Vitamin C Protects Against Hypochlorous Acid–Induced Glutathione Depletion and DNA Base and Protein Damage in Human Vascular Smooth Muscle Cells
Hypochlorous acid (HOCl), generated by myeloperoxidase released from activated macrophages, is thought to contribute to vascular dysfunction and oxidation of low density lipoproteins (LDLs) in atherogenesis. We have previously shown that HOCl exposure can cause chlorination and oxidation of isolated DNA and that vitamin C protects human arterial smooth muscle cells against oxidized LDL–mediated damage. We report in the present study that vitamin C attenuates HOCl-induced DNA base and protein damage and depletion of intracellular glutathione (GSH) and ATP in human arterial smooth muscle cells. Cells were pretreated in the absence or presence of 100 μmol/L vitamin C (24 hours) and then exposed to HOCl (0 to 500 μmol/L, 0 to 60 minutes) in the absence of vitamin C. Intracellular GSH and ATP levels were depleted by HOCl treatment, and gas chromatography–mass spectroscopy revealed a concentration- and time-dependent increase in DNA base oxidation and protein damage (measured as 3-chlorotyrosine). Pretreatment of smooth muscle cells with vitamin C significantly reduced the extent of HOCl-induced DNA and protein damage and attenuated decreases in intracellular ATP and GSH. Our findings suggest that physiological levels of vitamin C provide an important antioxidant defense against HOCl-mediated injury in atherosclerosis.
Myeloperoxidase (MPO) is expressed in human atherosclerotic lesions and released by phagocytic cells at sites of inflammation, catalyzing the formation of the potent chlorinating/oxidizing agent hypochlorous acid (HOCl) from H2O2 and chloride ions, with local concentrations of HOCl potentially exceeding 100 μmol/L.1 Up to 40% of H2O2 generated by activated phagocytes is used to form HOCl,2,3⇓ which can lead to the generation of other ‘reactive chlorine species,’ such as molecular chlorine (Cl2) and nitryl chloride (NO2Cl).4 Catalytically active MPO colocalizes with lipid-laden macrophages in human atherosclerotic lesions5 and appears to contribute to vascular cell dysfunction and modification of LDL in atherogenesis.5,6⇓ Elevated levels of proteins damaged by HOCl have been detected by using specific antibodies in advanced human atherosclerotic lesions.6 In addition, 3-chlorotyrosine, a biomarker of protein damage by reactive chlorine species, is elevated in tissue and LDL isolated from atherosclerotic intima.7
Despite accumulating evidence that many important biological molecules are oxidized/chlorinated by HOCl, such as sulfhydryl moieties, 8 ATP,8 plasma membrane ATPases,9 collagen,10 vitamin C,11 free amino acids,12 lipids and lipoproteins,7 DNA,13 and DNA repair enzymes,14,15⇓ the consequences of these events on vascular cells has not been fully investigated. Vitamin C attenuates the cytotoxic effects of oxidatively modified LDL,16 reverses vascular dysfunction in patients with coronary heart disease,17 and is involved in a redox couple with the glutathione (GSH) cycle.18 Moreover, low plasma vitamin C concentrations are implicated as an independent predictor of unstable coronary artery disease19 and with an increased severity of inflammation, atherosclerosis, and plaque stability.20
In this context, we have previously reported that pretreatment of human arterial smooth muscle cells with vitamin C protects cells against oxidized LDL–induced apoptosis16 and depletion of the key intracellular antioxidant, GSH.21 To our knowledge, the present study provides the first evidence in human cultured umbilical arterial smooth muscle cells (HUASMCs) that HOCl-induced DNA and protein damage and depletion of intracellular GSH and ATP levels are significantly attenuated by vitamin C supplementation. Moreover, vitamin C pretreatment protected human aortic smooth muscle cells from HOCl-induced GSH and ATP depletion.
