Cartilage specimens were obtained from OA patients who underwent total knee arthroplasty (64 ± 9 years, mean ± standard error, n = 34). Diagnoses were established according to American College of Rheumatology criteria 20. OA cartilage (femoral condyles and tibial plateaus) was obtained under aseptic conditions and carefully dissected from the underlying bone in each specimen. This project and the informed consent form were approved by the institutional Ethics Committee Board of the Hôpital du Sacré-Coeur de Montréal. OA chondrocytes were released from cartilage explants as described previously 18. Isolated chondrocytes were seeded at high density in culture flasks until confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/mL penicillin/100 mg/mL streptomycin (Invitrogen Canada Inc, Burlington, ON, Canada). First-passage chondrocytes were seeded in culture plates at 10⁵ cells/cm² and incubated for 48 hours in the above medium. Before the experiments, the medium was replaced by fresh medium containing 2% FBS and treated as indicated in the experimental protocols.

HNE-induced chondrocyte cytotoxicity was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay 21. Tests were performed in 96-well plates. Briefly, chondrocytes were incubated for 16 hours with increasing concentrations (0 to 30 μM) of HNE (Cayman Chemical Company, Ann Arbor, MI, USA) or with 30 μM HNE for increasing times of incubation in the presence or absence of 200 μM NAC (Sigma-Aldrich, Oakville, ON, Canada). To explore the signalling cascade in HNE-induced cell death, cells were incubated for 1 hour with the inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), 5-iodo-6-amino-1,2-benzopyrone, at 50 and 100 μM (INH₂BP; Sigma-Aldrich) or with anti-Fas/CD95 antibody at 20 μg/mL, followed by another incubation for 16 hours with 30 μM HNE. Then, the cells were incubated with 0.5 mg/mL MTT for 15 minutes at 37°C. Thereafter, 100 μL of solubilization solution (0.04 M HCl-isopropanol) was added. The amount of MTT formazan product was quantified by measuring of optical density at 570 nm with a microplate reader (BioTek Instruments, Winooski, VT, USA).

Apoptotic nuclear morphology was assessed by Hoechst 33258 incorporation (Sigma-Aldrich). Briefly, chondrocytes (10⁵ cells/cm²) were treated with or without 30 μM HNE for 16 hours. The cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes and then washed and stained with 10 μg/mL Hoechst 33258 in phosphate-buffered saline (PBS) at room temperature for 10 minutes. Hoechst-stained cells were analyzed by fluorescence microscopy.

Enzymatic caspase-8, -9, and -3 activities were measured with commercial kits. Chondrocytes (10⁵ cells/cm²) were treated with 30 μM HNE for increasing incubation times (0 to 16 hours). To measure caspase-8 and -9 activities, the cells were washed with PBS and resuspended in 100 μL of lysis buffer (R&D Systems, Minneapolis, MN, USA), left on ice for 10 minutes, and centrifuged. Protein concentration of the supernatants was measured according to the bicinchoninic acid method (Pierce, Rockford, IL, USA). Total proteins (50 μg) were reacted with 200 μM IETD-pNA or LEHD-pNA substrate in the presence of 100 μL of reaction buffer. To quantitate caspase-3 activity, the cells were washed with PBS and lysed in 100 μL of lysis buffer (Sigma-Aldrich), left on ice for 15 minutes, and centrifuged. Total proteins (5 μg) were reacted with 200 μM DEVD-pNA substrate in the presence of 100 μL of reaction buffer. After 16 hours of incubation at 37°C, p-nitroanilide release was measured at 405 nm for caspase-3, -8, and -9.

Chondrocytes (10⁵ cells/cm²) were treated with 30 μM HNE for increasing incubation times (0 to 16 hours). The protein expression of the antiapoptotic Bcl-2 was assayed in cell extracts with a Bcl-2 enzyme-linked immunosorbent assay (ELISA) kit (catalogue number QIA23; Calbiochem, now part of EMD Biosciences, Inc., San Diego, CA, USA) according to the manufacturer's instructions. The Bcl-2 level was expressed in units per milligram of protein.

