15 U/ml SOD. In all experiments, baseline (control medium alone) ·NO concentration was monitored for at least 15 min before addition of SIN-1.

Measurement of PAEC barrier function. The ability of monolayers to prevent the transendothelial passage of albumin was measured as an index of PAEC function as previously reported (23, 39). In brief, PAEC monolayers on polycarbonate filters attached to plastic ring wells were floated in 45 ml of RPMI 1640 medium, forming upper and lower chambers, respectively. Evans blue dye bound to 4% bovine serum albumin in RPMI 1640 plus 20 mM HEPES buffer was gently placed inside the luminal (upper) well of each chamber. The abluminal (lower) medium was continuously stirred and was maintained at 37°C by a circulating water bath. Before experimental interventions took place, samples (0.3 ml) were taken from the abluminal medium at 10-min intervals for 1 h and placed into a 96-well microtiter plate. After baseline albumin clearance had been measured for 1 h, ONOO- or SIN-1 was added to the luminal medium, and albumin clearance was monitored for an additional 2 h. The absorbance of the samples was read at 620 nm with a spectrophotometric plate reader. The raw absorbance data were transferred to a spreadsheet program on an IBM personal computer for analysis. The net abluminal sample absorbance was used to calculate the equivalent theoretical volume of luminal medium cleared to the abluminal space (39). The clearance of albumin (expressed in µl/min) was calculated by linear regression analysis of changes in absorbance over 1-h intervals. To determine the recovery of PAEC barrier function, PAEC monolayers treated with 500 µM SIN-1 or HBSS vehicle alone in RPMI medium for 2 h were washed and placed back into maintenance medium for 24 h before albumin clearance was determined for 1 h.

In selected experiments, after baseline barrier function was determined for 1 h as described, PAEC monolayers were treated with 500 µM SIN-1 with or without 100 µM c-PTIO, 15 U/ml SOD, or the combination of c-PTIO plus SOD for 2 h at 37°C. PAEC barrier function was then measured for 2 h. To determine whether ONOO- directly modified albumin to stimulate its clearance across PAEC monolayers, albumin was treated with 500 µM ONOO- for 1 h, dialyzed with 24-kDa MWCO tubing, and added to luminal medium above monolayers. Albumin clearance was then measured for 2 h.

Detection of nitrotyrosine by immunoprecipitation. PAEC in 100-mm dishes were treated for 2 h with 500 µM SIN-1, 500 µM ONOO-, or RPMI 1640 medium with 4% albumin and 20 mM HEPES alone. The cells were then solubilized in lysis buffer [30 mM potassium phosphate, pH 7.4, 150 mM NaCl, 10% (vol/vol) glycerol, 1 µM phenylmethylsulfonyl fluoride,10 µg/ml aprotinin, and 1% Triton X-100] as previously reported (34). Equal amounts of PAEC lysates were precleared with Sepharose and exposed to monoclonal anti-nitrotyrosine antibodies (Cayman Chemicals) overnight. The lysates were then combined with Sepharose and centrifuged, and proteins were eluted with a saturated nitrotyrosine solution. The proteins were then resolved using SDS-PAGE (4-12%). Samples were run in parallel with albumin (0.2% in phosphate buffer, pH 7.4), nitrated albumin (0.2% albumin in phosphate buffer, pH 7.4, treated with 500 µM ONOO- and vortexed immediately), and molecular weight markers. The proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked with 1% albumin and 1% goat serum in phosphate-buffered saline with Tween 20, and probed with monoclonal anti-nitrotyrosine antibodies (1:1,000). The membrane was then treated with horseradish peroxidase-conjugated goat anti-mouse antibody (1:5,000; Jackson ImmunoResearch, West Grove, PA), and bands were detected using enhanced chemiluminescence (ECL; Pierce, Rockford, IL). In selected experiments, nitrotyrosine immunoprecipitates were subjected to SDS-PAGE, followed by slot blotting with antibodies to actin (1:250; courtesy of Dr. Fred Pavalko) or beta -catenin (1:250; Zymed, San Francisco, CA).

Detection of oxidized proteins. After treatment with control, SIN-1, SIN-1 plus SOD (15 U/ml), or ONOO-, PAEC lysates were prepared as described in Detection of nitrotyrosine by immunoprecipitation, and oxidized proteins were detected with a commercially available kit (Oxyblot; Oncor). Briefly, oxidant-induced carbonyl side chains were derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by 2,4-dinitrophenylhydrazine (DNPH). The samples were then resolved with SDS-PAGE as described above. DNP residues were then detected with the use of polyclonal rabbit anti-DNP antibodies, followed by treatment with secondary antibodies and ECL as described above.