Calf thymus DNA, 6-azathymine, 2,6-diaminopurine, 8-bromoadenine, 5-hydroxyuracil (isobarbituric acid), 4,6-diamino-5-formamidopyrimidine (FAPy adenine), 2,5,6-triamino-4-hydroxypyrimidine, 5-(hydroxymethyl)uracil, and guanase were purchased from Sigma Chemical Co. 8-Hydroxyguanine and ethanethiol were purchased from Aldrich. 8-Hydroxyadenine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPy guanine) were synthesized by, respectively, treatment of 8-bromoadenine with concentrated formic acid (95%) at 150°C for 45 minutes with purification by crystallization from water and treatment of 2,5,6-triamino-4-hydroxypyrimidine with concentrated formic acid and purification by crystallization from water. Thymine glycol was synthesized by reaction of 5-methyluracil with OsO4 for 1 hour at 60°C, and excess OsO4 was removed by freeze-drying. Purity of standards (>99%) was assessed by mass spectrometry. 2-Hydroxyadenine, 5-hydroxycytosine, 5-hydroxyhydantoin, and 5-hydroxy-5-methylhydantoin (5-OH,Me hydantoin) were kind gifts from Dr M. Dizdaroglu (National Institute of Standards and Technology, Gaithersburg, Md). Silylation grade acetonitrile and bis(trimethylsilyl)trifluoroacetamide (BSTFA), containing 1% trimethylchlorosilane, (TMCS), and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) were obtained from Pierce Chemical Co.
Culture of Vascular Smooth Muscle Cells and HOCl Treatment
Human smooth muscle cells were cultured from umbilical artery (HUASMCs) or from aortic explants (HASMCs) in MCDB131 medium (GIBCO) supplemented with 10% (vol/vol) FCS, penicillin (100 U · mL−1), and streptomycin (100 μg · mL−1) at 37°C in a 5% CO2/95% air atmosphere.16,21⇓ Cells were confirmed as smooth muscle by their typical “hill and valley” morphology and positive immunofluorescent staining for smooth muscle α-actin. HUASMCs between passages 3 and 7 and HASMCs between passages 10 and 15 were used in all experiments. Cells were pretreated for 24 hours in the absence or presence of 100 μmol/L vitamin C before HOCl exposure. Hypochlorite concentration was quantified immediately before use spectrophotometrically at 290 nm [pH 12, ε=350 (mol/L)−1 · cm−1], and HOCl dilutions were prepared in Krebs buffer (mmol/L: NaCl 130, KCl 5.7, NaHCO3 25, NaH2PO4 1.0, d-glucose 5, CaCl2 1, and MgCl2 0.5, pH 7.4) immediately before an experiment to avoid scavenging of HOCl by antioxidant components of serum-containing culture medium and to mimic the release of HOCl near vascular smooth muscle. We use the term hypochlorous acid (pKa 7.46) to refer to an ≈50% ionized mixture of HOCl and OCl− (hypochlorite). The addition of HOCl (up to 500 μmol/L) did not significantly alter pH, and HOCl loss in buffer alone was calculated to be ≈10% per hour. Culture medium was removed from confluent smooth muscle cell monolayers, and cells were washed twice with Krebs buffer (37°C), followed immediately by the addition of HOCl (0 to 500 μmol/L, 0 to 120 minutes). After incubation at 37°C in 5% CO2 for defined periods, solutions were removed, cells were washed twice with PBS, and lysates were then prepared for subsequent determination of ATP, GSH, or damaged DNA and protein products.
