Do Oxidized Polyunsaturated Fatty Acids Affect Endoplasmic Reticulum Stress-Induced Apoptosis in Human Carotid Plaques?

Abstract

Macrophage apoptosis is involved in atherosclerotic plaque development. The aim of this study was to evaluate the interrelationship between macrophage apoptosis and the endoplasmic reticulum (ER) stress in the tissue around the necrotic core (TANC) and in the periphery (P) of the same carotid plaques derived from patients undergoing carotid endarterectomy. Apoptosis was significantly higher in TANC than in P (p<0.001). mRNA and protein expression of the protein kinase-like ER kinase (Perk) and the nuclear erythroid-related factor 2 (Nrf2)-related survival genes was significantly higher in P than in TANC (p<0.01), while CCAAT/enhancer-binding protein homologous protein (Chop) and the apoptosis-related genes were higher in TANC than in P (p<0.01). The TANC extract was characterized by significantly higher concentrations of oxidized derivatives of polyunsaturated fatty acids (PUFAs) than the P extract (p<0.01). When THP-1 cells were incubated with P or TANC extracts there was a dose-dependent increase of Perk and Nrf2 or of Chop and of the apoptosis-related genes, respectively (p<0.01). Our observations lead to the hypothesis that ER stress induced by oxidized derivatives of PUFAs may promote macrophage apoptosis in TANC and favor the expansion of the necrotic core of the plaques, a major feature responsible for its disruption and acute luminal thrombosis. Antioxid. Redox Signal. 21, 850–858.

Introduction

Macrophage apoptosis is an important characteristic of atherosclerotic plaque development (4). In advanced lesions, when phagocytes cannot dispose of the apoptotic cells in an efficient manner, they may become necrotic thereby contributing to necrotic core (NC) formation and to atherothrombotic disease (4). Endoplasmic reticulum (ER) stress is an important event during the initiation, development, and clinical progression of atherosclerosis (8). When ER stress occurs leading to the accumulation of unfolded proteins, the three ER transmembrane sensors are activated to initiate adaptive responses (8). These sensors include the inositol-requiring kinase 1, the transcriptional factor activating transcription factor 6, and the protein kinase-like ER kinase (Perk). All three sensors are maintained in an inactive state through the interaction of their N-terminus with the glucose-regulated protein 78 kDa (GRP78) (8). When unfolded proteins accumulate in the ER, GRP78 releases these sensors to allow their oligomerization and thereby initiates the unfolded protein response (UPR) (8). The basic leucine zipper nuclear erythroid-related factor 2 (Nrf2) is a direct Perk substrate and an effector of Perk-dependent cell survival (2). Nrf2 binds to the antioxidant response element (ARE) to activate transcription of genes encoding detoxifying enzymes, including heme-oxygenase-1 (Ho-1) and γ-glutamylcysteine synthetase (γ-Gcs) (2). Once the UPR fails to control the level of unfolded and misfolded proteins in the ER, ER-initiated apoptotic signaling is induced with the activation of the death factor, CCAAT/enhancer-binding protein homologous protein (Chop) (8). In this context, reduced apoptosis and plaque necrosis have been shown in mice lacking Chop (9), providing direct evidence for a causal link between the ER stress effector Chop and plaque progression.

Innovation

The tissue around the necrotic core (TANC) of carotid plaques is characterized by an abnormal amount of apoptotic cells. This phenomenon may be related to a sustained endoplasmic reticulum (ER) stress since there is an abundance of the CCAAT/enhancer binding protein homologous protein (Chop) and apoptosis-related gene expression in TANC, while the protein kinase-like ER kinase (Perk) and survival genes prevail in periphery (P). This specular expression may be driven by different concentrations of oxidized derivatives of polyunsaturated fatty acids that, by binding to human thromboxane A2 (TP) and G2A receptors, may determine the direction toward cell apoptosis or survival. So, ER stress may promote macrophage apoptosis in TANC and favor the expansion of necrotic core, a feature responsible for its disruption and luminal thrombosis.

In ER-stressed macrophages, Chop has been demonstrated to induce ER oxidase 1α, which activates inositol triphosphate receptor-mediated release of calcium into the cytosol (8) and the subsequent activation of a calcium/calmodulin-dependent protein kinase II, which triggers apoptosis through different mechanisms (8).

