Autophagy regulates trans fatty acid-mediated apoptosis in primary cardiac myofibroblasts.

Autophagy regulates trans fatty acid-mediated apoptosis in primary

cardiac myo

fibroblasts

Saeid Ghavami

, Ryan H. Cunnington

, Behzad Yeganeh

, Jared J.L. Davies

, Sunil G. Rattan

,

Krista Bathe

, Morvarid Kavosh

, Marek J. Los

, Darren H. Freed

, Thomas Klonisch

, Grant N. Pierce

,

Andrew J. Halayko

, Ian M.C. Dixon

⁎

a_{Department of Physiology, University of Manitoba, Canada} b_{Institute of Cardiovascular Sciences, University of Manitoba, Canada} c

Cardiac Sciences Program, St. Boniface General Hospital, University of Manitoba, Canada d

Department of Human Anatomy and Cell Science, University of Manitoba, Canada e

Department of Internal Medicine, University of Manitoba, Canada f

Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, MB, Canada g

Department of Clinical & Experimental Medicine, Integrative Regenerative Medicine Center, Linköping University, Sweden

a b s t r a c t

a r t i c l e i n f o

Article history: Received 12 July 2012

Received in revised form 19 September 2012 Accepted 21 September 2012

Available online 28 September 2012 Keywords: Myofibroblast Trans fat Vaccenic acid Elaidic acid Autophagy Apoptosis

Trans fats are not a homogeneous group of molecules and less is known about the cellular effects of individual members of the group. Vaccenic acid (VA) and elaidic acid (EA) are the predominant trans monoenes in ru-minant fats and vegetable oil, respectively. Here, we investigated the mechanism of cell death induced by VA and EA on primary rat ventricular myofibroblasts (rVF). The MTT assay demonstrated that both VA and EA (200μM, 0–72 h) reduced cell viability in rVF (Pb0.001). The FACS assay confirmed that both VA and EA induced apoptosis in rVF, and this was concomitant with elevation in cleaved caspase-9, -3 and -7, but not caspase-8. VA and EA decreased the expression ratio of Bcl2:Bax, induced Bax translocation to mitochondria and decrease in mitochondrial membrane potential (Δψ). BAX and BAX/BAK silencing in mouse embryonic fi-broblasts (MEF) inhibited VA and EA-induced cell death compared to the corresponding wild type cells. Transmission electron microscopy revealed that VA and EA also induced macroautophagosome formation in rVF, and immunoblot analysis confirmed the induction of several autophagy markers: LC3-β lipidation, Atg5–12 accumulation, and increased beclin-1. Finally, deletion of autophagy genes, ATG3 and ATG5 signifi-cantly inhibited VA and EA-induced cell death (Pb0.001). Our findings show for the first time that trans fat acid (TFA) induces simultaneous apoptosis and autophagy in rVF. Furthermore, TFA-induced autophagy is required for this pro-apoptotic effect. Further studies to address the effect of TFA on the heart may reveal significant translational value for prevention of TFA-linked heart disease.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The impact of diet on the incidence of cardiovascular disease is well

established, and nutrition may be responsible for ~40% of all

cardiovas-cular disease (Canadian government report-Health Canada issued in

1995). Recent data indicate that the majority of myocardial infarctions

(MI) are directly linked to modi

fiable environmental factors, including

diet

[1], in particular the relative intake of saturated fatty acids (SFA)

and polyunsaturated fats (PUFAs)

[2,3]. Trans fatty acids (TFA) are

un-saturated fatty acids with at least one double bond in the trans con

figu-ration that renders them structurally/chemically unstable compared to

saturated SFAs

[4]. Elaidic acid (18:1 trans-9) is the main TFA isomer in

hydrogenated vegetable oils and products containing hydrogenated

margarines or vegetable shortening including fried foods, cookies, and

crackers, and accumulates in atherosclerotic lesions and adipose tissue

of obese patient

[4

–6]

. Health professionals support removal or

reduc-tion of dietary TFAs for improved health

[7,8]. This notwithstanding,

myofibroblast; MEF, mouse embryonic fibroblasts; TFA, trans fatty acid; MI, myocardial infarction; PUFA, poly unsaturated fat; SFA, saturated fatty acids; PI, propidium iodide; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; ATG5, autophagy protein 5; ATG3, autophagy protein 3; BAD, the Bcl2-associated death pro-moter protein; BAK, BCL-2-antagonist/killer; BAX, the Bcl2-associated X protein; Bcl2, B-cell lymphoma 2; BID, BH3 interacting domain death agonist; Caspases, cysteine-dependent aspartate-directed proteases; ER, endoplasmic reticulum; LC-3, microtubule-associated protein light chain 3; TEM, transmission electron microscopy

⁎ Corresponding author at: Department of Physiology and the Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, 351 Tache Ave, Winnipeg, Manitoba, Canada R2H 2A6. Tel.: + 1 235 3419; fax: + 1 233 6723.

E-mail address:idixon@sbrc.ca(I.M.C. Dixon). 1_{These authors have equal senior authorship.}

0167-4889/$– see front matter © 2012 Elsevier B.V. All rights reserved.

http://dx.doi.org/10.1016/j.bbamcr.2012.09.008

Contents lists available at

SciVerse ScienceDirect

Biochimica et Biophysica Acta

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 / b b a m c r

some recent evidence indicates that speci

fic isomeric configuration in

different TFAs may exhibit different effect(s) on cardiovascular health

[7]. Vaccenic acid (18:1 trans-11) is unique in structure and source, as

it is enriched in dairy products and meats of ruminant species

[9].

Several recent in vitro studies indicate that saturated fatty acids

induce apoptosis (programmed cell death I) in somatic and cancer

cells of many different origins, including cardiomyocytes and

fibro-blasts

[10

–21]

. Though a recent report suggests a role for autophagy

in TFA-induced cell death of hepatocytes

[22], apoptosis is generally

accepted as the principal mechanism driving cell demise in health

and disease

[23

–26]

. Apoptosis can be initiated via extrinsic and/or

intrinsic pathways

[24,27], driven by the activation of caspases.

Macroautophagy is an evolutionarily conserved catabolic,

homeostat-ic process that can support cell survival or, if excessive, can drive cell

death. It consists of membrane isolation, autophagosome and

autolysosome formation

[28,29], the later driving breakdown of

mac-romolecules and organelles by lysosomal enzymes

[28,30,31].

Apo-ptosis and autophagy have many common regulators, and cross-talk

regulates cell fate in response to cellular stress. The complex

interac-tion of apoptotic and autophagic pathways necessitates the careful

consideration of their integrated control and impact on cell fate to

un-derstand cell death phenomena

[32,33].

Fibroblasts are a heterogeneous group of cells that exhibit distinct

differentiated phenotypes in different organs

[34]. In humans cardiac

fibroblasts represent the most numerous non-myocytes in the

myo-cardium, with these cells synthesizing and organizing collagens,

fi-bronectins and other interstitial components to maintain the

integrity of the cardiac extracellular matrix (matrix)

[35]. In the

cur-rent paper, we address the cell death effects of elaidic and vaccenic

TFAs on rat ventricular myo

fibroblasts (rVF), dissecting the roles of

apoptosis and autophagy and cross-talk between these pathways.