Measurement of Total and Reduced Vitamin C Levels
High-performance liquid chromatography (HPLC) of reduced and total ascorbic acid was based on the methods of Iriyama et al22 and Mitton and Trevithick.23 To allow for reduced vitamin C analysis, half the cell suspension (in saline) was diluted 1:2 with ice-cold 10% metaphosphoric acid (MPA), vortexed, and immediately snap-frozen and stored at −80°C. The culture medium (500 μL) was also diluted 1:2 with 10% MPA, snap-frozen, and stored at −80°C before analysis. The remaining cell suspension and additional aliquots of culture medium were immediately snap-frozen and stored at −80°C. The acidified cell suspension and culture medium were allowed to thaw on ice, and 100 μL HPLC-grade heptane was added to both samples. Mixed samples were centrifuged at 13 000g for 5 minutes at 4°C, and the lower layer was retained. The heptane extraction process was repeated until the cell lysate and medium were free of particulates and stored at 4°C. The nonacidified cell suspension or culture medium (400 μL) was thawed on ice, incubated with 50 μL of 1% dithiothreitol at 21°C, and acidified with 50 μL of 50% MPA, and heptane was extracted as described above. Aliquots of 20 μL were analyzed by HPLC with the use of a 4.6×250-mm, 5-μm ApexII ODS column with a 2-cm Bio300 guard (Jones Chromatography) and eluted with 0.2 mol/L K2HPO4-H3PO4 running buffer, pH 2.1, containing 0.25 mmol/L octane sulfonic acid at a flow rate of 1.0 mL/min. An amperometric electrochemical detector (EG & G) was used, with electrode set at 810 mV, a time constant of 5 seconds, a cathodic output, and sensitivity of 100 nA (cell lysate) or 0.5 μA (culture medium). Standards of reduced (ascorbic acid) and oxidized (dehydroascorbic acid) vitamin C were prepared in MPA and/or treated with dithiothreitol. Minimum detection was 0.1 μmol/L for the reduced vitamin C.
Determination of ATP and GSH
Trichloroacetic acid (6.5%, 4°C) was added to each sample (0.5 mL/0.5×106 cells), which were stored overnight at −80°C, thawed, and then kept at 4°C for analysis. ATP was determined by use of a bioluminescence assay.24 Acid sample extract (10 μL) or acid ATP standard (0 to 40 μmol/L) was added to 170 μL of a 1:1:1 mixture of 80 mmol/L MgSO4 · 7H2O/10 mmol/L KH2PO4/100 mmol/L Na2AsO4 (pH 7.4) and then 40 μL of luciferase extract. Bioluminescence was measured for 20 seconds by using a luminescence plate reader (Dynex MLX, Dynex Technologies). GSH was determined by using a fluorometric assay described by Hissin and Hilf.25 Briefly, 7.5 μL of acid sample extract or acid GSH standard (0 to 40 μmol/L) was added to 275 μL of 80 mmol/L phosphate buffer (pH 8.0) containing 5 mmol/L EDTA and then 15 μL of 0.1% (wt/vol) o-phthalaldehyde in methanol. Fluorescence was measured after 20 minutes (CytoFluor 4000, PerSeptive Biosystems) by using excitation and emission wavelengths of 350 and 420 nm, respectively. Protein concentrations were determined by Coomassie blue spectrophotometry after removal of residual trichloroacetic acid extract and solubilization of adherent cell protein by the addition of 1.0 mL of 1 mol/L NaOH.