Effects of ONOO- on PAEC cytoskeletal architecture. PAEC were seeded onto gelatinized glass coverslips in 35-mm dishes and grown to confluence. Coverslips were then incubated in RPMI 1640 medium containing 4% bovine serum albumin and 20 mM HEPES with vehicle, SIN-1, or ONOO- for 1-2 h at 37°C. After fixation with 5% paraformaldehyde in wash buffer (150 mM NaCl, 0.1% Na-azide, and 50 mM Tris · HCl, pH 7.6) for 10 min at room temperature, the coverslips were rinsed and then permeabilized for 3.5 min with 0.2% Triton X-100 in wash buffer. After coverslips were rinsed and blocked with 1% BSA in buffer for 1 h, they were treated with monoclonal anti-beta -catenin antibody (1:50 in 1% BSA/buffer; Transduction Laboratories, Lexington, KY) overnight at 4°C. After they were thoroughly washed, the coverslips were treated with FITC-labeled donkey anti-mouse IgG (1:50; Jackson ImmunoResearch) and rhodamine phalloidin (1:200; Molecular Probes) in 1% BSA/buffer. The coverslips were then rinsed and mounted on slides with SlowFade/glycerol (Molecular Probes). Fluorescence was observed by confocal microscopy (1024 System; Bio-Rad, Hercules, CA) with a ×60 oil objective by using a krypton-argon laser at 10% laser power with an iris aperture of 3 mm. Rhodamine and FITC fluorescence were each recorded for 10-17 sections at 0.5-mm intervals by using identical contrast and gain settings. The data were processed using MetaMorph (Universal Imaging, West Chester, PA). A minimum of six images from each treatment group was analyzed. Representative images were imported to PowerPoint for printing by Kodak dye sublimation (Rochester, NY).

Effect of ONOO--treated actin on PAEC barrier function. In an attempt to determine the direct effects of ONOO- on actin and PAEC barrier function, nonmuscle actin from human platelets (Cytoskeleton, Denver, CO) was treated with 500 µM ONOO- for 1 h. Treated actin was then dialyzed against four exchanges of 5 mM Tris · HCl, pH 8.0, 0.2 mM CaCl2, 0.2 mM ATP, and 0.5 mM dithiothreitol. Dialyzed ONOO--treated or control actin (1 mg/ml) was incubated for 20 min with an equal volume (5 µl) of Lipofectamine (GIBCO BRL, Gaithersburg, MD) and then added to the luminal medium above PAEC monolayers, and albumin clearance was measured as described in Measurement of PAEC barrier function.

Statistical analysis. In all experiments data were analyzed with analysis of variance or repeated-measures analysis of variance to determine the significance of treatment effects, followed by Bonferroni or Student-Newman-Keuls analysis to examine differences between individual treatment groups. The level of statistical significance was taken as P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SIN-1-induced PAEC cytotoxicity. Treatment with 10-500 µM ONOO- or 10-500 µM SIN-1 for 2 h in RPMI 1640 plus 4% albumin and 20 mM HEPES did not cause PAEC cytotoxicity assessed as %LDH release (Table 1). Phase-contrast microscopy confirmed no cytolytic alterations in PAEC morphology in ONOO-- or SIN-1-treated cells (not shown).

                              
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Table 1.   Lack of cytotoxicity in SIN-1- and ONOO--treated cells

Effect of ONOO- and SIN-1 on PAEC barrier function. After baseline albumin clearance was measured for 1 h, PAEC were treated with 1-500 µM SIN-1 (Fig. 1A) or bolus addition of ONOO- (Fig. 1B). As previously reported (23, 26), confluent PAEC present a substantial barrier to the transmonolayer clearance of albumin that is stable during the 3-h measurement period (Fig. 1B). SIN-1 failed to cause barrier dysfunction initially but caused significant barrier dysfunction during the second hour following its addition that demonstrated no significant concentration dependence between 1 and 500 µM. Bolus addition of ONOO- caused more extensive barrier dysfunction but, like SIN-1, had minimal effect during the first hour of treatment (Fig. 1B). Repeated dosing with a lower ONOO- concentration (150 µM) each minute for 4-8 min also caused significant barrier dysfunction after 2 h (Fig. 1B). In monolayers treated with 500 µM ONOO- for 2 h, followed by washing and incubation in maintenance medium for 24 h (Fig. 2, recovery), barrier dysfunction was only partially reversible. Although cysteine alone increased baseline albumin clearance, it significantly inhibited SIN-1-mediated barrier dysfunction (Fig. 2).