Cytoplasmic histone-associated DNA fragments were quantitated with a Cell Death Detection ELISA^PLUS kit (Roche Applied Science, Laval, QC, Canada) according to the manufacturer's recommendations. Briefly, chondrocytes (2 × 10⁶ cells) were treated for 16 hours with increasing HNE concentrations (0 to 30 μM) with or without 200 μM NAC. After incubation, the cells were lysed with lysis buffer for 30 minutes and centrifuged at 200 g for 10 minutes. The supernatant and a mixture of anti-histone-biotin and anti-DNA-peroxidase were added to streptavidin-coated microplates and incubated for 2 hours at room temperature. After adding the substrate, absorbance was measured at 405 nm.

To measure cytochrome c release, chondrocytes (1 × 10⁶ cells) were treated for 16 hours with increasing HNE concentrations (0 to 30 μM) with or without 200 μM NAC, washed with PBS, and resuspended in 1 mL of buffer (220 mM mannitol, 70 mM sucrose, 10 mM Hepes KOH, pH 7.4, 10 mM ethylenediaminetetraacetic acid [EDTA], 10% glycerol). The cell suspension was incubated on ice for 15 minutes and centrifuged at 10,000 g for 15 minutes. The pellet containing the mitochondria was resuspended in 200 μL of the above buffer and served to assess citric acid activity as described below. Cytochrome c level was measured in cytosolic fractions (supernatants) with a Cytochrome c ELISA kit (EMD Biosciences, Inc.) according to the manufacturer's directions. Cytochrome c level was expressed in nanograms per milligram of protein.

Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (mNADP⁺-ICDH) activity was assessed in mitochondrial fractions prepared, as described above, from chondrocytes treated with 30 μM HNE for 16 hours. The assay was performed in the presence of 5 mM isocitrate, 1 mM NADP, and 2 mM MgCl₂. Activities were expressed in units per milligram of protein, where 1 unit was defined as the amount of enzyme catalyzing the conversion of 1 μmol substrate per minute at 37°C.

ATP level was assessed in cellular extracts from chondrocytes treated with 30 μM HNE for 16 hours with an ATP Assay kit from EMD Biosciences, Inc. The results were expressed as picomoles per milligram of proteins.

Chondrocytes (2 × 10⁶ cells) were incubated for increasing time periods (0 to 16 hours) with 30 μM HNE. The cells were washed with PBS and centrifuged at 800 g for 5 minutes. Pellets were resuspended in buffer (0.4 M 2-[N-morpholino] ethanesulphonic acid, 0.1 M phosphate, 2 mM EDTA) and centrifuged at 10,000 g for 15 minutes. Glutathione (GSH) and oxidized GSH (GSSG) levels were quantified with a Glutathione Assay Kit (Cayman Chemical Company) according to the manufacturer's directions. Values were expressed as the GSSG/[GSSG+GSH] ratio.

Chondrocytes were incubated for increasing time periods (0 to 16 hours) with 30 μM HNE or with increasing concentrations of HNE (0 to 30 μM) for 16 hours with or without 200 μM NAC. Cellular protein extract (20 μg) or nuclear protein (5 μg), isolated as previously described 19, was subjected to discontinuous 4% to 12% SDS-PAGE as previously described 18. The primary antibodies used were rabbit anti-human PARP, rabbit anti-human apoptosis-inducing factor (AIF), rabbit anti-human Bax, rabbit anti-human caspase-3, -9, anti-Akt (EMD Biosciences, Inc.), rabbit anti-human caspase-8, anti-p53, and mouse anti-human Fas/CD95 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GSTA4-4 (Abnova, Taipei, Taiwan). After serial washes, primary antibodies were detected by goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Specific signals were visualized with enhanced chemiluminescence detection kits (Pierce).