Extraction of Cellular DNA and GC-MS Analysis
HUASMCs were lysed with 1 mL of buffer (0.1 mol/L NaCl, 20 mmol/L Tris-base [pH 8.0] containing 10 mmol/L EDTA and 0.5% SDS) and then incubated for 1 hour at 37°C with 15 and 10 U RNase A and T1, respectively. Samples were then incubated for 2 hours at 37°C with 30 U proteinase K. One milliliter of 6 mol/L NaCl was added to each sample, and the protein pellet was removed after centrifugation at 4000g for 10 minutes. The latter step was repeated, and the DNA was precipitated after the addition of 5 mL ice-cold ethanol. The DNA pellet was subsequently washed twice in 70% ethanol and then dissolved in water at 4°C. Extracted DNA (40 to 60 μg) containing the internal standards 6-azathymine and 2,6-diaminopurine (0.5 nmol) was lyophilized. Samples were hydrolyzed by the addition of 0.5 mL of 60% formic acid and by heating at 140°C for 45 minutes in an evacuated, sealed, hydrolysis tube; then they were cooled and lyophilized. Samples were derivatized in poly(tetrafluoroethylene)-capped glass vials after purging with nitrogen by adding 75 μL of a BSTFA (1% TMCS)/acetonitrile/ethanethiol (16:3:1 [vol/vol]) mixture at 23°C for 2 hours and analyzed by gas chromatography (GC)–mass spectrometry (MS), as described previously.13
Assay of Cell Viability (Mitochondrial Dehydrogenase Activity)
Confluent smooth muscle cells were pretreated in the absence or presence of vitamin C (100 μmol/L, 24 hours), washed with PBS, and then incubated with HOCl (0 to 500 μmol/L for 1 hour) in the absence of vitamin C. Mitochondrial dehydrogenase activity was used as an index of cell viability and assessed by using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide (MTT) assay.16
Analysis of Cell Protein Damage
Fluorescence measurements of protein-bound tryptophan were recorded by using a Hitachi F-4500 fluorescence spectrometer at excitation and emission wavelengths of 282 and 331 nm, respectively. The emission and excitation slits were set to 5 nm. Units were calculated as fluorescence intensity per milligram protein.
The concentration of free amine groups was monitored by using fluorescamine-amine fluorescence. Briefly, 15 μL of cell protein was added to 135 μL of 50 mmol/L phosphate buffer (pH 8.0) and then 100 μL of 0.015% (wt/vol) fluorescamine in acetonitrile added. Fluorescence was measured (CytoFluor 4000, PerSeptive Biosystems) by using excitation and emission wavelengths of 400 and 460 nm, respectively. Units were calculated as fluorescence intensity per milligram protein.
GC-MS Analysis of 3-Chlorotyrosine
An aliquot of cell protein (≈0.25 mg) or standard (tyrosine and Cl-tyrosine), containing the internal standard p-hydroxyphenylglycine (0.5 nmol) was lyophilized. Samples were hydrolyzed by the addition of 0.5 mL of 6 mol/L HBr containing 1% phenol and heated at 110°C for 24 hours, cooled, and lyophilized. Samples were derivatized after purging with N2 by adding 50 μL of acetonitrile and 50 μL MTBSTFA (1% tertbutyldimethylchlorosilane) at 50°C for 30 minutes and analyzed by GC-MS (Hewlett Packard 5890II gas chromatograph and 5971A mass spectrometer). The injection port and the GC-MS interface were kept at 250°C and 290°C, respectively. Separations were carried out on a fused silica capillary column (12 m×0.22-mm internal diameter) coated with cross-linked 5% phenylmethylsiloxane (film thickness 0.25 μm; BPX5, SGE). Helium was the carrier gas, with a flow rate of 0.93 mL/min. Derivatized samples (1 μL) were injected into the GC injection port by using a split ratio of 8:1. Column temperature was increased from 125°C to 175°C at 8°C/min after 2 minutes at 125°C and then from 175°C to 220°C at 30°C/min, held at 220°C for 1 minute, increased from 220°C to 290°C at 40°C/min, and held at 290°C for 2 minutes. Selected ion monitoring was performed by using the electron-ionization mode at 70 eV, with the ion source maintained at 180°C. Quantification of tyrosine and Cl-tyrosine was performed by monitoring ions at mass-to-charge ratios 302 and 350 for 4-hydoxyphenylglycine.
Data points are mean±SEM of experiments in at least 4 different smooth muscle cell cultures. ANOVA and the Student t test were carried out as appropriate, with a value of P<0.05 considered statistically significant.