Oxidized derivatives of arachidonic and linoleic acids have already been identified in human atherosclerotic plaques as markers of oxidative stress (7) and supposed to function as second hits to trigger apoptosis in macrophages exposed to low levels of ER stress (8). Some of these oxidized derivatives of arachidonic and linoleic acids such as 8-iso-prostaglandin-F2α (8-iso), the prototype of F2-isoprostanes, 9-hydroxyoctadecadienoic acid (9-HODE), and 15-hydroxyeicosatetraenoic acid (15-HETE) have also been shown to be mediators of important biological effects through their binding to the human thromboxane A2 (TP) receptor (1) and G2A receptor (6).

We have recently demonstrated that some oxidized derivatives of polyunsaturated fatty acids (PUFAs) contained in the tissue around the necrotic core (TANC) of human carotid plaques may have a role in the expansion of NC, by inducing defective efferocytosis (3). The aims of this study were to evaluate the interrelationship between macrophage apoptosis and the ER stress in TANC and in the periphery (P) of human carotid plaques and whether oxidized derivatives of PUFAs may have a role in promoting ER-induced macrophage apoptosis. Therefore, in TANC and in P of the same carotid plaques derived from patients undergoing carotid endarterectomy, we first quantitated apoptotic cells, the expression of Perk and Chop together with some genes correlated with apoptosis and Nrf2/ARE survival pathway, and the concentration of 8-iso, 9-HODE, and 15-HETE. Then, in the in vitro study, we evaluated the effect of TANC, P, and of normal artery (NA) extracts on the expression of Perk, Chop, and of apoptosis and Nrf2/ARE genes in macrophage-like THP-1 cells. Finally, we verified if 8-iso, 9-HODE, and 15-HETE increase the expression of Perk and Chop by binding to the TP and G2A receptors and the subsequent mobilization of intracellular Ca²⁺ (Ca²⁺i).

Apoptotic cells and expression of Perk, Chop, and of the genes correlated with Nrf2/ARE pathway and apoptosis in NA, and in P and TANC of human carotid plaques

Figure 1 shows a representative section of segments derived from P (Fig. 1A) and TANC (Fig. 1B) of the carotid plaques. Positively macrophage apoptotic-stained cells resulted much higher in TANC than in P sections (p<0.001) (Fig. 1C). The mRNA and protein expression of Perk, Chop, and of survival genes (Nrf2, Ho-1, and γ-Gcs) resulted significantly higher in P and TANC of human carotid plaques than in NA (p<0.01) (Fig. 2A, B); the same genes were significantly more expressed in P than in TANC (p<0.01), while only Chop was higher in TANC than in P (p<0.01) (Fig. 2A, B). The apoptosis-related genes such as caspase (Casp)3, Bcl-2-associated X protein (Bax), tumor suppressor protein 53 (P53), tumor necrosis factor receptor 1 (Tnfr1) were found to be significantly more expressed in P and TANC than in NA (p<0.01), while B-cell leukemia/lymphoma-2 (Bcl-2) expression was similar in NA, P, and TANC (Fig. 2C, D). Casp3, Bax, P53, and Tnfr1 resulted significantly more expressed in TANC than in P (p<0.01) (Fig. 2C, D). These results are in line with previous evidences that have indicated that UPR is chronically activated in cells of the atherosclerotic vascular wall and, in particular, in macrophages (8) and with the data of Myoishi et al. (5), who demonstrated that robust Chop expression and lesional apoptosis were more identified in advanced vulnerable plaques.

FIG. 1.

Apoptotic cells in the periphery (P) and in the tissue around necrotic core (TANC) of human carotid plaques. (A, B) are representative sections of segments derived from P and TANC (magnification 40×). (C) indicates the average number of positively stained cells in P and TANC. The number of apoptotic cells is expressed as a percentage of total number of the counted nuclei. *p<0.001 versus P.

FIG. 2.

