Hypothiocyanous acid is a potent inhibitor of apoptosis and caspase 3 activation in endothelial cells

Free Radical Biology & Medicine 49 (2010) 1054–1063

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Free Radical Biology & Medicine j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f r e e r a d b i o m e d

Original Contribution

Hypothiocyanous acid is a potent inhibitor of apoptosis and caspase 3 activation in endothelial cells Stephanie M. Bozonet ⁎, Amy P. Scott-Thomas, Péter Nagy, Margreet C.M. Vissers Free Radical Research Group, Pathology Department, University of Otago at Christchurch, Christchurch 8140, New Zealand

a r t i c l e

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Article history: Received 22 April 2010 Accepted 18 June 2010 Available online 13 July 2010 Keywords: Hypothiocyanous acid Caspase 3 Apoptosis Endothelial cell function Thiol oxidation Free radicals

a b s t r a c t Hypothiocyanous acid (HOSCN) is a common, thiol-specific oxidant with strong antibacterial activity. It is thought to be nontoxic to mammalian cells, although its ability to specifically target intracellular thiols may potentially cause cellular dysfunction. In this study we demonstrate specific effects of HOSCN on human endothelial cells, with exposure to high concentrations resulting in morphology changes unlike those seen with other oxidants. Effects were time- and dose-dependent and were accompanied by loss of total cell thiols and GSH and by inactivation of glyceraldehyde-3-phosphate dehydrogenase. High-dose exposure was cytotoxic, but lesser doses did not cause cell death, and apoptosis was not initiated by any concentration of HOSCN. In fact, initiation of apoptosis was blocked by minimal HOSCN exposure, with activation of caspase 3 and cleavage of the proenzyme being prevented. This was unlikely to be due to direct oxidation of the caspase 3 active-site cysteine and suggests alternative targeting of the caspase pathway. The survival of endothelial cells when HOSCN is present together with an inducer of apoptosis suggests that HOSCN differs from most other oxidants and could affect endothelial cell survival pathways in a way that may have an impact on vascular function. © 2010 Elsevier Inc. All rights reserved.

Hypothiocyanous acid (HOSCN) is an important antimicrobial oxidant generated from the preferential oxidation of thiocyanate (SCN−) by salivary peroxidase, lactoperoxidase (LPO), eosinophil peroxidase, and myeloperoxidase (MPO) [1–6]. In activated neutrophils the MPO–H2O2– (pseudo)halide system uses SCN−, Cl−, or Br− to produce HOSCN, hypochlorous acid (HOCl), or hypobromous acid (HOBr) [7,8]. Although Cl− is generally accepted as the physiological substrate for MPO, HOSCN is likely to be a major product even when the concentration of Cl− exceeds that of SCN− [4,5]. Similarly, eosinophil peroxidase has a higher affinity for SCN− than for other substrates and generates a considerable amount of HOSCN [2]. Also the rapid and favorable reactions of SCN− with HOCl (k = 2 × 107 M− 1 s− 1) [9] and HOBr (k = 2 × 109 M− 1 s− 1) [10] are likely to influence the generation of HOSCN by inflammatory cells. Therefore, the availability of SCN− will determine the amount of HOSCN produced. Plasma levels are dictated by dietary intake and are usually around 120 μM in humans [3], but in cigarette smokers they can reach 500 μM [11] and are likely to result in the enhanced vascular production of HOSCN in these individuals.

Abbreviations: Chaps, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate; GSH, glutathione; GSSG, oxidized glutathione disulfide; HBSS, Hanks’ balanced saline solution; HUVEC, human umbilical vein endothelial cells; LPO, lactoperoxidase; MPO, myeloperoxidase; PBS, phosphate-buffered saline; PI, propidium iodide; PS, phosphatidylserine; SFM, serum-free medium; TNB, 5-thio-2-nitrobenzoic acid. ⁎ Corresponding author. Fax: + 64 3 364 1083. E-mail address: [email protected] (S.M. Bozonet). 0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2010.06.028