2. Materials and methods

2.1. Materials and reagents

Cell culture plasticware was obtained from the Corning Costar

Compa-ny (Thermo Fisher Canada). Cell culture media, propidium iodide (PI),

rabbit anti-LC3

β, rabbit anti-beta actin,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), vaccenic acid, elaidic acid,

and vitamin C were obtained from Sigma (Sigma-Aldrich, Oakville,

Canada). Rabbit anti-cleaved caspase-7, -8, rabbit anti-Bak, Bax,

Bcl2, Atg12, Atg3, Atg5, cleaved caspase-3, and cleaved caspase-9

were purchased from Cell Signaling (MA, USA). 5,5

′,6,6′-Tetrachloro-1,1

′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), Mitotracker

Red, was obtained from Life Technologies Inc. (Burlington, Canada).

Caspase-Glo®-3/7, Caspase-Glo®-8 and Caspase-Glo®-9 assay were

pur-chased from Promega (WI, USA).

2.2. Primary cardiac

fibroblast preparation

Cardiac

_{fibroblasts were isolated as previously described}

[36,37].

Brie

fly, hearts from adult male Sprague–Dawley rats (150–200 g) were

subjected to Langendorff perfusion with DMEM-F-12 (GIBCO) followed

by serum-free MEM (SMEM; Life Technologies, Inc., Burlington, Canada).

Perfused hearts were digested with 0.1% wt/vol collagenase type 2

(Worthington) in SMEM for 20 min. Hearts were minced in dilute

collagenase solution (0.05% wt/vol collagenase type 2 in SMEM)

for a further 15 min before addition of growth media DMEM-F-12

supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin

(GIBCO-BRL), 100

μg/ml streptomycin (GIBCO BRL), and 1 μM ascorbic

acid (Sigma-Aldrich). Upon settling of large tissue pieces to the bottom

of a 50-ml tube, supernatant was centrifuged at 2000 rpm for 5 min.

Cell pellets were re-suspended in growth media and plated on 75-cm

culture

flasks. Cells were allowed to adhere for 2–3 h in a 5% CO

37 °C

in-cubator, then washed twice with phospho-buffered saline (PBS) followed

by the addition of fresh growth media. Media were changed the following

day, and cells were allowed to grow for 3

–4 days before passaging into

first passage (P1) myofibroblasts. P1 myofibroblasts were transferred to

DMEM/F12 media and after 24 h all cultures have been done in DMEM

medium. For all experiments, passages 3

–7 of rat cardiac myofibroblast

were used in DMEM/F12 complete media.

2.3. Cell viability assay

We measured the viability of cardiac myo

fibroblasts under various

treatment conditions, as described previously using MTT

[38,39].

Brie

fly, primary cardiac myfibroblasts, wild type murine embryonic

fibroblasts (Wt MEF), MEF Bax knock out (MEF Bax

_{), and MEF}

Bax/Bak double knock out (MEF Bax/Bak

) cells were treated with

vaccenic or elaidic acid (0

–400 μM, 0–72 h). Relative cell viability

(percent of control) was calculated using the equation: (mean OD of

treated cells / mean OD of control cells) × 100. For each time point,

the treated cells were compared with control cells that had been

treated with vehicle only (DMSO, 0.1% V/V). In experiments

investi-gating if vitamin C can modulate the cytotoxic effects of vaccenic

and elaidic acids, vitamin C (2.5 and 5 mM) was added to culture

media 4 h before the treatment and later the cells were co-treated

with vaccenic and elaidic acids (200

μM, 72 h).

2.4. Measurement of apoptosis by

flow cytometry

Apoptosis in our cell preparations was measured using the

Nicoletti method

[40,41]. Brie

fly, cells grown in 12-well plates were

treated with 200 and 400

μM vaccenic and elaidic acids for the

indicat-ed time intervals. After scraping, the cells were harvestindicat-ed by

centrifuga-tion at 1500 ×g for 5 min, washed once with phosphate-buffered saline,

and resuspended in hypotonic propidium iodide lysis buffer (1% sodium

citrate, 0.1% Triton X-100, 0.5 mg/ml RNase A, 40

μg/ml propidium

io-dide). Cellular nuclei were incubated for 30 min at 30 °C and

subse-quently analyzed by

flow cytometry. Nuclei to the left of the G1 peak

containing hypo-diploid DNA were considered apoptotic.

2.5. Luminescence caspase activity assays

Luminometric assays Caspase-Glo® 8, 9 and 3/7 (Promega, Canada,

Nepean, ON) were used to measure the proteolytic activity of

caspases-3/7 (DEVD-ase), 8 (IETD-ase), and 9 (LEHD-ase) as we have

previously done

[42].

2.6. Measuring mitochondrial membrane potential

Mitochondrial membrane potential was measured employing the

mitochondria-speci

fic cationic ratiometric dye JC-1 that undergoes

ΔΨ

-dependent aggregation in the mitochondria. Normally, JC-1

ex-ists as a green

fluorescent (540 nm, excitation 490 nm) monomer at

ΔΨ

b140 mV, but when ΔΨ

> 140 mV, then JC-1 aggregates and

emits red spectra

fluorescence (590 nm, excitation 540 nm). We

measured the

ΔΨ

of cardiac myo

fibroblasts under various treatment

conditions, as described previously

[43].

2.7. Analysis of cellular morphology

To assess cell viability based on gross cellular appearance

(chro-matin condensation and cell shrinkage) rat cardiac myo

fibroblast

cells were grown on 12-well plates and morphology was assessed

by phase contrast microscopy (Olympus CKX41) using a Olympus

In-finity 1 CCD digital camera to capture images.

2.8. Immunoblotting

We used Western blotting to detect cleaved caspase-8, -3, -9,

-Bcl2, Bid, Bax, LC3

β, Atg5–12, Atg3, Atg5, and β actin. Briefly, cells

were washed and protein extracts prepared in lysis buffer (20 mM

Tris

_{–HCl (pH 7.5), 0.5% Nonidet P-40, 0.5 mM PMSF, 100 μM}

β-glycerol 3-phosphate and 0.5% protease inhibitor cocktail). After a

high-speed spin (13,000 ×g for 10 min), the supernatant protein

con-tent was determined by Lowry protein assay and then proteins were

size-fractionated by SDS-PAGE and transferred onto nylon

mem-branes under reducing conditions. After blocking memmem-branes with

non-fat dried milk and Tween 20, blots were incubated overnight

with the primary antibodies at 4 °C. HRP-conjugated secondary

anti-body incubation was carried out for 1 h at room temperature. Blots

A

24 hrs

B

_{72 hrs}

Control

Vaccenic Acid

400 µM

Elaidic Acid

400 µM

Fluorescence Intensity

Cell Count

C

D

72 hrs

Fig. 1. Trans fats (vaccenic or elaidic acid) induce apoptosis in primary rat ventricular myofibroblasts. (A, B) Cardiac myofibroblasts were treated with vaccenic and elaidic acids (200 and 400μM) and cell viability was assessed 24 and 72 h thereafter by MTT assay. Control cells for each time point were treated with vehicle control (ethanol). Results are expressed as percentage of corresponding time point control and represent the means ± SD of 8 independent experiments in three different primary rat ventricular myofibroblast (***Pb0.001). (C) Examples of typical DNA histograms showing propidium iodide staining measured by flow cytometry for control and vaccenic and elaidic acid-treated rat ven-tricular myofibroblast. In each panel the region labeled “M2” indicates cells with sub-diploid DNA and the percentage of cells within those regions are indicated. Peaks for diploid (G1) and tetraploid (G2) nuclear staining in non-apoptotic cells are visible in the region labeled as“M1”. The cells were treated with vaccenic and elaidic acids and their correspon-dence control for 24 (top row) and 72 (bottom row) hours. (D) Percent sub-G1 rat ventricular myofibroblast abundance induced by vaccenic and elaidic acids (200 and 400 μM) or ethanol solvent control after 72 h. Results represent the means ± SD of 4 independent experiments in two different primary rat ventricular myofibroblast primary cell lines (statistical significance is indicated by ***Pb0.001 vs time-matched controls).

were then developed by enhanced chemiluminescence (ECL)

detec-tion (Amersham-Pharmacia Biotech). In experiments detecting Bak

oligomerization, non-reducing immunoblotting was used.