Expression of Perk, Chop, and of the genes correlated with Nrf2/ARE survival pathway and apoptosis in normal artery (NA), in periphery (P), and in tissue around the necrotic core (TANC) of human carotid plaques. mRNAs of Perk, Chop, Ho-1, γ-Gcs, and Nrf2 (A), Casp3, Bax, P53, Tnfr1, and Bcl-2 (C) were analyzed by quantitative real-time PCR. Normalized gene expression levels were given as the ratio between the mean value for the target gene and that for the β-actin in each sample. Figure also shows a representative western blot analysis for the indicated proteins and the average quantification obtained by densitometric analysis (B, D). Data on western blot analysis are expressed as the density ratio of target protein [1] to control (β-actin) [2] in arbitrary units. Data represent the mean±SD of measurements performed in triplicate. *p<0.01 versus NA; ^† p<0.01 versus P. Perk, protein kinase-like ER kinase; Chop, CCAAT/enhancer-binding protein homologous protein; Ho-1, heme-oxygenase-1; γ-Gcs, γ-glutamylcysteine synthetase; Nrf2, nuclear erythroid-related factor 2; Casp, caspase; Bax, Bcl-2-associated X protein; P53, tumor suppressor protein 53; Tnfr1, tumor necrosis factor receptor 1; Bcl-2, B-cell leukemia/lymphoma 2; ARE, antioxidant response element; ER, endoplasmic reticulum.

Quantitative analysis of the components of NA, P, and TANC extracts and their effect on the expression of Perk, Chop, and of the genes correlated with survival (Nrf2/ARE pathway) and apoptosis in THP-1 cells

The quantitative analysis of NA, P, and TANC extracts revealed that F2-isoprostanes (m/z 353.2), HETEs (m/z 319.2), and HODEs (m/z 295.3) were the major components of TANC and P extracts, their concentrations being significantly higher in TANC than in P (p<0.01) and NA (p<0.001) (Table 1); HETEs and HODEs also resulted higher in P than in NA (p<0.05) (Table 1).

Table 1.

Concentrations of Total F2-Isprostans, HETEs, and HODEs in NA, P, and in TANC of Carotid Plaques

	NA	P	TANC
Total F2-isoprostances	0.08±0.03	0.1±0.03	0.18±0.04^* ^,†
total HODEs	4.9±1.6	6.2±2.5^*	13.8±2.9^* ^,†
total HETEs	1.2±0.5	1.9±0.8^*	3.3±0.8^* ^,†

Data are expressed as mean±SD (ng compound/mg tissue).

p from<0.05 to<0.001 versus NA; ^† p<0.01 versus P.

HETEs, total hydroxyeicosatetraenoic acids; HODEs, total hydroxyoctadecadienoic acids; NA, normal artery; P, periphery; TANC, tissue around the necrotic core.

To understand if the oxidative derivatives of PUFAs present in the extracts were able to stimulate the expression of Perk and Chop and of the genes correlated with the Nrf2/ARE pathway and apoptosis, THP-1 cells were incubated with increasing amounts of NA, P, and TANC extracts. As shown in Figure 3, there was a dose-dependent increase of both Perk and Chop only after incubation with P (p<0.05) and TANC extracts (p<0.01). The rise, however, was much more evident for Perk when THP-1 cells were incubated with P, and for Chop when the cells were incubated with TANC (p<0.01). P also determined a dose-dependent increase of Nrf2 and Ho-1 (p<0.01), while TANC induced a less, but nondose-dependent increase (p<0.05). As for the genes correlated with apoptosis, both TANC and P dose dependently increased Bax and Tnfr1 expression (p from<0.001 to<0.01), but the rise was much higher by using TANC than P (p<0.001). The highest increase of Chop and of the genes correlated with apoptosis therefore was caused by TANC, while P determined the maximum increase of Perk and of the genes correlated with the Nrf2/ARE survival pathway. Even if preliminary, these results suggest that the concentrations of these oxidative derivatives may play a role in determining the direction toward apoptosis or survival of THP-1 cells.

FIG. 3.

Dose–response (20 and 40 μl/ml medium) effect of the extracts of normal artery (NA), periphery (P), and tissue around necrotic core (TANC) of human carotid plaques on the expression of Perk, Chop, Nrf2, Ho-1, Bax, and Tnfr1 in THP-1 cells. mRNA expression of Perk and Chop (A), Nrf2 and Ho-1 (C), Bax and Tnfr1 (E) was analyzed by quantitative real-time PCR after incubation of THP-1 cells with NA, P, and TANC for 24 h. Normalized gene expression levels were given as the ratio between the mean value for the target gene and that for the β-actin in each sample. Figure also shows a representative western blot analysis for the indicated proteins and the average quantification obtained by densitometric analysis (B, D, and F). Data on western blot analysis are expressed as the density ratio of target protein [1] to control (β-actin) [2] in arbitrary units. Data represent the mean±SD of measurements performed in triplicate in six different occasions. *p from<0.05 to<0.01 versus basal; ^† p<0.01 versus 20 μl; ^‡ p from<0.01 to<0.001 versus 20 μl P; ^§ p from<0.01 to<0.001 versus 40 μl P.