Vitamin C Levels in HUASMCs
HUASMCs were incubated for up to 24 hours in medium containing 100 μmol/L vitamin C, and medium and intracellular levels of ascorbic acid, dehydroascorbic acid, and total vitamin C were determined. At time 0, ≈80% of total vitamin C in the medium was present in the form of ascorbate with ≈20% in the form of dehydroascorbic acid (Table 1). After 24 hours, ascorbate in the culture medium was reduced to ≈20% of initial levels at time 0. Intracellular levels of total vitamin C reached a maximal value after 6 hours of treatment (5.0±0.1 μmol/L) and decreased to 3.3±0.7 μmol/L after 24 hours, with the majority of vitamin C in the form of ascorbic acid.
Effects of Hypochlorous Acid on Cell Viability
Mitochondrial dehydrogenase activity remained unchanged during the incubation of cells for 2 hours with medium lacking HOCl (data not shown). When cells were exposed for 60 minutes to HOCl, MTT activity was reduced to 86±4%, 79±3%, and 69±2% of control values in cells exposed to 100, 200, and 500 μmol/L HOCl, respectively. When cells were pretreated with vitamin C (100 μmol/L, 24 hours) and then challenged with HOCl (100 to 500 μmol/L) in the absence of vitamin C, the decrease in MTT activity induced by HOCl was attenuated (92±4%, 87±5% [P<0.05], and 77±4% [P<0.05] of control values in cells exposed to 100, 200, and 500 μmol/L, respectively).
Effects of HOCl on Intracellular ATP and GSH Levels
Intracellular levels of total GSH and ATP in HUASMCs remained relatively constant during incubation of cells with Krebs buffer for up to 2 hours (Figure 1C and 1D). Treatment of cells with HOCl induced a time-dependent depletion of GSH and ATP, with significant decreases observed within 10 minutes of exposure of the cells to 200 μmol/L HOCl (Figure 1C and 1D). Decreases in ATP levels induced by lower concentrations of HOCl were not accompanied by a significant loss of cell viability. GSH levels remained depressed over a 2-hour exposure period to 200 μmol/L HOCl, whereas ATP levels recovered after 10 minutes, reaching ≈70% of control values after 2 hours (Figure 1C). When concentration-dependent effects of HOCl were examined, GSH and ATP levels decreased significantly after 1-hour exposure of the cells to HOCl. GSH levels were as follows (nmol/mg protein): control 7.9±0.9, 50 μmol/L HOCl 5.9±0.8, and 200 μmol/L HOCl 3.9±0.8. ATP levels were as follows (nmol/mg protein): control 8.7±1.0, 50 μmol/L HOCl 5.7±0.8, and 200 μmol/L HOCl 5.2±1.1. Pretreatment of HUASMCs with vitamin C (100 μmol/L) for 24 hours prevented HOCl-mediated decreases in GSH and ATP levels (Figure 1A and 1B). To confirm that responses in HUASMCs also occurred in other smooth muscle cell types, we examined the cytoprotective action of vitamin C on cells derived from human aortic explants. Pretreatment of aortic smooth muscle cells with vitamin C (100 μmol/L, 24 hours) prevented HOCl (50 μmol/L, 1 hour)–induced depletion of intracellular GSH and ATP (data not shown).
HOCl-Induced DNA Base Damage
GC-MS analysis of DNA base damage revealed increases in several base-modified products and established a pattern of HOCl-induced cellular DNA base damage (Table 2). HOCl increased thymine glycol and 5-OH,Me hydantoin in a time-dependent (0 to 0.11±0.01 versus 0.50±0.05 nmol/mg DNA for control and for 120 minutes of 200 μmol/L HOCl, respectively) and concentration-dependent (0.10±0.01 versus 0.50±0.08 nmol/mg DNA for control and for 200 μmol/L HOCl, respectively) manner. Minor increases in other oxidized base lesions of cytosine and adenine were observed, but these were not accompanied by any increase in guanine oxidation, detected as 8-hydroxyguanine or FAPy guanine. Oxidized base lesions of uracil [5-Cl uracil, 5-hydroxyuracil, and 5-(hydroxymethyl)uracil] and adenine (8-Cl adenine and 8-hydroxyadenine) were either not detected or not modified by HOCl.