Oxidant generation by MPO has been implicated as a cause of tissue damage in numerous inflammatory diseases [12–18]. The extreme reactivity of HOCl and HOBr has made them a focus of interest: biomarkers of these oxidants have been found within atherosclerotic plaques [19–22] and a wealth of evidence now links the MPO–H2O2–halide system with cardiovascular disease [23–25] and poor postinfarct prognosis [8,26]. The presence of MPO and H2O2 in the extracellular milieu could result in oxidant generation in close proximity to the endothelium [23,24], and the cell surface protein cytokeratin 1 has been shown to facilitate the uptake of MPO by endothelial cells [27], making the intracellular generation of MPOderived oxidants likely. What effect this might have on vascular function is unknown, but this ongoing oxidant exposure could influence signaling pathways and may lead to impaired NO generation and alterations in endothelial cell function [27]. Unlike HOCl and HOBr, HOSCN has received comparatively little attention despite its being a significant biological oxidant. MPO-derived oxidants have a preferred reactivity for thiol groups and this is thought to account for their ability to affect cell signaling [28–31]. Accordingly, HOCl causes a dose-dependent loss of GSH and intracellular protein thiols in endothelial cells [32], which may also affect the activity of protein kinase pathways [33]. Exposure to HOCl or HOBr can initiate growth arrest and/or the induction of apoptosis or necrosis [34] and other studies have shown HOCl-induced activation of transcription factors including p53 [35] and nuclear factor (NF)-κB [36,37]. However, the reactivity of HOCl and HOBr is not confined to thiols and it is likely that many of their biological

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effects are due to reaction with methionine residues or phospholipid dienes or to the formation of reactive haloamines [2,6,28,38,39]. This pattern of reactivity could differ significantly from that of HOSCN, which is a weaker oxidant that reacts almost exclusively with thiols [40–42]. The probable generation of HOSCN makes it a likely contributor to the pathogenesis of vascular disease and an important target for study. Recent evidence points to a role for HOSCN in the development of atherosclerosis [43], and Wang and co-workers [30] have demonstrated that HOSCN is a potent inducer of endothelial cell tissue factor, which is instrumental in the progression of thrombosis. The pathogenic effects of HOSCN in vivo have also been linked to protein carbamylation in smoking-related diseases [44] and this may reflect the formation of relatively large quantities of HOSCN in smokers. Given the potential for HOSCN to affect endothelial cell function, we have investigated the reaction of this oxidant with cultured human umbilical vein endothelial cells (HUVEC) to determine its effects on intracellular thiol oxidation and cell death and survival pathways. Experimental procedures Reagents Cell culture medium including M199, penicillin/streptomycin, cosmic calf serum (CCS), endothelial cell growth factor (ECGF), and trypsin was supplied by Invitrogen (Carlsbad, CA, USA), as was the Apotarget annexin V/propidium iodide (PI) kit. Heparin (Multiparin) was acquired through the Christchurch Hospital Pharmacy and collagenase was from the Worthington Biochemical Corp. (Lakewood, NJ, USA). Polyvinylidene difluoride (PVDF) membrane and the Enhanced Chemiluminescence (ECL) Plus Western blotting detection system were from GE Healthcare (Buckinghamshire, UK). Complete protease inhibitors were supplied by Roche (Mannheim, Germany). Anti-caspase 3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Caspase substrate was supplied by the Peptide Institute (Osaka, Japan). Sodium cyanate (OCN−), sodium thiocyanate (NaSCN−), and catalase (from bovine liver) were from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals were reagent grade or better. Cell culture Umbilical cords were obtained with informed consent and endothelial cells isolated from veins by collagenase digestion (CLS Type 1, 100 U/ml) as previously described [45]. Cells were cultured in M199 supplemented with 20% CCS and ECGF (full medium), and cells were used between the second and the third passage. Serum-free medium (SFM) was prepared using M199 as above but excluding CCS and ECGF. For experimental use cells were confluent but without cobblestone morphology. For experiments involving Western blotting, cells were cultured in 10-cm plates and the oxidant was added in 5-ml volumes. For other experiments HUVEC were grown in 24-, 12-, or 6-well tissue culture plates and the oxidant was added in 1-, 1.5-, or 2-ml volumes, respectively. Preparation of HOSCN This was done enzymatically using bovine LPO at 4 °C. LPO (2 μM) was added to NaSCN− (7.5 mM) in 10 mM potassium phosphate buffer (pH 6.6), and 10 μl/ml H2O2 (75 mM) was added in four separate aliquots at 1-min intervals. The concentration of HOSCN was determined using 5-thio-2-nitrobenzoic acid (TNB) by measuring the change in absorbance at 412 nm, using the molar extinction coefficient for TNB (14,100 M− 1 cm− 1) and adjusting for the 1:2 stoichiometry of the reaction (HOSCN:TNB) [41]. Upon the first