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Fig. 1.   Effect of peroxynitrite (ONOO-) on pulmonary artery endothelial cell (PAEC) barrier function. PAEC barrier function was measured for 1 h before experimental interventions were introduced (baseline) as the transmonolayer albumin clearance (as described in METHODS). Next, 3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM) (A) or ONOO- (B) was added, as either a bolus (500 µM) or repeated doses (150 µM) each min for 4 or 8 min, to RPMI 1640 medium containing 4% albumin and 20 mM HEPES above PAEC monolayers. Albumin clearance was then measured for an additional 2 h (2nd and 3rd h). Values are means ± SE and represent the average albumin clearance from 3-6 experiments. *P < 0.05 vs. baseline.



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Fig. 2.   ONOO--mediated PAEC barrier dysfunction reversibility and inhibition by cysteine. PAEC barrier function was measured for 2 h after treatment with either 500 µM ONOO- or vehicle (0.3 M NaOH). Selected PAEC monolayers were then washed and incubated in maintenance medium for 24 h before barrier function was measured for 1 h (recovery). Other monolayers were pretreated with cysteine (5 mM) or vehicle before measurement of baseline barrier function and treatment with SIN-1 (as described in METHODS). Values are means ± SE and represent the average albumin clearance from 3-6 experiments. *P < 0.05 vs. control; dagger P < 0.05 vs. ONOO-; Dagger P < 0.05 vs. SIN-1.

In preliminary experiments, the ability of SIN-1 or ONOO- to alter Evans blue dye-albumin binding was examined. Evans blue dye bound to 4% albumin in RPMI 1640 medium containing 20 mM HEPES was treated with vehicle, SIN-1 (500 µM), or ONOO- (500 µM) for 2 h. These albumin preparations were then placed inside dialysis tubing (24-kDa MWCO) and dialyzed against RPMI 1640 medium. Although Evans blue dye alone readily diffused through the dialysis membrane, no Evans blue dye was released from albumin treated with vehicle, SIN-1, or ONOO- (not shown). Furthermore, albumin that was treated with ONOO- in the absence of PAEC, dialyzed, and then added to the luminal medium did not increase albumin clearance at 2 h compared with untreated albumin (albumin, 0.044 ± 0.001 µl/min; ONOO--treated albumin, 0.045 ± 0.003 µl/min).

Characterization of reactive species generated by SIN-1. To examine the kinetics of ONOO- generation, SIN-1-mediated DHR oxidation was measured under conditions identical to those employed in Fig. 1A. SIN-1-mediated DHR oxidation was both dose (1-500 µM) and time (15-120 min) dependent and was fully inhibited by the presence of 5 mM cysteine (not shown). As shown in Fig. 3A, SIN-1 caused little DHR oxidation during the first hour of treatment, similar to the time course of SIN-1-induced barrier dysfunction (Fig. 1). SIN-1-mediated DHR oxidation was partially inhibited by SOD (15 U/ml) or c-PTIO (100 µM) and more fully inhibited by the combination of c-PTIO and SOD. In contrast, catalase (50 U/ml) had no significant effect on SIN-1-mediated DHR oxidation, indicating that H2O2 was not the oxidizing agent (Fig. 3A). In the absence of SIN-1, PAEC caused low levels of DHR oxidation that changed little during the 2-h treatment interval studied. Figure 3B shows that under the conditions employed for measurements of barrier function, in the absence of SOD, SIN-1-mediated ·NO generation was nearly undetectable, consistent with its rapid reaction with O<UP><SUB>2</SUB><SUP>−</SUP></UP> to form ONOO-. In the presence of SOD, however, ·NO production by SIN-1 became detectable, due to SOD-mediated conversion of O<UP><SUB>2</SUB><SUP>−</SUP></UP> to H2O2. These findings are consistent with SOD-mediated reductions in SIN-1-induced ONOO- generation (Fig. 3A).


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Fig. 3.   Characterization of reactive species generated by SIN-1. The oxidation of dihydroxyrhodamine 123 (DHR; 50 µM) to rhodamine by SIN-1 (500 µM) was monitored at 30-min intervals for 2 h at 37°C in RPMI 1640 medium containing 4% albumin and 20 mM HEPES, pH 7.4, in the presence of PAEC (A). After DHR was added, the ·NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (c-PTIO; 100 µM), superoxide dismutase (SOD; 15 U/ml), catalase (Cat; 50 U/ml), or combinations of these reagents were added as indicated. Values are means ± SE and represent the mean absorbance from 3 determinations. At points where there are no error bars, the SE is too small to be graphically represented. *P < 0.05 vs. control; dagger P < 0.05 vs. SIN-1. To further determine whether SOD altered the generation of reactive species by SIN-1, ·NO concentrations were monitored in RPMI 1640 medium containing 4% albumin plus 20 mM HEPES in the presence or absence of 15 U/ml SOD just before and for 25 min after the addition of SIN-1 (500 µM) as described in METHODS. A representative tracing from 1 of 3 experiments is shown (B).