GSTA4-4 small interfering RNA (siRNA) and randomly sequenced siRNA as negative controls were purchased from Ambion (Austin, TX, USA). Wild-type and mutant GSTA4-4 expression plasmids were generously provided by Dr Sanjay Awasthi 17 (University of North Texas Health Science Center, Fort Worth, TX, USA). Subconfluent chondrocytes were transiently transfected by Lipofectamine 2000™ reagent (Invitrogen Canada Inc) according to the manufacturer's protocol. Briefly, transfections were conducted for 6 hours with DNA lipofectamine complexes containing 10 μL of lipofectamine reagent, 100 nM GSTA4-4 siRNA (a mixture of three siRNAs targeting the GSTA4-4 gene) or randomly sequenced siRNA, or 2 μg of DNA plasmid and 0.5 μg of pCMV-β-gal (as a control of transfection efficiency). After washing, experiments were performed in 2% FBS fresh medium supplemented or not supplemented with 30 μM HNE. Then, cell viability and GSTA4-4 expression were analyzed by the MTT method and Western blotting, respectively, as described previously. β-gal level was measured with ELISA kits from Roche Diagnostics Canada (Laval, QC, Canada).

Chondrocytes were cultured for 16 hours in 24-well plates at 5 × 10⁵ cells per well in 2% FBS/DMEM in the presence or absence of 30 μM HNE. The culture media were replaced by 2% FBS/glucose-free DMEM containing 10 μCi/mL 2-deoxy-D-[³H]-glucose. Then, plates were incubated for 20 minutes at 37°C. Subsequently, the media were aspirated and the cells were washed three times with cold PBS. They were then lysed with 400 μL/well of cell death lysis buffer (EMD Biosciences, Inc.) for 15 minutes. Volumes of 300 μL of cell lysates were transferred to scintillation vials, and radioactivity was measured by scintillation counting. The data are expressed as counts per minute (cpm) per milligram of proteins.

Results were expressed as the mean ± standard error of the mean of eight specimens, and assays were performed in three independent experiments. Statistical analysis was performed using the two-tailed paired Student t test, and a difference of less than or equal to 0.05 was considered significant.

To investigate the effect of HNE on chondrocyte apoptosis, we first documented HNE cytotoxicity by MTT assay. After 16 hours of incubation, up to 10 μM HNE did not alter cell viability, but 20 and 30 μM HNE was cytotoxic and significantly decreased cell viability by approximately 50% and 52%, respectively. Furthermore, pre-treatment with 200 μM NAC for 1 hour before adding 30 μM HNE (Figure 1a) completely prevented HNE-induced cell death. However, the addition of anti-Fas/CD95 (20 μg/mL) (Figure 1b) or the PARP inhibitor INH₂BP (50 and 100 μM) (Figure 1c) 1 hour before incubation with 30 μM HNE partially prevented HNE-induced cell death. Next, Hoechst 33342 staining was undertaken to examine the morphological changes occurring to chondrocytes. Upon exposure to 30 μM HNE, numerous chondrocytes exhibited characteristics typical of apoptosis with highly condensed nuclei (Figure 2). In contrast, very few apoptotic cells were observed in untreated cells.

Caspases play important roles in the terminal execution of apoptosis induced by various stimuli. Caspase-3, -8, and -9 activities were measured with commercial kits, and their protein cleavage was detected by Western blot analysis. Compared with untreated cells, HNE incubation resulted in a time-dependent increase of caspase-8 activity (Figure 3a). HNE significantly induced caspase-9 activity after 2, 4, and 8 hours of incubation and significantly induced caspase-3 activity after 4 and 8 hours of incubation. At 16 hours, both caspase-3 and -9 activities were reduced to the control level (Figure 3b, c). At the protein level, 20 and 30 μM HNE decreased pro-caspase-8, -9, and -3 levels after 16 hours of incubation, probably via the cleavage process of the pro-caspase (Figure 3d). In contrast, the addition of 200 μM NAC prevented the HNE-induced caspase activation.