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addition of H2O2 the yield of HOSCN accounted for around 90% of the H2O2 added, indicating that no other product was being generated; however, this declined after each subsequent addition of H2O2 and only around 40% of the final addition was converted to HOSCN. This is most likely due to cumulative inhibition of the LPO by H2O2 [46] or HSOCN [47] or a competing reaction of HOSCN with H2O2 to generate OCN−. Indeed we found that this reaction was highly favorable under the conditions used (unpublished observations). After the last aliquot of H2O2 0.01 mg/ml catalase was added to remove unreacted H2O2. LPO and catalase were removed by centrifugation (3700 rpm) at 4 °C using 10,000 Mw exclusion filters. Final solutions generally contained between 1.5 and 2 mM HOSCN. To study the effects of HOSCN breakdown products, a stock HOSCN solution of known concentration was left to degrade at room temperature overnight and the remaining concentration determined using TNB. There was no change in the absorbance of TNB upon addition of the “aged” HOSCN, indicating that no oxidant remained and that any long-lived breakdown products generated did not react with TNB. To validate the use of TNB for measuring the concentration of HOSCN, the concentration was also calculated by spectrophotometric analysis using a quartz cell with calibrated 5-cm pathlength at 376 nm, a wavelength at which there is a well-defined absorption maximum for HOSCN [48]. Treatment of HUVEC The administration of HOSCN in Hanks’ balanced saline solution (HBSS; 0.5 mM MgCl2, 1 mM CaCl2, and 5.5 mM glucose in phosphatebuffered saline, PBS) eliminated the possibility of secondary oxidant formation. HOSCN and OCN− were diluted to the appropriate concentrations (0, 50, 100, 200, or 300 μM) in HBSS, unless otherwise stated, and added to HUVEC. Equivalent concentrations of aged HOSCN were prepared from the original known concentration in the same way. The reaction of HOSCN with HUVEC was determined by measuring the amount of HOSCN remaining in the HBSS and was compared to HOSCN loss from equivalent wells without cells. Generally cells were treated for 0, 30, 60, 120, 180, 240, or 300 min and then any remaining HOSCN was quenched by the addition of 50 mM cysteine. Cells were washed in PBS and harvested or reincubated with fresh full medium. Any detached cells were reintroduced. Morphological changes were visualized directly using phase-contrast microscopy. Measurement of thiol oxidation Inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Activity was determined from measurement of NADH oxidation at 340 nm, in a coupled system with 3-phosphoglycerate phosphokinase, using a SpectraMAX 190 fluorescence plate reader (Molecular Devices, Victoria, Australia) with SoftmaxPro software [49]. GSH To measure GSH levels, cells were derivatized with monobromobimane (MBB; 1 mM in acetonitrile) and then lysed using trichloroacetic acid [50]. Proteins were removed by centrifugation and samples were analyzed by reverse-phase HPLC with fluorescence detection (excitation 394 nm, emission 480 nm) [51]. Total protein thiols Whole-cell lysates were derivatized with MBB and the protein was precipitated with 5% trichloroacetic acid. Pellets were resuspended in 1% sodium dodecyl sulfate (SDS) and the amount of thiol present was determined using a Hitachi fluorescence spectrophotometer (excitation 394 nm, emission 480 nm) [51].

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Assays for cell death Cell apoptosis and/or necrosis were monitored after exposure to HOSCN. When required, apoptosis was induced by incubating the cells for 5 h in SFM, a strong inducer of apoptosis. Apoptosis and necrosis were monitored using the Apotarget annexin V FITC apoptosis kit, which assesses phosphatidyl serine (PS) exposure and membrane integrity to identify apoptotic and necrotic cells. After exposure to HOSCN, cells were harvested and washed in PBS and then incubated with annexin V and PI according to the manufacturer's instructions. The fluorescence of up to 10,000 cells was analyzed using a Cytomics FC 500 flow cytometry system (Beckman Coulter). Binding of annexin V to PS exposed on the cell surface indicates early stage apoptosis, and additional binding of PI indicates loss of membrane integrity or necrosis [52]. Caspase 3 activity assay Caspase 3 activity was measured as described previously using AcDEVD-AMC [53] in a 96-well plate or a 100-μl quartz cuvette (in vitro assay). Formation of the fluorescent cleavage product, 7-amino-4methylcoumarin (AMC), was detected at 460 nm (with excitation at 390 nm). SDS–PAGE and Western blotting for cleaved caspase 3 After HOSCN treatment, cells were washed in PBS and lysed using buffer containing 40 mM Hepes, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Complete protease inhibitors, and 1% Chaps at pH 7.6. DNA was removed by centrifugation and lysates were stored at −20 °C. Controls were prepared from untreated cells and cells incubated with SFM for 5 h. Proteins were concentrated by methanol/chloroform precipitation; 1 vol lysate was added to 4 vol methanol (MeOH), 1 vol chloroform, and 3 vol H2O, with mixing after each addition. The samples were centrifuged at 13,000 rpm for 5 min and the aqueous layer was removed. Four volumes of MeOH was added and then mixed, and the centrifugation was repeated. Proteins were resuspended in buffer containing 10% glycerol, 3% SDS, 62.5 mM Tris–HCl (pH 6.8), 10% dithiothreitol (DTT), and bromophenol blue. After boiling for 5 min proteins were separated by 15% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) then transferred to PVDF membrane (0.2 μm) and probed with anti-caspase 3 antibody (1:1000 in 5% milk in Tris-buffered saline with 0.05% Tween 20) overnight at 4 °C. Secondary antibody was a goat anti-rabbit horseradish peroxidase conjugate diluted to 1:20,000. Caspase 3 bands were detected using the ECL Plus Western blotting detection system and Quantity One software. Statistical analysis Unless otherwise stated, results are means ± SE of a minimum of three experiments and statistical analyses were carried out using the Student t test with significance determined as P ≤ 0.05. Results Generation of HOSCN We generated HOSCN in vitro by the LPO-catalyzed oxidation of SCN− with H2O2 and, because of the complex chemistry involved in its generation and breakdown, we monitored its formation and stability to ensure that the active compound in our cell experiments was indeed HOSCN. When a 1 mM solution of HOSCN in HBSS was incubated at 37 °C, ~20% of the oxidant remained after 180 min, as measured using either TNB or spectrophotometric absorbance at 376 nm (Fig. 1A). The overlapping kinetic traces for the decomposition of HOSCN measured by the two methods indicate that decomposition products of the oxidant