Pretreatment of HUASMCs with vitamin C (100 μmol/L, 24 hours) significantly attenuated thymine oxidation induced by HOCl (200 μmol/L, 1 hour), whereas the minor increases in oxidized base lesions of cytosine and adenine were not significantly affected by vitamin C pretreatment (Figure 2).
Modification of Protein by HOCl
HOCl induced a time- and concentration-dependent depletion of tryptophan and amino groups in cellular proteins (Figure 3A and 3B), confirming previous findings in isolated LDL.26 Under control conditions without HOCl, protein levels of tryptophan and free amines did not decrease significantly over 1 hour (data not shown). Pretreatment of cells with vitamin C (100 μmol/L for 24 hours) before HOCl exposure (200 μmol/L, 60 minutes) protected cell protein against the HOCl-induced loss of tryptophan and free amines (Figure 3C and 3D).
HOCl also caused a time- and concentration-dependent formation of 3-chlorotyrosine (Figure 4), highlighting a specific biomarker of HOCl-induced vascular smooth muscle cell damage. Significant increases in chlorotyrosine levels were observed after exposure of smooth muscle cells to 50 to 500 μmol/L HOCl (data not shown), similar to findings in isolated proteins.27 Pretreatment of HUASMCs with vitamin C (100 μmol/L, 24 hours) markedly attenuated HOCl (200 μmol/L, 60 minutes)–induced increases in chlorotyrosine levels (Figure 4, inset).
We report the first evidence in human vascular smooth muscle cells that physiologically relevant HOCl concentrations (≤200 μmol/L) cause significant DNA base damage, with thymine-derived oxidation products, thymine glycol, and 5-OH,Me hydantoin increased to the greatest extent. DNA base damage was accompanied by HOCl-induced depletion of intracellular levels of GSH and ATP. Pretreatment of cells with vitamin C protected against HOCl-induced rapid protein and DNA damage, loss of intracellular GSH and ATP, and decreased mitochondrial dehydrogenase activity, highlighting the cytoprotective actions of intracellular vitamin C against smooth muscle cell damage. This may contribute to the reported protective effect of vitamin C in atherogenesis.
HOCl-Induced Depletion of GSH and ATP and Smooth Muscle Cell Viability
MPO, detected within atherosclerotic lesions,5 provides the only mechanism for generating physiological levels of HOCl in humans. HOCl rapidly oxidizes thiols and thioethers and reacts with amines to form chloramines,28 yet there is limited information on their damage to vascular endothelial and smooth muscle cells. HOCl-induced modifications to the extracellular facing thiol moieties of membrane-bound ATPases would disturb ion homeostasis.9 Recent studies have shown that NO2− reacts with HOCl to produce a potent chlorinating and nitrating species, NO2Cl,4 which can oxidize and nitrate human LDL29 and chlorinate tyrosyl residues.4 This is of particular relevance for atherosclerotic lesions, in which elevated levels of NO2− may also be generated from inducible NO synthase in activated macrophages.30
Our findings of HOCl-induced smooth muscle cell damage are consistent with those reported previously for endothelial, respiratory tract, and red blood cells,31–35⇓⇓⇓⇓ in which the reactions of HOCl with cell thiols were suggested to modulate cell processes similar to the actions of H2O2 and peroxynitrite.31–34⇓⇓⇓ GSH is rapidly depleted in cells exposed to HOCl because of the formation of mixed protein disulfides and a sulfonamide.8 Without formation of oxidized glutathione (GSSG), GSH becomes a sacrificial rather than a recyclable antioxidant and may provide only limited protection for protein thiols against HOCl.