2.9. Immunocytochemistry, confocal imaging and electron microscopy

To carry out immunocytochemistry, MEF Bax-GFP cells were

grown overnight on coverslips and then treated with vaccenic and

elaidic acids (200

μM) for 72 h prior to fixation (4%

paraformalde-hyde/120 mM sucrose) and permeabilization (3% Triton X-100).

Thereafter, mitochondria were stained with Mitotracker Red CMXRos

(Molecular Probes; 200 nM). The

fluorescent images were then

ob-served and analyzed using an Olympus FluoView multi-laser confocal

microscope. For transmission electron microscopy, cells were

fixed

(2.5% glutaraldehyde in PBS (pH 7.4) for 1 h at 4 °C) and post-

fixed

(1% osmium tetroxide) before embedding in Epon. TEM was

performed with a Philips CM10 instrument at 80 kV using ultra-thin

sections (100 nm on 200 mesh grids) stained with uranyl acetate

and counterstained with lead citrate

[39].

2.10. Statistical analysis

The results were expressed as mean ± SE and statistical

differ-ences were evaluated by one-way or two-way ANOVA followed by

Tukey's or Bonferroni's post hoc test, using Graph Pad Prism 5.0.

P

_{b0.05 was considered significant. For all experiments data was}

col-lected in triplicate from at least three cell lines unless otherwise

indicated.

3. Results

3.1. Trans fats (vaccenic and elaidic acids) induce intrinsic apoptosis and

cell death in primary rat cardiac myo

fibroblasts

We used a broad range of physiologically relevant concentrations of

vaccenic acid (VA) and elaidic acid (EA) (0, 200 and 400

μM) and treated

primary rat cardiac myo

fibroblasts cells for up to 72 h to identify their

impact of cell viability and death. The MTT assay indicated that both VA

and EA induce signi

ficant cell death in a concentration-related manner

at 24 and 72 h (Fig. 1A and B) (P

_{b0.001). In separate experiments}

using FACs assessment of sub-Go nuclear population abundance, we

con-firmed that loss of viability was associated with significant apoptotic cell

death in primary rat cardiac myo

fibroblasts (Pb0.001 at 72 h) (

Fig. 1C

and D). To further discriminate whether apoptosis was linked to intrinsic

or extrinsic effector pathways, we next assessed caspase cleavage and

activation. Both VA and EA activated caspase-3, con

firming induction

of apoptosis, and this was associated with activation of caspase-9

(intrin-sic pathway marker)

[24,44,45]

but not extrinsic pathway markers

[24,46,47], caspase-8 (Fig. 2A

–D) or Bid cleavage (data not shown).

We conclude that vaccenic acid and elaidic acid both selectively

induce caspase-dependent intrinsic apoptosis in primary rat cardiac

myo

fibroblasts.

3.2. Trans fats induce autophagy in primary rat cardiac myo

fibroblasts

LC3 (microtubule-associated protein light chain 3), the

mammali-an equivalent of yeast Atg8, exists in two forms: cytosol-localized

LC3-I, and its lipidated proteolytic derivative LC3-II (18 and 16 kDa,

respectively), which localizes to autophagosomal membranes, and

thus represents a sensitive marker for autophagosome formation

[38,48,49]. In the current study we showed that both VA and EA

(200

μM, 0–72 h) induce LC3β lipidation and LC3β II formation, and

Atg5

–12 formation (

Fig. 3A) in primary rat cardiac myo

fibroblasts.

Autophagic

flux was confirmed using an inhibitor of lysosome–

autophagosome fusion, Ba

filomycin A1 (Baf-A1 at 10 nM). Treatment

with Baf-A1 increased LC3

β II formation in the presence of either

VA or EA (Fig. 3B), con

firming TFA treatment indices de novo

autophagosome formation and subsequent turnover in the absence

of Baf-A1. Transmission electron microscopy further con

firmed

autophagosome formation in both VA- and EA-treated cells (Fig. 3C

and D). Finally, another feature of autophagy, lysosomal activation,

indicated by increased in lysotracker red staining, was also induced

by both VA and EA (200

μM, 72 h) (

Fig. 3E).

Cleaved-Caspase-9 37 kD Cleaved-Caspase-3 17-19 kD β-Actin Time hrs 0 36 48 72 36 48 72 VA EA Control Vaccinic Acid (200 μm) Elaidic Acid (200 μm) Control Vaccinic Acid (200 μm) Elaidic Acid (200 μm) Control Vaccinic Acid (200 μm) Elaidic Acid (200 μm) 4000 10000 8000 6000 4000 2000 0 8000 6000 4000 2000 0 3000 NS NS 2000 1000 0 36 hrs 72 hrs Time (hrs) 36 hrs 72 hrs Time (hrs) 36 hrs 72 hrs Time (hrs) Caspase-8 Activity (Luminescence Intensity) Caspase-8 Activity (Luminescence Intensity) Caspase-8 Activity (Luminescence Intensity)

A

C

D

B

Fig. 2. Both vaccenic and elaidic acids induce rat ventricular myofibroblast apoptosis via activation of mitochondrial pathway. (A) Immunoblot detection of cleaved caspase-7, and caspase-3 in total cell lysates of primary cultured rat ventricular myofibroblast. Cells were treated with vaccenic and elaidic acids (200 μM) for up to 48 h. For all lanes β-actin was used as protein loading control. The blots shown are typical of three experiments completed using different cultures of rat ventricular myofibroblasts. (B, C, D) Effects of vaccenic or elaidic acid (200μM) treatment (up to 72 h) on caspase-8, caspase-9, and caspase-3/-7 enzymatic activity, as detected by Caspase-Glo®

luminometric assay. Caspase activity normalized to that measured for solvent-only treated cultures is represented on the Y-axis. The data represent mean ± SD of duplicate experiments performed on 3 different rat ventricular cell lines (statistical significance is indicated by ***Pb0.001 vs untreated controls).