Role of TP and G2A receptors on the expression of Perk, Nrf2, Bax, and Chop induced by TANC extract in THP-1 cells

To examine whether the fatty acid oxidative products present in plaque extract stimulated THP-1 cells by binding to TP or G2A receptors, we first incubated THP-1 cells with TANC extract, 8-iso, and TP receptor agonist U46619 and TP receptor antagonist SQ29512. Figure 4A and B shows that all these stimuli determined a significant increase of mRNA and protein expression of Bax, Chop, Perk, and Nrf2 (p from<0.01 to<0.001). The rise induced by TANC and 8-iso was significantly reduced by SQ29512, a specific TP antagonist (p<0.01). Then, we incubated THP-1 cells with TANC and with a mixture of 9-HODE and 15-HETE and looked at the effect of preincubation with the G2A-specific antibody. Figure 4C and D shows that the mRNA and protein expression of Bax, Chop, Perk, and Nrf2 expression was significantly induced by TANC and by a mixture of 9-HODE and 15-HETE (p from<0.01 to<0.001). The preincubation of the cells with an anti-G2A-specific antibody determined a significant decrease of the expression of Bax, Chop, Perk, and Nrf2 (p<0.01). TP receptors are membrane-bound, G-protein-coupled, seven-transmembrane receptors distributed widely in the cardiovascular systems. In addition to platelets and endothelial cells, TP receptors are also expressed in other cell types involved in atherothrombosis, such as smooth muscle cells (1). By binding to TP receptors, F2-isoprostanes have already been reported to promote endothelial cell activation, that is, adhesion molecule expression, vascular smooth muscle cell contraction, and platelet aggregation, thereby accelerating the progression of atherosclerotic lesions (1). The G2A receptor is a G-protein-coupled receptor that has been shown to function as a receptor for oxidized free fatty acids (6). HODEs and HETEs have recently been shown to increase the expression of Adam17 in THP-1 cells, an enzyme involved in defective efferocytosis (3). On the basis of these results, we are tempted to speculate that some oxidative derivatives of PUFAs present in TANC may increase the expression of Chop, Perk, and of the genes correlated with apoptosis by binding to TP and G2A receptors.

FIG. 4.

Effect of tissue around necrotic core (TANC), 8-iso, U46619, 9-HODE, 15-HETE, and of TP and G2A receptor antagonists on the expression of Bax, Chop, Perk, and Nrf2 in THP-1 cells. Cells were preincubated with SQ29512 (10 μM) (A), anti-G2A-specific antibody (Ab) (2 μg/ml), and Ab control (ctrl) (2 μg/ml) (C) for 1 h and then incubated for 24 h with the TANC extract (40 μl/ml medium) (A, C), 8-iso (10 μM), U46619 (1 μM) (A), and with a mixture of 9-HODE and 15-HETE (10 μM) (C). Normalized gene expression levels were given as the ratio between the mean value for the target gene and that for the β-actin in each sample. Figure also shows a representative western blot analysis for the indicated proteins and the average quantification obtained by densitometric analysis (B, D). Data on western blot analysis are expressed as the density ratio of target protein [1] to control (β-actin) [2] in arbitrary units. Data represent the mean±SD of measurements performed in triplicate. *p from<0.01 to<0.001 versus control; ^† p from<0.01 to<0.001 versus TANC alone; ^‡ p<0.01 versus 8-iso alone; ^§ p<0.01 versus 9-HODE/15-HETE alone. 8-iso, 8-iso-prostaglandin-F2α; 9-HODE, 9-hydroxyoctadecadienoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; TP, human thromboxane A2.

Table 2.