Fig. 1. Stability of HOSCN solutions and consumption by HUVEC. (A) Time course of the breakdown of a 1 mM HOSCN solution at 37 °C, monitored using both TNB (○) and spectrophotometric analysis (●) at 376 nm using a cuvette with 5-cm pathlength. (B) The decomposition of HOSCN (100 (□) and 300 μM (○) in HBSS) was monitored over time at 37 °C using TNB as above. (C) Solutions of HOSCN (50 (■), 100 (□), 200 (●), and 300 μM (○)) in HBSS were added to HUVEC in a 1-ml volume in 24-well plates, supernatants were sampled at the indicated times, and the amount of oxidant (nanomoles) consumed by HUVEC was calculated using TNB by comparison with control wells (no cells). Results are the means ± SE of (B) five and (C) four experiments.

do not react with TNB and that the reaction with TNB is therefore a reliable measure of the amount of HOSCN in solution. Together these findings enabled us to determine a half-life of ~85 min with a first-order rate constant of 6.6× 10− 3 M− 1 min− 1 for the decomposition of a 1 mM stock solution of HOSCN at 37 °C and [SCN−] of 7.5 mM (Fig. 1A). However, the stability of HOSCN varied with the initial concentration of the solution, and lower concentrations that would be used for treatment of cells remained more stable over time (Fig. 1B). As it was established that the decomposition of HOSCN is a first-order reaction, the half-life of the oxidant should remain the same regardless of the starting concentration. However, we have found that unreacted SCN− may catalyze the decomposition of HOSCN (unpublished observations). Therefore, dilution of the stock solution will affect the decomposition of HOSCN because of dilution of the catalyst. Finally, we confirmed that the breakdown products of HOSCN (aged HOSCN), SCN−, and OCN− do not react with TNB (not shown). Rate of reaction of HOSCN with HUVEC MPO-derived oxidants have been shown to be variably cellpermeative and their rates of reaction with cellular components differ

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concentration; cells consumed approximately 30 and 15 nmol of HOSCN, respectively, in 180 min.

Oxidation of cellular thiols by HOSCN

Fig. 2. Oxidation of total cell protein thiols by HOSCN. HUVEC were treated with increasing concentrations of HOSCN in HBSS for 3 h and the level of total protein thiols was measured using fluorescence detection after derivatization with MBB. Results are expressed as a percentage of thiols present in control cells treated with HBSS. The means ± SE of three experiments are shown. *Statistically significant difference (P ≤ 0.05).

significantly [6,34,39,49,50,54]. We found that the reaction of HOSCN with HUVEC was time- and concentration-dependent (Fig. 1C). In our 1-ml reaction volume, 100,000 cells exposed to 300 nmol HOSCN consumed a maximum of ~50 nmol within 60 min, whereas with 50 nmol much less (~15 nmol) reacted with the cells over the entire 180-min time course (Fig. 1C). Cells treated with either 200 or 300 μM HOSCN eventually consumed the same amount of oxidant, although the rate of consumption differed. With 100 and 50 μM HOSCN the amount that reacted with cells was directly proportional to the initial