Effect of c-PTIO and SOD on SIN-1-mediated PAEC barrier dysfunction. Because SOD and c-PTIO attenuated SIN-1-mediated ONOO- generation and DHR oxidation, the ability of these interventions to modulate SIN-1-mediated PAEC barrier dysfunction was examined. After baseline albumin clearance was measured for 1 h, PAEC monolayers on polycarbonate filters were exposed to 500 µM SIN-1 for 2 h in RPMI 1640 plus 4% albumin and 20 mM HEPES, in the presence or absence of c-PTIO (100 µM), SOD (15 U/ml), or both c-PTIO and SOD. Treatment with SIN-1 for 2 h caused significant barrier dysfunction (Fig. 4), consistent with the findings reported in Fig. 1. SIN-1-mediated barrier dysfunction was not attenuated by either c-PTIO or SOD individually and was significantly exacerbated by the combination of c-PTIO and SOD (Fig. 4). We postulated that exacerbation of SIN-1-mediated barrier dysfunction was caused by SOD-induced generation of H2O2 combined with c-PTIO-induced removal of barrier protective effects of ·NO (20). Therefore, in separate studies, PAEC monolayers were treated with SIN-1 alone, the combination of SIN-1, c-PTIO, and SOD (as described above), or SIN-1, c-PTIO, and SOD plus catalase (50 U/ml). In these studies (n = 3), barrier dysfunction 2 h after the addition of SIN-1, c-PTIO, and SOD was 199.2% of the baseline value, whereas the addition of catalase reduced barrier dysfunction to 144.2% of the baseline value.


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Fig. 4.   Effect of SOD and/or c-PTIO on SIN-1-mediated PAEC barrier dysfunction. Albumin clearance was measured for 1 h as an index of PAEC barrier function (baseline). SIN-1 (500 µM) alone or in combination with 100 µM c-PTIO (SIN-1/c-PTIO), 15 U/ml SOD (SIN-1/SOD), or c-PTIO plus SOD (SIN-1/c-PTIO/SOD) was then added to the luminal medium, and the transmonolayer clearance of albumin was measured for an additional 2 h (2nd and 3rd h). Values are means ± SE and represent the mean albumin clearance from 6 experiments. *P < 0.05 vs. 1st h; dagger P < 0.05 vs. SIN-1 3rd h and SIN/SOD 3rd h.

Detection of ONOO--induced protein modifications. PAEC were treated with 500 µM ONOO- or SIN-1 for 2 h and then collected as described in METHODS. Lysates were immunoprecipitated with monoclonal anti-nitrotyrosine antibodies, and equal amounts of protein were then resolved with Western blotting and probed with the same anti-nitrotyrosine antibody. Compared with PAEC treated under control conditions, treatment with either SIN-1 or ONOO- increased the number of proteins with nitrotyrosine epitopes (molecular mass ranging from ~20 to 125 kDa) (Fig. 5A). In control cells, nitrotyrosine epitopes were detected in proteins with molecular masses of ~41 and 125 kDa with faint bands at ~36 and 200 kDa (Fig. 5A, arrowheads). In ONOO-- and SIN-1-treated PAEC, roughly eight additional proteins displayed evidence of tyrosine nitration (Fig. 5A, arrows). The anti-nitrotyrosine immunoprecipitates were also probed with antibodies to actin and beta -catenin. These studies (Fig. 5B) show that proteins with molecular masses of ~40 and 84 kDa react with both anti-nitrotyrosine and actin or beta -catenin antibodies, respectively. Whole cell lysates prepared from PAEC treated with control, SIN-1, SIN-1 plus SOD, or ONOO- for 2 h were also examined for evidence of protein oxidation. Compared with control conditions in which oxidized proteins were detected with molecular masses of ~42, 84, and 209 kDa (Fig. 6, arrowheads), treatment with SIN-1, ONOO-, or SIN-1 plus SOD increased the oxidation of PAEC proteins as evidenced by the detection of more DNPH epitopes in proteins with molecular masses of ~21, 30, 33, 53, and 130 kDa, along with increased intensity of staining in the 42- and 209-kDa bands (Fig. 6, arrows). The presence of SOD had little effect on SIN-1-induced PAEC protein oxidation, suggesting that the observed oxidation was mediated by reactive species other than O<UP><SUB>2</SUB><SUP>−</SUP></UP> . Because this concentration of SOD failed to completely prevent ONOO- generation, the current studies cannot discriminate whether SIN-1-mediated protein oxidation was caused by ONOO-, H2O2, other reactive compounds, or combinations of these species. The comparable pattern of protein oxidation detected in SIN-1, ONOO-, and SIN-1 plus SOD suggests that ONOO- may be responsible.