Characteristics of the Subjects and of Atherosclerotic Plaques (n=41)

Age (mean±SD)	74 (±6.2)
Sex (male/female)	25/16
Cigarette smoking, n (%)	10 (24.4)
Diabetes, n (%)	9 (21.9)
Dyslipidemia, n (%)	15 (36.6)
Hypertension, n (%)	23 (56.1)
Treatments, n (%)
ACE inhibitors and/or AT1 receptor blockers and/or calcium antagonists	22 (53.7)
Oral antidiabetics	8 (19.5)
Statins	16 (39.0)
Aspirin	24 (58.5)
Clinical presentation, n (%)
Asymptomatic	41 (100)
Plaque type, n (%)
Fibrous cap atheroma	26 (63.4)
Thin fibrous cap atheroma	15 (36.6)

ACE, angiotensin-converting enzyme; AT, angiotensin.

Effect of TANC extract, 8-iso, 9-HODE, and 15-HETE on Ca²⁺i

We found that TANC extract dose dependently increased Ca²⁺i in THP-1 cells. As shown in Figure 5A, TANC extract evoked a dose-dependent Ca²⁺i mobilization in THP-1 cells (p<0.01) that was significantly higher than that induced by P and NA extracts (p<0.01). The Ca²⁺i mobilization was significantly reduced when THP-1 cells were incubated with the TP antagonist SQ29512 or with the anti-G2A-specific antibody (p<0.01) (Fig. 5B). We also observed a dose-dependent Ca²⁺i mobilization after incubation of THP-1 cells with increasing concentrations of the TP agonist U46619, 8-iso, and the mixture of 9-HODE and 15-HETE (Fig. 5C, D) (p<0.01). Again, the TP antagonist SQ29512 and the anti-G2A-specific antibody reduced Ca²⁺i mobilization (p<0.01). The fact that both the TP and G2A receptors, once activated by different ligands, are known to cause a rapid increase of Ca²⁺i (1, 6) and the demonstration that specific antagonists of the receptors counteract this phenomenon as well as the expression of Perk and Chop, may preliminarily indicate that the oxidized derivatives of PUFAs contained in TANC may promote apoptosis also through these receptors.

FIG. 5.

Effect of tissue around necrotic core (TANC), periphery (P), and normal artery (NA) extracts, 8-iso, 9-HODE, 15-HETE, U46619, and of TP and G2A receptor antagonists on Ca²⁺i concentration in THP-1 cells. Cells were preincubated for 1 h with SQ29512 (10 μM) (B–D), anti-G2A-specific antibody (Ab) (B, D), and then cells were stimulated with the TANC (A, B) and P extracts of carotid plaques (40 μl/ml medium) and with the extract derived from a NA (A). As controls, U46619 (1 μM) (C), 8-iso (10 μM), and a mixture of 9-HODE and 15-HETE (20 μM) were lso used (D). The Ca²⁺i measurements were performed by monitoring the intensity of Fura2 fluorescence. Data represent the mean±SD of measurements performed in triplicate. By two-way analysis of variance, there was a significant effect for the agonists (p<0.01) and for the concentrations (p<0.01) with a significant agonist/concentration interaction (p<0.01) on the fluorescence rati. *p<0.001, ^† p<0.01 versus 0. Ca²⁺i, intracellular Ca²⁺. 8-iso, 8-iso-prostaglandin-F2alpha; 9-HODE, 9-hydroxyoctadecadienoic acid; 15-HETE, 15-hydroxyeicosatetraenoic acid; TP, human thromboxane A2.

Notes

Materials and methods

All the investigations conform to the Declaration of Helsinki. The study was approved by the Ethics Committee of the University of Verona and all participants provided written consent.

Collection of human specimens

We collected 107 carotid plaque specimens from 117 asymptomatic consecutive patients undergoing carotid endarterectomy according to the present guidelines. After removal from the carotid artery, the plaques were placed in chelex-100-treated phosphate-buffered saline (PBS) containing EDTA (0.75 mg/ml), BHT (0.02 mg/ml), and RNA stabilization reagent (RNALater; Qiagen, Hilden, Germany). The plaques were examined macroscopically to individualize the site of the maximum plaque thickening and then transversely dissected into two segments. One of the two segments was further dissected in three specimens for histology, immunohistochemistry, and protein expression; the other segment was used to prepare TANC and P extracts. In brief, after removing NC, the segment close to NC was used to obtain TANC extract, while P was derived from the periphery of the same human carotid plaques. The segments were blotted dry and immediately stored at −80°C. NA extracts were collected from 10 patients undergoing splenectomy.