Pretreatment of HUASMCs (data not shown) with physiological concentrations of vitamin C36 and within the optimal range for l-ascorbic acid uptake into vascular smooth muscle cells18 attenuated the depletion of GSH and ATP by HOCl. Under our experimental conditions, supplementation of HUASMCs with 100 μmol/L vitamin C increased intracellular vitamin C levels 3-fold (Table 1). The cytoprotective action of vitamin C extends our previous findings that vitamin C spares endogenous adaptive antioxidant defense mechanisms in HUASMCs exposed to oxidized LDL.21 Vitamin C protects LDL against most of HOCl-mediated modifications, probably because of the rapid reaction rate of HOCl with vitamin C.11
HOCl-Induced DNA Base Damage
HOCl has been shown to attack nucleotides and individual DNA bases in vitro13,37,38⇓⇓ and to inactivate various DNA repair enzymes,14,15⇓ processes that could contribute to the increased risk of atherogenesis. The application of GC-MS has enabled us to identify for the first time the pattern of DNA damage in vascular smooth muscle cells exposed to HOCl. This unique pattern mirrors that reported by us for isolated DNA,13 indicating that hydroxyl radicals or other reactive oxygen species were not major contributors to DNA damage and may not be the only in vivo mediators of DNA base modification. Pretreatment of HUASMCs with vitamin C markedly attenuated HOCl-induced DNA oxidation (Figure 2). Vitamin C has also been shown to inhibit oxidative DNA damage in isolated and cultured cells exposed to UV-visible light and H2O2.39,40⇓ We have shown previously that pretreatment of HUASMCs with vitamin C increases cellular antioxidant defenses by raising intracellular GSH levels,21 which may in part explain the inhibition of DNA and protein damage afforded by vitamin C in the present study.
Recent evidence suggests that DNA damage may also contribute to the development of atherosclerosis.41 For example, vascular smooth muscle cells extracted from the abdominal aorta of atherosclerotic patients have been reported to reveal a high degree of DNA base damage when assayed with the use of 8-hydroxy-2′-deoxyguanosine as an indicator of oxidative DNA damage,41 although the present study demonstrates that this lesion is not generated by HOCl.
HOCl-Induced Chlorination of Smooth Muscle Cell Proteins
3-Chlorotyrosine has been measured as a specific marker of chlorination of LDL in vitro6 and as a molecular fingerprint of MPO activity and protein chlorination in vivo.6,7⇓ Elevated levels of 3-chlorotyrosine in human atherosclerotic aortas and in LDL isolated from atherosclerotic lesions7 support the hypothesis that MPO plays an important role in the oxidative modification of lipoproteins, which may contribute to vascular disease. We have measured this marker of reactive chlorine species–mediated protein damage in human vascular smooth muscle cells exposed to HOCl and established that the formation of 3-chlorotyrosine is dependent on the concentration and time of exposure to HOCl. We cannot eliminate the possibility that residual chloride in cell protein samples contributed to chlorination during acid hydrolysis of proteins and that it elevated the background levels of 3-chlorotyrosine. However, it is worth noting that inasmuch as control and treated smooth muscle cell extracts were processed similarly, this would only lead to an underestimate of the relative increase in tyrosine chlorination.
The present study has identified a number of biomarkers of vascular cell oxidative injury and provides evidence that vitamin C affords protection against HOCl-induced damage. Evidence from previous studies documents vitamin C–mediated protection against LDL-mediated smooth muscle cell apoptosis,16 oxidative stress–induced GSH depletion,21 and endothelial dysfunction in patients with coronary artery disease.19 In accord with our present finding that vitamin C provides an important antioxidant defense against HOCl-mediated smooth muscle cell injury, recent studies in human umbilical vein endothelial cells also suggest a cytoprotective role for intracellular vitamin C against HOCl-mediated apoptosis.42
↵*These authors contributed equally to the present study.
Received December 6, 2001; revision accepted February 12, 2002.
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