3.3. Bcl2 family proteins are involved in VA- and EA-induced apoptosis in

primary rat cardiac myo

fibroblasts

In healthy cell stasis Bcl2 is localized in the mitochondrial

mem-brane, being tightly associated with Bax (a proapoptotic protein),

and preventing release of apoptogenic factors from the mitochondrial

intermembrane space to the cytoplasm

[50

–53]

. During apoptosis the

balance of pro- and anti-apoptotic proteins shifts, leading to

mito-chondrial damage, decrease in mitomito-chondrial membrane potential,

and release of apoptogenic factors. Both VA and EA affect relative

Bcl2 and Bax abundance in primary rat cardiac myo

fibroblasts, to

favor apoptosis (Fig. 4A and B). In another study we used BAX knock

out (BAX KO) mouse embryonic

fibroblasts (MEFs) and assessed the

effect of VA and EA, compared to wild type MEFs, and showed that

BAX KO MEFs exhibit a signi

ficantly decreased cytotoxic response to

VA and EA (Fig. 4C, P

_{b0.001). Subcellular localization of Bax to}

mito-chondria is required to promote apoptotic cell death, thus we used a

GFP-Bax over-expressed MEF cell line and assessed the impact of

treatment with VA and EA; both trans fats induced Bax translocation

to mitochondria after 48 h (Fig. 4D). In additional experiments using

rat cardiac myo

_{fibroblasts we also observed that VA and EA}

signifi-cantly decreased mitochondrial membrane potential (

Δψ) after 48 h

(P

b0.001) (

Fig. 4E), a time line that correlated with changes in Bax/

Bcl2 expression (Fig. 4A and B) and Bax translocation to mitochondria

(Fig. 4D).

Trans fat exposure has been associated with generation of

cyto-toxic reactive oxygen species (ROS)

[54], thus we next determined

whether ROS played a role in EA and VA-induced cell death of rat

cardiac myo

fibroblasts. We used vitamin C (5 mM) as a ROS

scaven-ger

[55,56], and found that it signi

ficantly inhibited VA and

EA-induced loss of rat cardiac myo

fibroblast viability (

Fig. 4F and

G) (P

b0.05). We also confirmed the involvement of Bcl2 family

pro-teins in VA and EA-induced cell death using BAX/BAK KO MEFs. Of

note MEFs lacking pro-apoptotic Bcl2 proteins (BAX/BAK KO MEFs)

were refractory to the cytotoxic effects exerted by VA and EA

(Fig. 4H

–J).

VA

EA

LC3 I

LC3 II

Atg5-12

-Actin

A

B

Autophagosome

Autophagolysosme

Control

Vaccenic Acid

C

0

24

36

48

24

36

48

Fig. 3. Both vaccenic and elaidic acids induce autophagy in primary rat ventricular myofibroblasts. (A) Western blot analysis of cell lysates from primary rat ventricular myofibroblast cells. Cells were treated with 200 μM vaccenic or elaidic acid for the indicated time periods, and then immunoblotted using the indicated specific antibodies. Both vaccenic and elaidic acids induce LC3β lipidation and Atg5–12 conjugation. Equal loading among lanes was confirmed using β-actin. (B) Western blot analysis of cell lysates from primary rat ventricular myofibroblast cells. Cells were pre-treated with Bafilomycin A1 (10 nM, 4 h) and then co-treated with 200 μM of either vaccenic or elaidic acid for indicated durations, and then immunoblotted using the LC3β antibody. Bafilomycin A induced lapidated LC3β in both vaccenic and elaidic acid treatment in rat ventricular myofibroblast. Equal loading was confirmed using β-actin. (C, D) Rat ventricular primary myofibroblasts were either left untreated (top panel left) or they were treated with 200μM vaccenic (B) or elaidic acid (C) (top panel right and lower panels) for 72 h. Cells were then imaged by transmission electron microscopy. Magnification: 4.6×103_{. Structures} identified as autophagosomes or autophagolysosome are indicated within the dotted margin. The scale bar represents 2 μm in all the top row and left panel in the middle. The scale bar in the right side panel in the middle row represents 2μm. The lower row shows enlarged images of autophagosome and autophagolysosomes. (D) Rat ventricular myofibroblasts treated with vaccenic and elaidic acids (200 μM, 72 h) showed increased in Lysotracker Red staining, a marker of lysosomal activation.

Elaidic Acid

Control

Late

autophagolysosome

Autophgosome

D

E

3.4. Autophagy is necessary for VA and EA induced cell death and

apopto-sis induction

Data from several studies indicate that apoptotic and autophagic

cellular processing may be interdependent in some settings but can

be simultaneously regulated and initiated by a common trigger in

other cases, thus resulting in different cellular outcomes

[39,57,58].

Therefore we used ATG3 KO and ATG5 KO MEFs to compare the

cyto-toxic effects of EA and VA in cells that are de

ficient in proteins that are

required for autophagy to occur

[38]. Our results indicate that absence

of ATG5 and ATG3 signi

ficantly inhibited the cytotoxic effects of trans

fats (Fig. 5A and B). Moreover, we showed that lack of ATG3 and

ATG5 decreased activation of caspase-3 and caspase-7 otherwise

in-duced by trans fat exposure (Fig. 5C and D). Collectively, these

Bcl-2

Bax

-Actin

A

B

D

C

Control

Vacinic Acid

Elaidic Acid

observations demonstrate an essential role for autophagy in trans

fat-induced cell death and apoptosis.

4. Discussion

Our experiments show that trans fats (VA and EA) induce an

intrinsic apoptotic pathway in cardiac myo

fibroblasts. VA and

EA-induced apoptosis is regulated by Bcl2 family proteins and we

found that autophagy is required for VA and EA-dependent apoptosis

activation in myo

fibroblasts.

Dietary TFA is composed of varying amounts of elaidic acid (EA)

and vaccenic acid (VA) isomers. VA is derived from milk, yoghurt,

cheese, butter and from meats of ruminants

[59,60]. Several studies

have identi

_{fied a link between the ingestion of TFAs and coronary}

heart disease

[61

–64]

. TFAs are relatively rare in nature, derived

solely from the diet, and are in abundance in processed foods

H

E

F

G

Fig. 4. Both vaccenic and elaidic acids induce apoptosis in rat ventricular myofibroblasts via altering the balance between Bcl2 pro- and anti-apoptotic proteins. (A) Immunoblot detection of Bcl2 and Bax in total cell lysates of primary cultured rat ventricular myofibroblasts. Cells were treated with vaccenic and elaidic acids (200 μM) for up to 48 h. Both acids decrease Bcl2 expression while increasing Bax expression in rat ventricular myofibroblasts. For all lanes β-actin was used as protein loading control. Blots are typical of three experiments completed using different cultures of rat ventricular myofibroblasts (B). Densitometry analysis showed a significant decrease in the ratio of Bcl2/Bax (statistical significance is indicated by ***Pb0.001 vs controls). (C) Wild type and BAX−/−_{mouse embryo myofibroblasts (MEF) were treated with either vaccenic or elaidic acid (200 μM for} 48 h) and cell viability was assessed 48 h thereafter using the MTT assay. Control cells for each time point were treated with the solvent control (ethanol). Results are expressed as percentage of corresponding time point control and represent the means ± SD of 6 independent experiments (***, Pb0.001). (D) MEF GFP-Bax expressing cells were treated with vaccenic and elaidic acids (200μM, 48 h) and the cells were stained with Mitotracker red after 48 h of treatment. Both acids induce Bax translocation to mitochondria. (E) Effects of vaccenic and elaidic acid treatment (200μM, 48 h) on mitochondrial trans-membrane potential (ΔΨm). After vaccenic and elaidic acid treatment rat ventricular myofibroblast cells were loaded with JC-1 dye and the potential-dependent accumulation in the mitochondria (reducedΔΨmindicated by a decrease in Red:Greenfluorescence) measured directly (spectrofluorometry). Data represent the average values from duplicates of three independent experiments. ***Pb0.001 compared to time-matched solvent-only treated controls. (F and G) Histogram showing effects of Vitamin C on vaccenic and elaidic acid (200μM, 48 h) induced cell death in rat ventricular myofibroblast. Cells pre-treated with indicated concentration of Vitamin C for 4 h and then co-treated with vaccenic and elaidic acids with indicated concentration and time point. Data represent the average values from three independent experiments. *Pb0.05 compared to time-matched vehicle-or Vitamin C only controls. (H and I) MEF BAX/BAK+/+