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Fig. 5.   ONOO--induced nitrotyrosine formation. After treatment under control conditions or with SIN-1 (500 µM), or ONOO- (500 µM) for 2 h, PAEC were solubilized in lysis buffer, and proteins containing nitrotyrosine epitopes were immunoprecipitated as described in METHODS. Immunoprecipitated proteins were then resolved using SDS-PAGE (4-12%). The proteins were transferred to polyvinylidene difluoride membranes and probed with monoclonal anti-nitrotyrosine antibodies (A) or antibodies to actin or beta -catenin (B). The membranes were then treated with horseradish peroxidase-conjugated goat anti-mouse antibody (1:5,000), and bands were detected using enhanced chemiluminescence. Results are representative of 3 separate experiments. Arrowheads indicate nitrotyrosine epitopes in control PAEC, and arrows indicate additional nitrotyrosine epitopes in SIN-1- or ONOO--treated PAEC.



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Fig. 6.   ONOO--induced protein oxidation. After treatment under control conditions or with SIN-1 (500 µM), ONOO- (500 µM), or SIN-1 (500 µM) plus SOD (15 U/ml) for 2 h, PAEC were solubilized in lysis buffer, treated with 2,4-dinitrophenylhydrazine, and resolved using SDS-PAGE. Oxidized proteins were detected with a commercially available kit as described in METHODS. Results are representative of 3 separate experiments. Arrowheads indicate oxidized epitopes in control PAEC, and arrows indicate additional oxidized epitopes in SIN-1- or ONOO--treated PAEC.

Effect of ONOO- on PAEC cytoskeletal architecture. The cytoskeletal architecture of vascular ECs plays an important role in the regulation of EC shape and barrier function. To examine the effects of ONOO- on PAEC cytoskeletal organization, PAEC were stained with rhodamine phalloidin to examine the structure of filamentous (F)-actin and with beta -catenin antibodies to examine the distribution of this protein, which links the actin cytoskeleton to the adherens junction complex (19). Identical, representative fields obtained by confocal immunofluorescence microscopy are shown for F-actin (Fig. 7) and beta -catenin (Fig. 8). The F-actin staining of control PAEC shows the typical, regular polygonal arrangement of cultured EC with a fine weblike network of cellular actin (Fig. 7A). The F-actin is most prominent in dense peripheral bands that define the cell borders (Fig. 7A, arrow). Treatment with ONOO- increased F-actin content and caused reorganization into more parallel arrays of actin fibers, typical of a contractile state (Fig. 7B, arrowhead). Similar F-actin reorganization was observed 1 h after treatment with 100 µM SIN-1 (Fig. 7C). Two hours after the addition of 100 µM SIN-1, partial dissolution of the thick parallel actin filaments and diminution of total actin staining were observed, consistent with ongoing actin reorganization (Fig. 7D). Comparable but more accelerated alterations in actin fibers were seen after treatment with higher concentrations of SIN-1. For instance, treatment with 500 µM SIN-1 caused total dissolution of the dense peripheral bands and apparent reorganization of actin into parallel fibers within 1 h (Fig. 7E), comparable to the alterations observed after treatment with 100 µM SIN-1 for 2 h. Compared with the 100 µM concentration, 500 µM SIN-1 also caused a more rapid and extensive progression of actin depolymerization, manifest as loss of rhodamine staining intensity after treatment for 1 and 2 h (Fig. 7F). In addition, the higher dose of SIN-1 caused a dramatic change in cell shape to a contractile, spindle shape (Fig. 7F, open arrow). Thus ONOO- caused activation of PAEC to a contractile state, followed by dissolution of total filamentous polymerized actin that was roughly proportional to the dose and duration of ONOO- to which the cells were exposed.


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Fig. 7.   ONOO--induced actin reorganization. Confluent PAEC grown on glass coverslips were treated with vehicle (0.3 NaOH) for 2 h (A), ONOO- (500 µM) for 2 h (B), SIN-1 (100 µM) for 1 h (C), SIN-1 (100 µM) for 2 h (D), SIN-1 (500 µM) for 1 h (E), or SIN-1 (500 µM) for 2 h (F) and then fixed, stained for F-actin, and examined using the confocal fluorescent microscopy techniques described in METHODS. Representative fields from each group are shown (magnification, ×60). Control PAEC (A) are characterized by a band of actin surrounding the periphery of the cell (arrow). Exposure of the cells to ONOO- (B) resulted in increased F-actin content and partial reorganization into parallel fibers (arrowhead). Similar increases in F-actin and reorganization were observed in PAEC treated with 100 µM SIN-1 for 1 h (C). Further SIN-1 exposure (D) resulted in some dissolution of the thick parallel actin filaments, diminution of total actin staining, and paracellular gap formation (double arrows). Higher doses of SIN-1 resulted in total dissolution of the dense peripheral bands, organization of actin into parallel fibers, and loss in total F-actin within 1 h (E). The loss of F-actin was progressive over 2 h (F) and was associated with a change in cell shape to a contractile spindle shape (open arrow).