As HOSCN is particularly reactive with thiols we investigated the effects of HOSCN exposure on total cell protein thiol levels and on two highly susceptible intracellular thiols, GAPDH and GSH [32,49,55,56]. A relatively high dose of HOSCN was required to significantly affect the total level of protein thiols (Fig. 2). We found that around 80–90% of the protein thiols were oxidized by exposure to 300 μM HOSCN, and around 40% were oxidized with 200 μM HOSCN. This difference occurred despite the cells having consumed approximately the same amount of oxidant (Fig. 1C). This may indicate increased permeabilization of the cells by prolonged exposure to 300 μM HOSCN and selective targeting of thiols by HOSCN or may reflect the lack of recovery under conditions of more extreme oxidative stress. HOSCN also affected GAPDH activity, with the level of inactivation reflecting HOSCN consumption (Figs. 1C and 3A). In cells treated with 300 μM HOSCN the maximal consumption of ~ 50 nmol coincided with ~90% inactivation of GAPDH (Fig. 3A), and less GAPDH inactivation was observed in cells exposed to 200 μM HOSCN (around 25% reduction after 180 min; Fig. 3A). Lower concentrations of HOSCN had no effect on the activity of GAPDH. GAPDH inactivation after 1 h exposure to HOSCN was recovered by reincubating the cells in full medium. The inactivation caused by 200 μM was fully reversed within 60 min (Fig. 3B). Inactivation caused by 300 μM HOSCN was also partially reversible, with levels returning to around 70% of initial activity within 60 min (Fig. 3B), but no activity was recovered after a longer exposure to 300 μM oxidant (not shown). Monitoring GSH also revealed a time- and concentration-dependent decrease after exposure to HOSCN (Fig. 3C): 300 μM HOSCN

Fig. 3. Oxidation of intracellular thiols by HOSCN. HUVEC were treated with HOSCN in HBSS: 50 (■), 100 (□), 200 (●), and 300 μM (○) for up to 180 min. (A and B) GAPDH and (C and D) GSH were measured. GAPDH activity was measured from the rate of NADH oxidation at 340 nm during the “reverse” reaction of the enzyme. The amount of GSH was determined by reverse-phase HPLC with fluorescence detection after derivatization with MBB. Recovery of GAPDH (B) and GSH (D) was measured after 1 h exposure to the oxidant followed by incubation in fresh medium for 5 h. Results are expressed as a percentage of the GAPDH activity or GSH present in control cells treated with HBSS. Average control levels of GSH were 2.13 ± 0.43 nmol per 100,000 cells. The means± SE of at least three experiments are shown.

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Fig. 4. Morphology changes upon exposure to HOSCN. Cells were either untreated (control) or treated with the indicated concentrations of HOSCN in HBSS. Morphology was monitored by phase-contrast microscopy at 1 and 3 h and then again after overnight incubation in fresh full medium (★). Black arrows indicate distinct points of adhesion to the plate surface and white arrows indicate areas of blebbing and plasma membrane rupture. Images are representative of at least three experiments.

caused substantial loss (around 80% by 180 min), with less oxidation at 200 μM and virtually no effect on GSH at b200 μM (Fig. 3C). After exposure to ≤200 μM HOSCN, the small drop in GSH levels could be reversed by a 60-min incubation in full medium (Fig. 3D). However there was minimal recovery of GSH levels after exposure to 300 μM HOSCN (Fig. 3D), even after overnight incubation in full medium (not shown). Considering the oxidation of cell thiols by HOSCN, the amount of oxidant taken up by the cells corresponds to the amount of loss from the total thiol pool (sum of protein thiols and GSH). For example, when 50 nmol of HOSCN reacted with cells, around 50 nmol of protein thiols and 2 nmol of GSH were lost. This observation supports the suggestion that HOSCN enters the cells and reacts specifically with thiol groups.

and 300 μM HOSCN resulted in marked morphological changes atypical of the effects seen with other oxidants (Figs. 4C–J). The cells appeared to shrink from the plate surface but did not detach (Figs. 4C and G), as is common for other oxidants, including those generated by MPO [34]. Cells exposed to 300 μM HOSCN remained attached despite showing distinct blebbing that suggested rupture of the plasma membrane (Fig. 4H). After oxidant exposure for up to 3 h, the cells almost fully recovered their usual morphology after overnight incubation with full medium (Figs. 4E, F, and I), but cells treated with 300 μM for 3 h did not recover, and after 24 h extreme morphology changes were seen (Fig. 4J). Most notably the cells remained attached to the surface. Morphology changes did not show features typical of apoptosis, indicating that HOSCN may induce an alternative form of cell death.