and MEF BAX/BAK−/−cells were treated with 200μM vaccenic (H) and elaidic (I) acids for 36 and 72 h and then photographed under phase contrast microscopy settings. Magnified area highlights the effects of acids on the cells. MEF BAX/BAK−/−_{showed no changes in morphology after treatment with vaccenic and elaidic acids compared to correspondence control. (J) MTT assay is provided in a histogram which} shows the effects of vaccenic and elaidic acids (200μM, 72 h) on MEF BAX/BAK+/+

and MEF BAX/BAK−/−cells. BAX/BAK ablation was associated with a significant decrease in vaccenic- and elaidic acid-induced cell death. Data represent the average values from duplicates of three independent experiments (statistical significance is indicated by ***Pb0.001 vs time-matched controls).

consumed in developed nations

[65]. With a focus on these foods as a

source of TFA, Health Canada has issued public warnings advising against

the ingestion of TFA

[66]. This notwithstanding, the data linking elevated

TFAs to heart disease are indirect

—and thus their role in the pathogenesis

of heart disease remains in the focus of intense research. The hypothesis

that reduction of all TFA intake reduces heart disease requires rigorous

scienti

fic testing

[67]. Whether the large number of variables in the

epi-demiological evidence is the source of confusion or as TFA effects may be

masked or exacerbated by other risk factors is a confounding factor, the

need for studies to address the speci

fic effects of TFAs directly has

be-come a pressing topic for research.

The current study provides evidence that both VA and EA can induce

cell death in rat ventricular myo

fibroblasts. VA and EA-induced cell death

includes both apoptosis and autophagy mechanisms. Apoptosis is a

signi

ficant factor for normal development of the organisms and for

main-tenance of their homeostasis

[68]. Apoptosis is a well-characterized

programmed cell-death pathway that is highly conserved during

evolu-tion, and requires specialized machinery that involves proteases known

as caspases

[69]. Furthermore, the Bcl2 protein family, including the

anti-apoptotic members, Bcl2 and Bcl-xL and also the pro-apoptotic

members Bax and Bad, are central regulators of apoptosis by connecting

signals of survival and cell death that are generated within or outside

the cell

[70,71]. The imbalance between and anti-apoptotic Bcl2

pro-teins as well as their localization are the essential apoptosis initiators and

regulators

[72,73]. On the other hand it has been shown that a decrease in

mitochondrial membrane potential

[74,75]

and an increase in cellular

I

J

Fig. 5. Inhibition of autophagy decreases vaccenic and elaidic acid induce cell death in rat ventricular myofibroblast. (A and B) MEF ATG5+/+

, MEF ATG5−/−, MEF ATG3+/+ , and MEF ATG3−/−_{cells were treated with 200μM vaccenic and elaidic acids for 48 h and cell viability was measured using MTT assay. ATG5 (A) and ATG3 (B) KO was associated with a significant} de-crease in vaccenic and elaidic acid-induced cell death. Data represent the average values from duplicates of three independent experiments (statistical significance is indicated by ***Pb0.001 vs time-matched controls). (C and D) Immunoblot detection of cleaved caspase-7 and cleaved caspase-3 in total cell lysates derived from MEF ATG5+/+

(C), MEF ATG5−/−(C), MEF ATG3+/+ (D), and MEF ATG3−/−(D). Cells were treated with vaccenic and elaidic acids (200μM) for up to 84 h. ATG5 and ATG3 KD decrease caspase-7 and caspase-3 cleavages. For all lanes β-actin was used as protein loading control. Blots are typical of three experiments completed using different cultures of different MEF cell lines.

reactive oxygen species (ROS)

[76,77]

can trigger apoptotic cell death in

different models.

Reactive oxygen species production may trigger and accompany

the activation of the mitochondrial apoptotic pathway

[33,41].

The Bcl2 family serves as a checkpoint upstream of mitochondrial

dysfunction

[38]. Bcl2 may prevent reactive oxygen species

genera-tion and control the mitochondrial permeability by opposing the

ef-fect of Bax, thereby blocking cytochrome c release

[41]. Under

normal conditions, Bax exists as a soluble monomer in cytosol.

How-ever, upon stimulation, Bax translocates to mitochondria and the

level of mitochondrial Bcl2 decreases

[78]. Our study shows that

both VA and EA induce mitochondrial caspase-dependent apoptosis

in rVF. VA and EA cause an imbalance between Bax and Bcl2 and

also drive Bax mitochondrial translocation. On the other hand vitamin

C (a general reactive oxygen species scavenger) protects rVF treated

with VA and EA and con

_{firmed a leading role of ROS in TFA-induced}

cell death. MEF BAX and BAX/BAK double knock out shows signi

ficant

resistance toward TFA-induced cell death, which substantiates the

es-sential role of Bcl2 pro-apoptotic protein in TFA-induced cell death.

These data highlight that TFA induces classical, intrinsic pathway

mi-tochondrial apoptotic cell death in rVF.

Evidence obtained from various models supports the idea that both

apoptosis and autophagy can be involved in speci

fic cell death

mecha-nisms depending on the circumstances

[33,38,39,79,80]. Under stress

or cellular damage including starvation, oxidative stress, nutrient

depri-vation and the withdrawal of growth factors, autophagy is induced to

provide the energy necessary to support changes in metabolism or to

aid in the removal of damaged organelles to ensure the survival of the

cell. However, under some conditions, including severe mitochondrial

damage or endoplasmic reticular stress, autophagy can also lead to

ap-optosis and/or alternative pathways of cell death

[38,81,82]. Treatment

of rVF with TFA resulted in LC3 lipidation, Atg5

–Atg12 conjugation,

and autophagosome formation, con

firming a role for autophagy in

TFA-induced cell death in this system. Recently, interest in the

mecha-nistic relationship between apoptosis and autophagy in cell death has

increased

[83]. For certain types of apoptotic stimulation, induction of

autophagy is essential for apoptosis to occur

[84]. Under these

condi-tions, inhibition of autophagy may delay or even inhibit subsequent

ap-optosis

[84,85]. Conversely, autophagy can also act as a protective

mechanism against apoptotic cell death, in which case, blocking

autophagy can enhance apoptosis

[33,86]. Using MEF ATG3 and ATG5

KO cells a signi

_{ficant decrease in TFA-induced cell death and apoptosis}

(caspase-3, and -7 cleavages) in rVF was observed. This would support

an essential function for TFA-induced autophagy in TFA-provoked

apo-ptosis and cell death.

Elucidation of the molecular mechanism(s) of interplay between

autophagy and apoptosis upon TFA-treatment exceeds the objectives

of the current paper. However we hypothesize that VA and EA may

initially affect mitochondrial metabolism, which in turn may lead to

lowered energy production. This then could serve as a powerful

trig-ger for the induction autophagy. Increased mitochondrial metabolism

that ensues may lead to the hyperproduction of reactive oxygen

spe-cies that cause damage in mitochondria and other organelles

[87,88].