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Fig. 8.   beta -Catenin distribution in PAEC treated with ONOO- or SIN-1. Confluent PAEC grown on glass coverslips were treated with vehicle (0.3 N NaOH) for 2 h (A), ONOO- (500 µM) for 2 h (B), SIN-1 (100 µM) for 1 h (C), SIN-1 (100 µM) for 2 h (D), SIN-1 (500 µM) for 1 h (E), or SIN-1 (500 µM) for 2 h (F), as in Fig. 7. These representative fields are identical to the fields shown in Fig. 7, but FITC fluorescence reveals staining for beta -catenin. Representative fields from each group are shown (magnification, ×60). Control PAEC (A) show regular staining of catenin inside the adjacent cells (arrow). ONOO- treatment (B) resulted in decreased total catenin staining at the cell edges and discontinuity at some borders (arrowhead). SIN-1 (100 µM) also resulted in progressive loss of catenin staining with noted discontinuity along cell borders (C and D). Higher doses of SIN-1 (E and F) resulted in accelerated disorganization of catenin, progressive loss at the cell borders, and overall changes in cell morphology (F, open arrow).

ONOO- also caused dose- and time-dependent changes in beta -catenin distribution (Fig. 8). In control PAEC monolayers (Fig. 8A), the predominant staining of beta -catenin at PAEC borders (arrow) is consistent with the participation of this protein in adherens junctions and cell-cell attachment. Treatment with ONOO- and SIN-1 caused generalized thinning of catenin staining and discontinuity at some cell borders, consistent with compromise of the adherens junction tethering (Fig. 8B, arrowhead). Comparable to the effects on actin fibers (Fig. 7), higher doses of SIN-1 (500 µM; Fig. 8, E and F) caused more rapid disorganization of beta -catenin and progressive loss of catenin staining at the cell borders concomitant with modulation of cell shape to a more spindle morphology (Fig. 8F, open arrow).

Effect of actin treated with ONOO- on PAEC barrier function. To more directly address ONOO- effects on cytoskeletal protein function, we treated nonmuscle actin with ONOO- in vitro and then introduced it into PAEC monolayers with liposomes as described in METHODS. These ONOO- treatment conditions caused nitration of nonmuscle actin as confirmed by immunoblotting (not shown). As shown in Fig. 9, compared with baseline, empty liposomes increased albumin clearance slightly, and liposomes containing native nonmuscle actin had little effect on PAEC barrier function, whereas liposomes containing ONOO--treated actin caused significant increases in albumin clearance. None of these treatment conditions caused cytotoxicity (LDH release or phase-contrast microscopy, not shown).


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Fig. 9.   Effect of actin treated with ONOO- on PAEC barrier function. Nonmuscle actin was treated under control conditions or with ONOO- (500 µM) for 1 h, dialyzed to remove any oxidizing species, and then introduced into PAEC monolayers with liposomes as described in METHODS. After baseline albumin clearance was measured for 1 h, PAEC monolayers were treated with empty liposomes (Lipo), liposomes containing nonmuscle actin (Lipo/Actin), or liposomes containing ONOO--treated actin (Lipo/Nitrated Actin). Albumin clearance was then measured for 2 h. Values are means ± SE and represent the average albumin clearance from 3 experiments. *P < 0.05 vs. baseline.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Evidence for protein nitration and oxidation in lung tissue, plasma, and bronchoalveolar lavage fluid from patients with ARDS (15, 22, 29, 31) suggests that ONOO- may participate in the pathogenesis of lung injury. However, direct evidence establishing ONOO- and nitrotyrosine formation as mediators of vascular pathology as opposed to markers of inflammation has been lacking. Therefore, the current study examined the effects of ONOO- on endothelial barrier function as a relevant physiological parameter of vascular endothelial function, impairment of which contributes to vascular leakiness, interstitial edema, and organ dysfunction. The direct extrapolation of these in vitro studies to human pathophysiology remains limited by our lack of understanding of relevant concentrations of ONOO- generated in vivo. Therefore, the goal of the present investigation was to identify novel targets of ONOO- reactivity that might allow more focused investigations in future in vivo studies.