The Bcl-2 family is involved in apoptosis by regulating membrane permeability and induces cytochrome c release from mitochondria into the cytosol 222324. To investigate the effects of HNE on Bcl-2 and Bax expression, cells were treated with 30 μM HNE for increasing incubation times (0 to 16 hours) and then ELISA and Western blot experiments were performed. The level of the anti-apoptotic protein Bcl-2 was significantly decreased after 4 hours of incubation with 30 μM HNE (Figure 4a). In contrast, HNE at this concentration increased the apoptotic protein Bax after 4 and 8 hours of incubation and remained elevated at 16 hours of incubation (Figure 4b). We then analyzed the effect of HNE on cytochrome c release from mitochondria to the cytosol. As shown in Figure 4c, the cytochrome c level in cytosolic fractions significantly increased in chondrocytes treated with 20 or 30 μM HNE for 16 hours (Figure 4c). However, the pre-incubation of chondrocytes with 200 μM NAC prevented HNE-induced DNA fragmentation, PARP activation, and AIF translocation to the nucleus.

Nuclear damage is very important in cell death. Therefore, we further studied the role of HNE in DNA fragmentation, PARP activation, and AIF translocation to the nucleus. Chondrocytes were exposed to HNE in the presence or absence of 200 μM NAC. The extent of nuclear DNA fragmentation was measured quantitatively by ELISA. As shown in Figure 5a, the level of cytoplasmic histone-associated DNA fragments was increased when DNAs were extracted from chondrocytes after exposure to 20 and 30 μM HNE for 16 hours compared with the control. In contrast, cells pre-treated with 200 μM NAC were protected against HNE-induced DNA fragmentation. PARP activation and AIF translocation to the nuclei during apoptosis are implicated on a large scale in DNA fragmentation and peripheral chromatin condensation. As shown in Figure 5b, 30 μM HNE induced PARP cleavage and AIF translocation in the nuclei after 4 hours of incubation. Moreover, pre-treatment with 200 μM NAC blocked PARP cleavage and AIF translocation.

To study the molecular mechanisms involved in the induction of cell death by HNE, we examined Akt activity as well as Fas/CD95 and p53 protein expression in chondrocytes after different incubation times. Western blot analysis indicated that 30 μM HNE elicited Fas/CD95 and p53 protein expression but, in contrast, reduced survival signalling, including the phosphorylated form of Akt (Figure 6). Total Akt was unchanged.

In the next series of experiments, we determined whether the redox status and energy metabolism of chondrocytes could be implicated in part in HNE-induced cell death. As illustrated in Figure 7, 30 μM HNE decreased the reduced form of GSH and the reducing equivalent, NADPH, needed for its regeneration (Figure 7a), after 16 hours of incubation. We additionally established that HNE, at this concentration, evoked a significant diminution of energy depletion through inhibition of mNADP⁺-ICDH activity (Figure 7b), glucose uptake (Figure 7c), and intracellular ATP synthesis (Figure 7d).

GSTA4-4 is a known aldehyde-detoxifying enzyme as has been shown by previous studies 1617. To assess the functional consequences of GSTA4-4 inhibition versus overexpression in chondrocytes, the cytotoxicity of 30 μM HNE was evaluated by MTT cytotoxicity assay. First, the ablation of GSTA4-4 with GSTA4-4 siRNA in isolated chondrocytes augmented the HNE-induced cell mortality as measured by MTT assay at 4, 8, and 16 hours of incubation (Figure 8a). These results indicate that GSTA4-4 offers a significant protection against HNE-induced DNA damage in chondrocyte cells and that siRNA ablation of this enzyme augments the HNE-induced cell death. The increase in cell mortality in transfected chondrocytes with GSTA4-4 siRNA would be attributed to the inhibition of GSTA4-4 expression by more than 80% as compared with control siRNA (Figure 8b). Second, we tested whether the increased HNE-metabolizing capacity conferred on these cells by transfection of GSTA4-4 expression vectors could reverse the cytotoxic effects of HNE. Our data showed that GSTA4-4 overexpression provided cell resistance to direct HNE cytotoxicity (Figure 8c). Western blot analysis of cell extracts with the polyclonal antibody against GSTA4-4 revealed a strong band in cellular extracts of chondrocytes transfected with GSTA4-4 and a weak signal in untransfected cells (Figure 8d).