A hypothetical sequence of events is supported by acquired

experi-mental data in this project e.g., the protective effect of vitamin C (an

antioxidant) and overall slowed kinetics of VA- and EA-induced cell

death.

In conclusion, we observed that moderate concentrations of vaccenic

and elaidic trans fatty acids led to marked apoptotic death of primary rat

cardiac myo

fibroblasts, and that apoptosis by this stimulus is dependent

upon activation of autophagy.

Acknowledgements

SG was supported by Parker B Francis Fellowship in Respiratory

Dis-eases. BY was supported by postdoctoral fellowship from Manitoba

Health Research Council (MHRC). RHC was supported by an MHRC/

CIHR studentship. JJLD is supported by an Institute of Cardiovascular

Sciences studentship. This work was supported by operating grants

from the Canadian Institutes for Health Research (IMCD) as well as

the St. Boniface General Hospital and Research Foundation. AJH is

supported through the Canada Research Chairs (CRC) Program.

References

[1] S. Yusuf, S. Hawken, S. Ounpuu, T. Dans, A. Avezum, F. Lanas, M. McQueen, A. Budaj, P. Pais, J. Varigos, L. Lisheng, Effect of potentially modifiable risk factors as-sociated with myocardial infarction in 52 countries (the INTERHEART study): case–control study, Lancet 364 (2004) 937–952.

[2] S.M. Grundy, Cholesterol and Atherosclerosis, JB Lippincott Company, Philadel-phia, 1990.

[3] M.H. Moghadasian, J.J. Frohlich, B.M. McManus, Advances in experimental dyslipidemia and atherosclerosis, Lab. Invest. 81 (2001) 1173–1183.

[4] D. Mozaffarian, M.B. Katan, A. Ascherio, M.J. Stampfer, W.C. Willett, Trans fatty acids and cardiovascular disease, N. Engl. J. Med. 354 (2006) 1601–1613. [5] J.W. Bortolotto, C. Reis, A. Ferreira, S. Costa, C.C. Mottin, A.A. Souto, R.M. Guaragna,

Higher content of trans fatty acids in abdominal visceral fat of morbidly obese individuals undergoing bariatric surgery compared to non-obese subjects, Obes. Surg. 15 (2005) 1265–1270.

[6] E. Stachowska, B. Dolegowska, D. Chlubek, T. Wesolowska, K. Ciechanowski, P. Gutowski, H. Szumilowicz, R. Turowski, Dietary trans fatty acids and composition of human atheromatous plaques, Eur. J. Nutr. 43 (2004) 313–318.

[7] S.K. Gebauer, T.L. Psota, P.M. Kris-Etherton, The diversity of health effects of indi-vidual trans fatty acid isomers, Lipids 42 (2007) 787–799.

[8] T. Leth, H.G. Jensen, A.A. Mikkelsen, A. Bysted, The effect of the regulation on trans fatty acid content in Danish food, Atheroscler. Suppl. 7 (2006) 53–56. [9] C.M. Bassett, A.L. Edel, A.F. Patenaude, R.S. McCullough, D.P. Blackwood, P.Y.

Chouinard, P. Paquin, B. Lamarche, G.N. Pierce, Dietary vaccenic acid has antiatherogenic effects in LDLr−/− mice, J. Nutr. 140 (2010) 18–24.

[10] H. Malhi, S.F. Bronk, N.W. Werneburg, G.J. Gores, Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis, J. Biol. Chem. 281 (2006) 12093–12101. [11] J.Y. Kong, S.W. Rabkin, Palmitate-induced apoptosis in cardiomyocytes is

mediat-ed through alterations in mitochondria: prevention by cyclosporin A, Biochim. Biophys. Acta 1485 (2000) 45–55.

[12] R. Mishra, M.S. Simonson, Saturated free fatty acids and apoptosis in microvascu-lar mesangial cells: palmitate activates pro-apoptotic signaling involving caspase 9 and mitochondrial release of endonuclease G, Cardiovasc. Diabetol. 4 (2005) 2. [13] I. Kharroubi, L. Ladriere, A.K. Cardozo, Z. Dogusan, M. Cnop, D.L. Eizirik, Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mecha-nisms: role of nuclear factor-kappaB and endoplasmic reticulum stress, Endocri-nology 145 (2004) 5087–5096.

[14] U. Wenzel, A. Nickel, H. Daniel, Increased carnitine-dependent fatty acid uptake into mitochondria of human colon cancer cells induces apoptosis, J. Nutr. 135 (2005) 1510–1514.

[15] S. Hardy, Y. Langelier, M. Prentki, Oleate activates phosphatidylinositol 3-kinase and promotes proliferation and reduces apoptosis of MDA-MB-231 breast cancer cells, whereas palmitate has opposite effects, Cancer Res. 60 (2000) 6353–6358.

[16] M.B. Paumen, Y. Ishida, M. Muramatsu, M. Yamamoto, T. Honjo, Inhibition of car-nitine palmitoyltransferase I augments sphingolipid synthesis and palmitate-induced apoptosis, J. Biol. Chem. 272 (1997) 3324–3329.

[17] J.E. Ulloth, C.A. Casiano, M. De Leon, Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differen-tiated PC12 cells, J. Neurochem. 84 (2003) 655–668.

[18] L.L. Listenberger, D.S. Ory, J.E. Schaffer, Palmitate-induced apoptosis can occur through a ceramide-independent pathway, J. Biol. Chem. 276 (2001) 14890–14895. [19] R. Urade, M. Kito, Phospholipid metabolism in V79-R membranes composed of phospholipid molecular species containing trans-monoenoic fatty acids, J. Biochem. 96 (1984) 821–828.

[20] A. Miller, E. McGrath, C. Stanton, R. Devery, Vaccenic acid (t11–18:1) is converted to c9, t11–CLA in MCF-7 and SW480 cancer cells, Lipids 38 (2003) 623–632. [21] E. Oie, T. Ueland, C.P. Dahl, P. Bohov, C. Berge, A. Yndestad, L. Gullestad, P. Aukrust,

R.K. Berge, Fatty acid composition in chronic heart failure: low circulating levels of eicosatetraenoic acid and high levels of vaccenic acid are associated with disease severity and mortality, J. Intern. Med. 270 (2011) 263–272.

[22] S. Sharma, J.E. Mells, P.P. Fu, N.K. Saxena, F.A. Anania, GLP-1 analogs reduce hepato-cyte steatosis and improve survival by enhancing the unfolded protein response and promoting macroautophagy, PLoS One 6 (2011) e25269.

[23] I. Rashedi, S. Panigrahi, P. Ezzati, S. Ghavami, M. Los, Autoimmunity and apoptosis —therapeutic implications, Curr. Med. Chem. 14 (2007) 3139–3151.

[24] S. Ghavami, M. Hashemi, S.R. Ande, B. Yeganeh, W. Xiao, M. Eshraghi, C.J. Bus, K. Kadkhoda, E. Wiechec, A.J. Halayko, M. Los, Apoptosis and cancer: mutations within caspase genes, J. Med. Genet. 46 (2009) 497–510.

[25] A. Kleinridders, H.M. Pogoda, S. Irlenbusch, N. Smyth, C. Koncz, M. Hammerschmidt, J.C. Bruning, PLRG1 is an essential regulator of cell proliferation and apoptosis during vertebrate development and tissue homeostasis, Mol. Cell. Biol. 29 (2009) 3173–3185.