Morphology changes upon exposure to HOSCN

HOSCN does not induce apoptosis

When examined by phase-contrast microscopy, no effect was seen on cell morphology with 50 (not shown) or 100 μM oxidant, even at extended incubation times (Figs. 4A and B). However, exposure to 200

Flow cytometry analysis confirmed that exposure to ≤200 μM HOSCN for 1 or 3 h did not affect cell viability, with very little uptake of either annexin V or PI (Fig. 5A). However, very few intact cells could

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Fig. 6. Inactivation of caspase activity by HOSCN in vitro. Lysates were prepared from cells incubated with SFM for 7 h to induce apoptosis. These were treated with HOSCN (4, 10, 20, 40, 100, or 200 μM final concentration) for 1 min, and then caspase activity was determined in the presence or absence of 5 mM DTT. Results are expressed as % of control lysate not treated with HOSCN. Open bars, minus DTT; hatched bars, plus DTT (n = 3).

Fig. 5. HOSCN does not induce apoptosis. Cells were either untreated (control) or incubated with the indicated concentrations of HOSCN in HBSS. (A) Cells were incubated in HBSS for 1 or 3 h and then washed and full medium was replaced for 2.5 h. The number of nonviable cells was determined by flow cytometry using annexin V and PI fluorescence (representative of at least five experiments). (B) Cells were incubated in HBSS for 1 or 3 h and then harvested and the levels of caspase 3 activity were assayed using DEVD-AMC. Results are the means ± SE of four experiments. *Statistically significant difference from cells in HBSS (P ≤ 0.05). (C) Cells were incubated in HBSS for 5 h and the amount of caspase 3 cleavage was assessed by SDS–PAGE and Western blotting with antibodies to uncleaved and cleaved caspase 3. For a negative control (− ve) cells were untreated and for a positive control (+ ve) cells were incubated with SFM for 5 h to induce apoptosis. Arrow indicates cleaved caspase 3 band (n = 3).

be harvested after 3 h with 300 μM HOSCN and the majority were PIpositive (Fig. 5A), confirming loss of cell viability and suggesting membrane rupture. Long-term viability was not affected by oxidant exposure below 200 μM (trypan blue exclusion of 65 and 90% of cells exposed to 200 and 50 μM HOSCN, respectively, measured more than 72 h after the removal of oxidant). When cells were exposed to HOSCN in HBSS no caspase 3 activation was detected with any oxidant dose (Fig. 5B). In fact, inhibition of caspase activity was seen after short-term exposure (1 h) to even the lowest concentration of HOSCN, implying that the basal level of caspase 3 activity was inhibited. Apoptosis is induced in

HUVEC incubated in HBSS for several hours, with activation of caspase 3 [52], and this was also observed in our control cells (Fig. 5B). This activation was prevented by all doses of HOSCN, suggesting that it is able to block caspase 3 activation (Fig. 5B). Caspase 3 has been shown to be inactivated by oxidation of the active-site cysteine [53]. Therefore we investigated whether HOSCN directly inhibited the activity of the processed form or prevented cleavage and activation of the proenzyme. Western blotting with antibodies to both the proenzyme (inactive) and the cleaved (active) forms of caspase 3 revealed no processing of the protein in the presence of HOSCN (Fig. 5C), suggesting that oxidant exposure prevents caspase 3 cleavage and activation. To determine whether HOSCN can oxidize and directly inactivate activated caspase 3 we added HOSCN to extracts of apoptotic HUVEC. Increasing concentrations of oxidant decreased caspase 3 activity, and this was reversible by the addition of DTT (Fig. 6). As all previous caspase 3 assays contained 5 mM DTT in the reaction buffer, these results suggest that direct inhibition of the active site cysteine is unlikely to be the reason caspase 3 activity is undetectable in cells exposed to HOSCN. Low concentrations of HOSCN prevent apoptosis in HUVEC The above results suggest that HOSCN prevents caspase 3 activation and the induction of apoptosis. We therefore monitored the effect of HOSCN on caspase 3 activation in the presence of SFM, a strong inducer of apoptosis in HUVEC [52], and found that HOSCN inhibited activation in a concentration-dependent manner (Fig. 7A). Significant inhibition was seen with low concentrations of HOSCN that caused no loss of GAPDH activity or GSH nor a change in morphology. Lack of caspase 3 activity was mirrored by a lack of processing of procaspase 3 (Fig. 7B). To pursue this further we investigated whether HOSCN could block apoptosis when added after exposure to SFM. Addition of HOSCN to HUVEC for 2 h inhibited caspase 3 activity in cells pretreated for 5 h to induce apoptosis (Fig. 7C), and no cleaved form of the enzyme was present in any of the samples from HOSCN-treated cells (Fig. 7D). Recovery of caspase 3 activity after HOSCN exposure After HOSCN exposure, some recovery of GAPDH activity and GSH was possible (Figs. 3B and D). However, caspase 3 activity was more profoundly affected by exposure to HOSCN than were these other