ONOO- added as a bolus or generated continuously by SIN-1 caused significant PAEC monolayer barrier dysfunction that was not associated with PAEC cytotoxicity as determined by LDH release and morphological examination. Furthermore, ONOO--mediated barrier dysfunction was partially reversible and attenuated by cysteine, a known scavenger of ONOO- (6). Although recent reports have suggested that SIN-1 can generate a variety of reactive species (32), several lines of evidence in our study confirm that SIN-1 generated ONOO- under the conditions employed. First, SIN-1-induced DHR oxidation was attenuated by SOD and c-PTIO, indicating that scavenging of either O<UP><SUB>2</SUB><SUP>−</SUP></UP> or ·NO attenuated ONOO- formation. Furthermore, catalase failed to attenuate SIN-1-induced DHR oxidation, indicating that H2O2 did not account for SIN-1 reactivity. Second, addition of SOD to SIN-1 containing medium increased the amount of ·NO that was released into the culture medium bathing PAEC during measurements of barrier function, indicating that SIN-1 generates both O<UP><SUB>2</SUB><SUP>−</SUP></UP> and ·NO. Finally, treating PAEC with SIN-1 caused similar patterns of protein nitrotyrosine formation, as did bolus addition of ONOO-. Together, these results demonstrate that SIN-1 generated ONOO- under the conditions employed in the measurement of PAEC barrier function.

Although scavenging either ·NO or O<UP><SUB>2</SUB><SUP>−</SUP></UP> partially inhibited ONOO- formation and DHR oxidation by SIN-1, neither c-PTIO nor SOD attenuated SIN-1-mediated barrier dysfunction, and the addition of both c-PTIO and SOD exacerbated SIN-1-induced PAEC barrier dysfunction (Fig. 4). c-PTIO, a potent scavenger of ·NO, can, under certain conditions, modulate reactions of ONOO- and ·NO by other mechanisms that remain to be completely defined (40). Under conditions employed in the current study, neither c-PTIO nor SOD completely prevented DHR oxidation (Fig. 3) or attenuated barrier dysfunction (Fig. 4) caused by SIN-1. These findings suggest that SIN-1 produced ONOO- that was detectable only after 1 h (Fig. 3), accounting for the delayed development of barrier dysfunction. However, barrier dysfunction caused by bolus addition of ONOO- was also delayed and developed only after 1 h of treatment. Coupled with the poor correlation between barrier dysfunction and SIN-1 concentration (Fig. 1A), these findings suggest that ONOO- might alter endothelial function through cumulative effects on cytoskeletal dynamics that are more dependent on time than ONOO- concentration. Alternatively, the inability of ·NO or O<UP><SUB>2</SUB><SUP>−</SUP></UP> scavenging to attenuate SIN-1-mediated barrier dysfunction may relate to the complex interactions between reactive oxygen and nitrogen species in the regulation of endothelial barrier function. For instance, one could speculate that in the SIN-1 plus c-PTIO treatment group, barrier dysfunction was mediated by O<UP><SUB>2</SUB><SUP>−</SUP></UP> and/or H2O2 in the absence of ·NO, and in the SIN-1 plus SOD treatment group, barrier dysfunction was mediated by H2O2 that was generated from the dismutation of O<UP><SUB>2</SUB><SUP>−</SUP></UP> by SOD. The generation of H2O2 by the combination of SIN-1 and SOD is suggested by 1) attenuation of SIN-1/c-PTIO/SOD-induced barrier dysfunction with catalase and 2) persistence of oxidized proteins in PAEC treated with SIN-1 plus SOD (Fig. 6). The significant increase in SIN-1-mediated barrier dysfunction caused by the combination of both c-PTIO and SOD likely derives from the previously described barrier disruptive effects of H2O2 (23) plus the removal of the barrier protective effects of ·NO during H2O2-mediated oxidative stress (20). Together, the data presented in Figs. 1-4 show that both bolus additions of ONOO- and the continuous generation of ONOO- with SIN-1 cause PAEC monolayer barrier dysfunction that is partially reversible, unassociated with cytotoxicity, and comparable in extent to barrier dysfunction induced by ROS.