[26] J. Kang, B. de Brito Bezerra, S. Pacios, O. Andriankaja, Y. Li, V. Tsiagbe, H. Schreiner, D.H. Fine, D.T. Graves, Aggregatibacter actinomycetemcomitans infection enhances

apoptosis in vivo through a caspase-3 dependent mechanism in experimental periodontitis, Infect. Immun. 80 (2012) 2247–2256.

[27] J.S. Fridman, S.W. Lowe, Control of apoptosis by p53, Oncogene 22 (2003) 9030–9040.

[28] B. Levine, G. Kroemer, Autophagy in the pathogenesis of disease, Cell 132 (2008) 27–42.

[29] S.M. Alavian, S.R. Ande, K.M. Coombs, B. Yeganeh, P. Davoodpour, M. Hashemi, M. Los, S. Ghavami, Virus-triggered autophagy in viral hepatitis—possible novel strategies for drug development, J. Viral Hepat. 18 (2011) 821–830.

[30] C.D. Pacheco, A.P. Lieberman, The pathogenesis of Niemann–Pick type C disease: a role for autophagy? Expert Rev. Mol. Med. 10 (2008) e26.

[31] R.J. Xavier, A. Huett, J.D. Rioux, Autophagy as an important process in gut homeo-stasis and Crohn's disease pathogenesis, Gut 57 (2008) 717–720.

[32] E. Morselli, L. Galluzzi, O. Kepp, J.M. Vicencio, A. Criollo, M.C. Maiuri, G. Kroemer, Anti- and pro-tumor functions of autophagy, Biochim. Biophys. Acta 1793 (2009) 1524–1532.

[33] S. Ghavami, M.M. Mutawe, D. Schaafsma, B. Yeganeh, H. Unruh, T. Klonisch, A.J. Halayko, Geranylgeranyl transferase 1 modulates autophagy and apoptosis in human airway smooth muscle, Am. J. Physiol. Lung Cell. Mol. Physiol. 302 (2012) L420–L428.

[34] I. Maestre, J. Jordan, S. Calvo, J.A. Reig, V. Cena, B. Soria, M. Prentki, E. Roche, Mitochondrial dysfunction is involved in apoptosis induced by serum with-drawal and fatty acids in the beta-cell line INS-1, Endocrinology 144 (2003) 335–345.

[35] K.T. Weber, Y. Sun, J. Diez, Fibrosis: a living tissue and the infarcted heart, J. Am. Coll. Cardiol. 52 (2008) 2029–2031.

[36] J. Hao, B. Wang, S.C. Jones, D.S. Jassal, I.M. Dixon, Interaction between angiotensin II and Smad proteins infibroblasts in failing heart and in vitro, Am. J. Physiol. Heart Circ. Physiol. 279 (2000) H3020–H3030.

[37] R.H. Cunnington, B. Wang, S. Ghavami, K.L. Bathe, S.G. Rattan, I.M. Dixon, Antifibrotic properties of c-Ski and its regulation of cardiac myofibroblast pheno-type and contractility, Am. J. Physiol. Cell Physiol. 300 (2011) C176–C186. [38] S. Ghavami, M. Eshragi, S.R. Ande, W.J. Chazin, T. Klonisch, A.J. Halayko, K.D.

McNeill, M. Hashemi, C. Kerkhoff, M. Los, S100A8/A9 induces autophagy and apoptosis via ROS-mediated cross-talk between mitochondria and lysosomes that involves BNIP3, Cell Res. 20 (2010) 314–331.

[39] S. Ghavami, M.M. Mutawe, P. Sharma, B. Yeganeh, K.D. McNeill, T. Klonisch, H. Unruh, H.H. Kashani, D. Schaafsma, M. Los, A.J. Halayko, Mevalonate cascade regulation of airway mesenchymal cell autophagy and apoptosis: a dual role for p53, PLoS One 6 (2011) e16523.

[40] I. Nicoletti, G. Migliorati, M.C. Pagliacci, F. Grignani, C. Riccardi, A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry, J. Immunol. Methods 139 (1991) 271–279.

[41] S. Ghavami, M.M. Mutawe, K. Hauff, G.L. Stelmack, D. Schaafsma, P. Sharma, K.D. McNeill, T.S. Hynes, S.K. Kung, H. Unruh, T. Klonisch, G.M. Hatch, M. Los, A.J. Halayko, Statin-triggered cell death in primary human lung mesenchymal cells involves p53-PUMA and release of Smac and Omi but not cytochrome c, Biochim. Biophys. Acta 1803 (2010) 452–467.

[42] S. Ghavami, M. Eshraghi, K. Kadkhoda, M.M. Mutawe, S. Maddika, G.H. Bay, S. Wesselborg, A.J. Halayko, T. Klonisch, M. Los, Role of BNIP3 in TNF-induced cell death–TNF upregulates BNIP3 expression, Biochim. Biophys. Acta 1793 (2009) 546–560.

[43] S. Ghavami, C. Kerkhoff, W.J. Chazin, K. Kadkhoda, W. Xiao, A. Zuse, M. Hashemi, M. Eshraghi, K. Schulze-Osthoff, T. Klonisch, M. Los, S100A8/9 induces cell death via a novel, RAGE-independent pathway that involves selec-tive release of Smac/DIABLO and Omi/HtrA2, Biochim. Biophys. Acta 1783 (2008) 297–311.

[44] J. Garibal, E. Hollville, B. Renouf, C. Tetaud, J. Wiels, Caspase-8-mediated cleavage of Bid and protein phosphatase 2A-mediated activation of Bax are necessary for Verotoxin-1-induced apoptosis in Burkitt's lymphoma cells, Cell. Signal. 22 (2010) 467–475.

[45] C.Y. Chen, C.Y. Chang, H.J. Liu, M.H. Liao, C.I. Chang, J.L. Hsu, W.L. Shih, Apoptosis induction in BEFV-infected Vero and MDBK cells through Src-dependent JNK activation regulates caspase-3 and mitochondria pathways, Vet. Res. 41 (2010) 15.

[46] P. Marimuthu, K. Kaur, U. Kandalam, V. Jasani, N. Bukhari, M. Nguyen, A. Abdul, F.F. Pervez, A. Rathinavelu, Treatment of ovarian cancer cells with nutlin-3 and resveratrol combination leads to apoptosis via caspase activation, J. Med. Food 14 (2011) 46–52.

[47] T. Liu, L.Y. Wu, J.K. Choi, C.E. Berkman, Targeted photodynamic therapy for pros-tate cancer: inducing apoptosis via activation of the caspase-8/-3 cascade path-way, Int. J. Oncol. 36 (2010) 777–784.

[48] Y. Kabeya, N. Mizushima, T. Ueno, A. Yamamoto, T. Kirisako, T. Noda, E. Kominami, Y. Ohsumi, T. Yoshimori, LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing, EMBO J. 19 (2000) 5720–5728.

[49] N. Mizushima, A. Yamamoto, M. Hatano, Y. Kobayashi, Y. Kabeya, K. Suzuki, T. Tokuhisa, Y. Ohsumi, T. Yoshimori, Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells, J. Cell Biol. 152 (2001) 657–668.

[50] X. Liu, C.N. Kim, J. Yang, R. Jemmerson, X. Wang, Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c, Cell 86 (1996) 147–157.

[51] R.M. Kluck, E. Bossy-Wetzel, D.R. Green, D.D. Newmeyer, The release of cyto-chrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis, Sci-ence 275 (1997) 1132–1136.