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thiol-containing compounds (Fig. 5B). After HOSCN exposure for 1 h there was complete recovery of basal caspase 3 activity by reincubation in full medium for 5 h (Fig. 8A), but after a longer exposure with the oxidant the loss of activity could not be reversed (Fig. 8B). To eliminate the possibility that decomposition products of HOSCN (particularly OCN−) that might be generated over time or in the

Fig. 8. Recovery of basal caspase 3 activity after HOSCN exposure. HUVEC were treated with the indicated levels of HOSCN in HBSS for either (A) 1 or (B) 3 h and then washed, and full medium was added for 5 h. After the cells were harvested, caspase 3 activity was measured using DEVD-AMC. Results are presented as a percentage of caspase 3 activity in cells incubated with HBSS. Results are the means ± SE of (A) seven and (B) five experiments. *P ≤ 0.05, statistically significant difference from cells in HBSS.

reaction of HOSCN with H2O2 could be responsible for the loss of caspase 3 activity, we exposed HUVEC to solutions of aged HOSCN or reagent OCN− for 3 h. The level of caspase 3 activity in cells exposed to either the aged solution (Fig. 9A) or OCN− (Fig. 9B) was similar to that observed in cells treated with HBSS alone, and no morphology changes were observed with either treatment (not shown). This

Fig. 7. Low concentrations of HOSCN prevent apoptosis in HUVEC. Cells were treated with the indicated concentrations of HOSCN in SFM for 5 h and then harvested and (A) assayed for caspase 3 activity using DEVD-AMC or (B) analyzed by SDS–PAGE and Western blotting with antibodies to uncleaved and cleaved caspase 3. (Arrow indicates cleaved caspase 3; negative control (−ve), untreated cells; positive control (+ve), cells incubated with SFM for 5 h to induce apoptosis, n=3). To determine whether HOSCN could inhibit caspase 3 after induction of apoptosis HUVEC were incubated with SFM for 5 h and then with HOSCN (secondary treatment) for 2 h. Harvested cells were then analyzed (C) using DEVD-AMC for caspase 3 activity and (D) for cleavage of caspase 3. Cleaved caspase 3 was found in cells initially incubated with SFM for 5 h (+ve), but not in cells that were then exposed to HOSCN for 2 h. For (A) and (C) the results are the means±SE of four experiments. *P≤0.05, statistically significant difference.

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Fig. 10. Only high concentrations of HOSCN prevent subsequent caspase 3 activation. HUVEC were exposed to varying concentrations of HOSCN in HBSS (primary treatment) for 2 h and then washed, and apoptosis was initiated with SFM for 5 h. Cells were then harvested for caspase 3 activity assay using DEVD-AMC (means ± SE of three experiments shown).

Fig. 9. Effects of HOSCN breakdown products and cyanate on caspase 3 activation. (A) HUVEC were harvested 3 h after treatment with the indicated concentrations of fresh or decomposed HOSCN in HBSS and the level of caspase 3 activity was determined. (B) Reagent OCN− (hatched bars) did not inhibit caspase 3 activation initiated by incubation in HBSS (black bar). HOSCN (white bars) is shown in comparison. Results are the means ± SE of three experiments. *P ≤ 0.05, statistically significant difference from cells in HBSS.

indicates that neither decomposition products nor OCN− was responsible for the loss of activity observed previously. Lower concentrations of HOSCN do not prevent subsequent caspase 3 activation To determine whether the inhibition of caspase 3 activation was reversible, we examined the ability of HOSCN-treated HUVEC to undergo apoptosis once the oxidant was removed. This revealed that caspase 3 inhibition by ≤200 μM HOSCN was fully reversible, but that exposure to 300 μM for 2 h prevented the subsequent induction of maximal activity (Fig. 10). Discussion In this study we show that the mild oxidant HOSCN can have profound effects on endothelial cell function and that these effects are dependent on the timing and duration of oxidant exposure. The ability of HUVEC to undergo apoptosis after short-term exposure to HOSCN was effectively blocked by low concentrations of the oxidant. That this level of HOSCN exposure caused neither morphology changes nor detectable cell thiol oxidation suggests selective targeting of the