Histology, immunohistochemistry, and apoptosis detection

Segments for histology and immunohistochemistry were fixed in paraformaldehyde 4% for 2–4 h and embedded in paraffin as previously described (3). Sections of each segment were stained with hematoxylin–eosin for histological analysis to evaluate NC size, fibrous cap thickness and rupture, and presence of calcification. The carotid plaques were classified histologically as previously described (3). In this study, only carotid plaques with a fibrous cap atheroma or a thin fibrous cap atheroma, markedly infiltrated by macrophages and an underlying NC, were taken into consideration (n=41). Details of the main characteristics of the patients undergoing endarterectomy and of the plaques considered are described in Table 1. Immunostaining of CD68-positive cells was performed as previously described (3). Slices were incubated with 2% normal serum, 0.3% Triton X-100, 1% bovine serum albumin for 20 min, and then with the primary antibody for 1 h at room temperature (PG-M1; Dako, Aachen, Germany). The sections were then incubated with a complementary secondary antibody for 1 h and then with avidin–biotin for 30 min. Diaminobenzidine was used as chromogen and sections were counterstained with hematoxylin.

Detection of apoptotic cells in paraffin-embedded sections of each segment (P and TANC) was performed with the ApopTag Peroxidase In situ Oligo Ligation (ISOL) detection kit (Chemicon International, Inc., Darmstadt, Germany) following the manufacturer's instruction. Briefly, the tissue sections were deparaffinized with proteinase K. The endogenous peroxidase activity was quenched with 3% H₂O₂ and incubated with an equilibration buffer. Working strength terminal deoxynucleotidyl transferase was then applied for 1 h at 37°C. The slices were then stained with an anti-digoxigenin peroxidase conjugate for 30 min at room temperature. After washing, the peroxidase substrate diaminobenzidine was added. The reaction was stopped when the color developed. Slices were extensively washed with H₂O and mounted with a glass coverslip in an Aqua-Mount medium (Lerner Laboratories, New Haven, CT). Apoptosis was expressed as a percentage of the total number of nuclei.

Preparation of NA, P, and TANC extracts

NA, P, and TANC extracts were prepared as previously described (1). Briefly, the segments, still frozen, were immediately placed in the chelex-100-treated PBS buffer containing EDTA (0.75 mg/ml) and BHT (0.02 mg/ml). The samples were homogenized using an Ultra-Turrax T8 blade homogenizer (IKA Labortechnik, Staufen, Germany) for 5 min and the fine tissue powders derived from TANC and P of all plaques and from all normal spleen arteries were put together. The powder was extracted as previously described (3). Since no hydrolysis of cholesterol ester and triglycerides was performed, only the free forms of oxidative derivatives of PUFAs were extracted (3). 8-iso, 9-HODE, 15-HETE, and their deuterated internal standards were purchased from Cayman Chemical Company (Ann Arbor, MI).

Evaluation of components of NA, P, and TANC extracts

Mass spectrometry analysis of NA, P, and TANC extracts was performed on an ion trap mass spectrometer (model 1100; Agilent Technologies, Waldbronn, Germany) by reverse-phase HPLC using an Alltech Vydac C18 column (100×0.3 mm, 3 μm ODS) (Deerfield, IL, USA) equipped with a guard column at 37°C; the sample was eluted in the isocratic mode using methanol/ammonium acetate (10 nM) 95:5 v:v at a flow rate of 25 μl/min as previously described (3). Analytical quantification of F2-isoprostanes, HODEs, and HETEs was obtained by relating the peak areas with 8-iso, 9-HODE, and 15-HETE and with areas of the deuterated internal standards (Cayman Chemical Company).

Cell cultures

The macrophage-like THP-1 cell line was cultured and differentiated as previously described (3). THP-1 cells were incubated with NA, P, and TANC extracts to evaluate the expression of Perk, Chop, and apoptosis genes. In some experiments, THP-1 cells were preincubated with SQ29512 (a TP receptor antagonist) (10 μM) and with U46619 (a TP receptor agonist) (1 μM) (Sigma-Aldrich, Milan, Italy), G2A receptor-specific antibody (2 μg/ml) (SC-9692; Santa Cruz, Heidelberg, Germany), or normal goat IgG (2 μg/ml) for 1 h, and incubated with TANC extract, 8-iso (10 μM), and a mixture of 9-HODE and 15-HETE (10 μM). Early apoptosis and cell viability were determined using the AnnexinV-FITC Kit (Bender MedSystems GmbH, Vienna, Austria) and 7-amino-actinomycin D (7-AAD) (BD Biosciences, Buccinasco, Italy) by flow cytometry. Endotoxin contamination of cell cultures was routinely excluded with the chromogenic Limulus amebocyte lysate assay (Sigma-Aldrich).