The exact mechanisms by which ONOO- causes PAEC barrier dysfunction remain to be defined. At physiological pH, ONOO- nitrates tyrosine residues in actin, a cytoskeletal protein critical for maintenance of EC shape and barrier function (43). In addition to the actin cytoskeleton, adherens junctions are a critical structure in cell-cell adhesion and the formation of the endothelial barrier (9). Adherens junctions are composed of complexes of membrane-spanning cadherin proteins and intracellular catenins. The extracellular domain of cadherin links to the cadherin expressed on adjacent cells via Ca2+-dependent binding. The intracellular domain of cadherin binds to beta -catenin, which links to alpha -catenin to attach the actin cytoskeleton to the adherens junction complex (42). The current study provides several lines of evidence that ONOO- alters these cytoskeletal proteins to disrupt endothelial barrier function. Treatment with either SIN-1 or ONOO-, under conditions identical to those causing barrier dysfunction, resulted in PAEC protein nitration and oxidation. Alterations in the structure of PAEC actin and beta -catenin detected with confocal immunofluorescent microscopy provide additional evidence for these cytoskeletal proteins serving as protein targets or effectors in ONOO--induced endothelial barrier dysfunction. The barrier disruptive effects of cytoskeletal proteins nitrated with tetranitromethane, a powerful oxidizing and nitrating agent, were previously reported in cultured EC (12). The current study demonstrated that actin treated with ONOO-, but not native actin, caused barrier dysfunction when introduced into PAEC with liposomes (Fig. 9). We speculate that nitrated actin is incorporated into endogenous actin and alters the dynamics of actin polymerization and depolymerization, thereby changing cell shape and causing barrier dysfunction. The barrier disruptive effect of ONOO--induced nitration of actin and beta -catenin is further supported by results showing that proteins immunoprecipitated from ONOO--treated PAEC with a monoclonal anti-nitrotyrosine antibody reacted with antibodies to actin and beta -catenin (Fig. 5B). These results indicate that ONOO- directly alters EC cytoskeletal proteins, leading to EC barrier dysfunction. Although we have identified actin and beta -catenin as relevant targets for the barrier disruptive effects of ONOO-, other nitrated and oxidized proteins may well contribute to ONOO--induced EC barrier dysfunction. The identification of these additional ONOO--altered proteins constitutes an area of active investigation in our laboratory.

In addition to directly oxidizing or nitrating PAEC cytoskeletal proteins, ONOO- could potentially activate intracellular signaling pathways that regulate endothelial barrier function. For instance, oxidants stimulate tyrosine phosphorylation in cultured EC (45). Tyrosine phosphorylation of adherens junction proteins disrupts the functional integrity of the adherens junction complex (46). ONOO- can either activate tyrosine kinases or inhibit tyrosine kinase-mediated signaling through tyrosine nitration (16). However, in preliminary studies we found that inhibiting tyrosine kinases had no effect on SIN-1-mediated PAEC barrier dysfunction (data not shown), suggesting that ONOO--mediated tyrosine kinase activation does not constitute a major pathway mediating ONOO--induced endothelial barrier dysfunction. Although other cellular effects of ONOO- could also contribute to PAEC barrier dysfunction, including ONOO--mediated activation of PARS and subsequent NAD+ depletion and inhibition of mitochondrial respiration as reported in human umbilical vein endothelial cells (44), or to ONOO--induced alterations in EC calcium (11), our results suggest that the direct effects of ONOO- on key cytoskeletal proteins causes barrier dysfunction.

In summary, ONOO-, either as a bolus or generated continuously by SIN-1, caused significant PAEC barrier dysfunction that was not associated with cytotoxicity. This barrier dysfunction was partially inhibited by cysteine, suggesting that it is mediated by ONOO-. Several proteins from ONOO--treated PAEC displayed evidence of nitration and oxidation. Particularly relevant to endothelial barrier function, ONOO- caused nitration of actin and beta -catenin, suggesting that these cytoskeletal protein modifications contribute to barrier dysfunction. Actin treated with ONOO- and introduced into PAEC also caused significant barrier dysfunction. Together, these findings demonstrate that ONOO- modifies cytoskeletal proteins, resulting in changes in cell shape and subsequent barrier dysfunction. Additional studies are required to clarify other proteins that are nitrated and/or oxidized by ONOO- to define how these modifications relate to protein structure and function and to determine the relative contribution of oxidation and tyrosine nitration to alterations in protein and cell function. The current study expands the list of proteins that are modified by ONOO- and identifies novel cytoskeletal targets in the lung parenchyma as candidates for ONOO- reactivity.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the expert technical assistance of Shehnaz Khan, Delbert Bauzon, and Dean Kleinhenz.


    FOOTNOTES

This work was supported by the Roudebush Veterans Affairs Medical Center Research Service, National Heart, Lung, and Blood Institute (NHLBI) Grant PO1-HL-58064, NHLBI Training Grant T32-HL-07774, and the American Diabetes Association.

Address for reprint requests and other correspondence: C. Michael Hart, Division of Pulmonary and Critical Care Medicine, Atlanta Veterans Affairs Medical Center (151-P), 1670 Clairmont Rd., Decatur, GA 30033 (E-mail: michael.hart.3{at}med.va.gov).

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 10 July 2000; accepted in final form 30 April 2001.


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