apoptotic pathway. Inhibition of apoptosis coincided with a lack of caspase 3 activation and cleavage and the inhibition was not reversible by DTT, suggesting that caspase 3 itself may not be the target. Given the preference of HOSCN for thiol groups [40–42], this effect is likely to be dependent on the oxidation of particular unidentified thiols. Thiol oxidation is postulated to be a major contributor to oxidative stress, yet the effects of HOSCN on HUVEC differed significantly from those seen with other oxidants. Exposure to MPO-generated oxidants or H2O2 generally results in detachment from the substratum and growth arrest, apoptosis, or necrotic cell death, depending on the concentration of oxidant [32,34]. The morphology of HOSCN-treated cells, which remained attached to the support matrix at distinct foci, differed markedly from the “popcorn-like” appearance of apoptotic cells [32,34]. This suggests that the manner of cell death differs from that seen with other oxidants and that detachment of cells from the substratum involves more than thiol oxidation. This could reflect differences in cell targets or extracellular matrix protein oxidation [57] and indicates that the cell focal adhesion sites are unaffected by HOSCN. Low-level exposure to MPO-derived oxidants and chloramines causes thiol oxidation, with loss of GAPDH and GSH and changes in signaling pathways and NF-κB activation [33,35–37]. Many of these effects are thought to occur via thiol or methionine oxidation, but the different effects of HOSCN suggest that thiol oxidation alone cannot account for all oxidative stress responses. Our finding that GAPDH was reversibly inactivated by HOSCN suggests the formation of a disulfide or a sulfenyl thiocyanate [9]. The irreversible loss of GSH, however, suggests that oxidation by HOSCN does not form GSSG or mixed disulfides. HOSCN does not generate glutathione sulfonamide [58] and the fate of the lost GSH remains unknown, though it may be exported from the cell. Interestingly, loss of GSH has been implicated in the specific ability of HOSCN to kill parasitic organisms [43]. It was also notable that a higher concentration of HOSCN compared to other MPO-derived oxidants was required to affect GAPDH and GSH [32,40,49,55,56]. This, together with our

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observation that most of the HOSCN entering the cell reacted with protein thiols, suggests a different thiol specificity of this oxidant. Similar results have been reported [59,60], and it was recently suggested that tryptophan residues are also slightly susceptible to oxidation by HOSCN in vitro and that this may correlate with protein unfolding [61]. In agreement with earlier studies demonstrating the need for a high dose of HOSCN to kill cultured human cells [43], our results indicate that HOSCN is likely to be less cytotoxic than other MPOderived oxidants, with prolonged exposure to 300 μM being necessary to induce a form of cell death that was independent of caspase 3 activity. This dose of oxidant could be achieved in a physiological setting in which there is likely to be prolonged exposure to an oxidant stress. However, the inhibition of apoptosis with as little as 15 nmol HOSCN suggests selective targeting of this pathway and is more likely to affect vascular function. These findings are consistent with other studies showing that the addition of SCN− inhibits H2O2-mediated apoptosis in HL60 human leukemia cells, probably through preferential generation of HOSCN by MPO [62], and that HOSCN induces a form of caspase-independent cell death in a murine macrophage-like cell line [60]. We noted that basal caspase 3 activity in control cells was also very susceptible to HOSCN. This basal activity is uncharacterized and may not reflect activated caspase. A similar effect has been referred to by others, with a reported decrease in basal caspase activity in a control cell population treated with HOSCN (unpublished results mentioned in [43]). Although it remains unclear which part of the apoptotic process is targeted, we have shown that HOSCN inhibits caspase 3 activation and may therefore lead to the inappropriate survival of damaged cells. It seems unlikely that caspase 3 is itself the oxidant target, as we showed that although it can be inactivated by HOSCN, this was reversible in the presence of DTT, which is routinely added to the assay buffer. However, caspase 3 activation is dependent on a number of upstream caspases and these may be important targets. Our results also indicate that the presence of HOSCN results in accelerated turnover of activated caspase 3, with rapid disappearance of the cleaved protein. Identification of the critical HOSCN target will be the focus of future studies. Whether inhibition of apoptosis occurs as a result of up-regulation of prosurvival pathways or whether the cells are diverted into a caspase-independent cell death is unclear. HOSCN can affect signaling pathways [30,31,43], and we found that all cells treated with lower doses of HOSCN appeared to remain viable after an apoptotic stimulus. Recovery from mitochondrial outer membrane permeabilization has been shown to occur in the absence of caspase activity when ATP levels are elevated, possibly because of a glycolysisdependent switch to autophagy [63,64]. We are currently investigating whether a similar mechanism occurs with HOSCN. In conclusion, we have shown that HOSCN exposure can potentially influence cell stress responses and may therefore have a role in determining disease outcome. Far from being a benign antibacterial oxidant, the generation of HOSCN by MPO in the blood vessel wall could affect cell signaling responses and inhibit the process of apoptosis, resulting in the inappropriate survival of damaged cells. These effects have important health implications for HOSCN generation as a cause of endothelial dysfunction in cardiovascular damage, as well as for its potential involvement in the prevention of apoptosis during oncogenic transformation.

Acknowledgments We thank Dr. William Titulaer for technical assistance with FACS analysis and Professor Christine Winterbourn for critical reading of the manuscript. This study was supported by a University of Otago Post-Doctoral Scholarship to S.M.B.

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