RNA isolation and quantitative real-time PCR

Fresh, frozen tissue samples were cut and transferred into the rotor-stator homogenizer with the QIAzol Lysis buffer and magnetic beads to obtain tissue lysates. Total RNA isolation was performed with the RNeasy plus Universal Mini Kit on QiaCube instrument (Qiagen) according to the manufacturers' instructions. The RNA concentration and quality were evaluated with the RNA 6000 Nano LabChip kit by using the Bioanalyzer 2100 microcapillary electrophoresis system (Agilent Technologies, Inc., Santa Clara, CA). Reverse transcription was performed using the IScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The relative expression levels of genes of Perk, Nrf2, Ho-1, γ-Gcs, Chop, Casp3, Bax, P53, Tnfr1, and Bcl-2 were performed in triplicate using the QuantiTect Primer Assay and QuantiTect SYBR Green PCR Kit (Qiagen) on the MyiQ Thermal Cycler (Bio-Rad). QuantiTect Hs-ACTB Assay (Qiagen) was used as normalizer.

Western blotting analysis

Western blotting analysis was performed as previously described (3). The following primary antibodies against the indicated proteins were used: Perk (ab65142; Abcam, Cambridge, United Kingdom), Nrf2 (sc-13032; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), Ho-1 (sc-10789; Santa Cruz Biotechnology, Inc.), γ-Gcs (ab41463; Abcam), Chop (ab11419; Abcam), Casp3 (AAS-103; Stressgen Bioreagents Corp., Victoria, BC, Canada), Bax (AAS-040; Stressgen Bioreagents Corp.), P53 (ab26; Abcam), Tnfr1 (ab19139; Abcam), Bcl-2 (AAS-070; Stressgen Bioreagents Corp.), and β-actin (sc-130656; Santa Cruz Biotechnology, Inc.). As secondary antibodies, appropriate horseradish peroxidase-conjugated antibodies were used and the bands visualized according to a standard chemiluminescence protocol (Immobilon Western; Millipore, Billerica, MA). Protein expression was quantified using the VersaDoc Imaging System (Bio-Rad).

Calcium measurement

Ca²⁺i measurement in THP-1 cells was made by monitoring the intensity of Fura2 (Molecular Probes, Eugene, OR) fluorescence. THP-1 cells were washed twice in PBS, resuspended in the modified Ca⁺⁺/Mg⁺⁺-free Hank's buffered salt solution containing 10 mM HEPES, pH 7.6, and 0.1% bovine serum albumin (HBSS buffer) at 10⁷ cells/ml and incubated in the dark with 5 μM Fura2/AM for 45 min at 37°C. Subsequently, the cells were collected by centrifugation (600 g, 5 min), washed once in an equal volume of HBSS buffer, resuspended in HBSS buffer at 10⁷ cell/ml, and kept at room temperature in the dark until use. Fura2 fluorescence was recorded in THP-1 cells at 37°C with gentle stirring using a Shimadzu RF-5000 spectrofluorometer at excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. The rapid, transient rise and fall in Ca²⁺ levels in response to ligand stimulation were interpreted as receptor-mediated Ca²⁺i mobilization. The calibration of the signal was performed in each sample by adding 0.2 Triton X-100 to obtain the maximal fluorescence (F_max) and adding 1 mM EGTA to obtain the minimal fluorescence (F_min).

Statistical analysis

The data are presented as mean±SD. Statistical analysis was performed by two-tailed unpaired Student's t-test and by one- or two-way analysis of variance for repeated measures followed by the post hoc Tukey test for multiple comparisons using the SYSTAT program and statistical software manual (SYSTAT, Inc., Evanston, IL) for Macintosh. Statistical significance was inferred at p-values<0.05.

Footnotes

Acknowledgments

The authors thank the past and present members of our laboratory for their contribution to various aspects of histology and immunohistochemistry. Especially, the authors thank Stefania Manfro for the excellent histological preparations.

Abbreviations Used

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