[52] J. Yang, X. Liu, K. Bhalla, C.N. Kim, A.M. Ibrado, J. Cai, T.I. Peng, D.P. Jones, X. Wang, Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked, Science 275 (1997) 1129–1132.

[53] S. Ghavami, B. Yeganeh, G.L. Stelmack, H.H. Kashani, P. Sharma, R. Cunnington, S. Rattan, K. Bathe, T. Klonisch, I.M. Dixon, D.H. Freed, A.J. Halayko, Apoptosis, autophagy and ER stress in mevalonate cascade inhibition-induced cell death of human atrialfibroblasts, Cell Death Dis. 3 (2012) e330.

[54] M. Ahs, A. Prasad, Z. Aminov, D.O. Carpenter, Mechanisms of cell death of thymo-cytes induced by polyunsaturated, monounsaturated and trans-fatty acids, J. Cell. Biochem. 112 (2011) 3863–3871.

[55] M.G. Traber, J.F. Stevens, Vitamins C and E: beneficial effects from a mechanistic perspective, Free Radic. Biol. Med. 51 (2011) 1000–1013.

[56] L. Liu, Z. Zhang, D. Xing, Cell death via mitochondrial apoptotic pathway due to acti-vation of Bax by lysosomal photodamage, Free Radic. Biol. Med. 51 (2011) 53–68. [57] S.M. Gorski, S. Chittaranjan, E.D. Pleasance, J.D. Freeman, C.L. Anderson, R.J.

Varhol, S.M. Coughlin, S.D. Zuyderduyn, S.J. Jones, M.A. Marra, A SAGE approach to discovery of genes involved in autophagic cell death, Curr. Biol. 13 (2003) 358–363.

[58] G.M. Fimia, M. Piacentini, Regulation of autophagy in mammals and its interplay with apoptosis, Cell. Mol. Life Sci. 67 (2010) 1581–1588.

[59] M.H. Raitt, W.E. Connor, C. Morris, J. Kron, B. Halperin, S.S. Chugh, J. McClelland, J. Cook, K. MacMurdy, R. Swenson, S.L. Connor, G. Gerhard, D.F. Kraemer, D. Oseran, C. Marchant, D. Calhoun, R. Shnider, J. McAnulty, Fish oil supplementation and risk of ventricular tachycardia and ventricularfibrillation in patients with im-plantable defibrillators: a randomized controlled trial, JAMA 293 (2005) 2884–2891. [60] J.P. Chin, A.P. Gust, P.J. Nestel, A.M. Dart, Marine oils dose-dependently inhibit va-soconstriction of forearm resistance vessels in humans, Hypertension 21 (1993) 22–28.

[61] A.T. Erkkila, S.L. Booth, F.B. Hu, P.F. Jacques, J.E. Manson, K.M. Rexrode, M.J. Stampfer, A.H. Lichtenstein, Phylloquinone intake as a marker for coronary heart disease risk but not stroke in women, Eur. J. Clin. Nutr. 59 (2005) 196–204. [62] F.B. Hu, W.C. Willett, Optimal diets for prevention of coronary heart disease, JAMA

288 (2002) 2569–2578.

[63] F.B. Hu, M.J. Stampfer, J.E. Manson, E. Rimm, G.A. Colditz, B.A. Rosner, C.H. Hennekens, W.C. Willett, Dietary fat intake and the risk of coronary heart disease in women, N. Engl. J. Med. 337 (1997) 1491–1499.

[64] G.F. Watts, B. Lewis, P. Jackson, V. Burke, E.S. Lewis, J.N. Brunt, D.J. Coltart, Relationships between nutrient intake and progression/regression of coronary atherosclerosis as assessed by serial quantitative angiography, Can. J. Cardiol. 11 (Suppl. G) (1995) 110G–114G.

[65] S.K. Gebauer, J.M. Chardigny, M.U. Jakobsen, B. Lamarche, A.L. Lock, S.D. Proctor, D.J. Baer, Effects of ruminant trans fatty acids on cardiovascular disease and can-cer: a comprehensive review of epidemiological, clinical, and mechanistic studies, Adv. Nutr. 2 (2011) 332–354.

[66] K.C. Fitzpatrick, Regulatory issues related to functional foods and natural health products in Canada: possible implications for manufacturers of conjugated linoleic acid, Am. J. Clin. Nutr. 79 (2004) 1217S–1220S.

[67] J.L. Gerberding, Safer fats for healthier hearts: the case for eliminating dietary artificial trans fat intake, Ann. Intern. Med. 151 (2009) 137–138.

[68] Z. Mallat, A. Tedgui, Apoptosis in the vasculature: mechanisms and functional im-portance, Br. J. Pharmacol. 130 (2000) 947–962.

[69] M. Djavaheri-Mergny, M.C. Maiuri, G. Kroemer, Cross talk between apoptosis and autophagy by caspase-mediated cleavage of Beclin 1, Oncogene 29 (2010) 1717–1719. [70] C. Borner, The Bcl-2 protein family: sensors and checkpoints for life-or-death

decisions, Mol. Immunol. 39 (2003) 615–647.

[71] J.M. Adams, S. Cory, Life-or-death decisions by the Bcl-2 protein family, Trends Biochem. Sci. 26 (2001) 61–66.

[72] F. Tzifi, C. Economopoulou, D. Gourgiotis, A. Ardavanis, S. Papageorgiou, A. Scorilas, The role of BCL2 family of apoptosis regulator proteins in acute and chronic leukemias, Adv. Hematol. 2012 (2012) 524308.

[73] O. Teijido, L. Dejean, Upregulation of Bcl2 inhibits apoptosis-driven BAX inser-tion but favors BAX relocalizainser-tion in mitochondria, FEBS Lett. 584 (2010) 3305–3310.

[74] X. Chen, J.Y. Wong, P. Wong, E.H. Radany, Low-dose valproic acid enhances radio-sensitivity of prostate cancer through acetylated p53-dependent modulation of mitochondrial membrane potential and apoptosis, Mol. Cancer Res. 9 (2011) 448–461.

[75] T. Singh, S.D. Sharma, S.K. Katiyar, Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo, PLoS One 6 (2011) e27444.

[76] M. Khan, C. Ding, A. Rasul, F. Yi, T. Li, H. Gao, R. Gao, L. Zhong, K. Zhang, X. Fang, T. Ma, Isoalantolactone induces reactive oxygen species mediated apoptosis in pan-creatic carcinoma PANC-1 cells, Int. J. Biol. Sci. 8 (2012) 533–547.

[77] K. Nys, H. Maes, G. Andrei, R. Snoeck, M. Garmyn, P. Agostinis, Skin mild hypoxia enhances killing of UVB-damaged keratinocytes through reactive oxygen species-mediated apoptosis requiring Noxa and Bim, Free Radic. Biol. Med. 52 (2012) 1111–1120.

[78] Q. Li, Q. Zhang, M. Wang, S. Zhao, J. Ma, N. Luo, N. Li, Y. Li, G. Xu, J. Li, Eicosapentaenoic acid modifies lipid composition in caveolae and induces trans-location of endothelial nitric oxide synthase, Biochimie 89 (2007) 169–177. [79] C. Assuncao Guimaraes, R. Linden, Programmed cell deaths. Apoptosis and

alter-native deathstyles, Eur. J. Biochem. 271 (2004) 1638–1650.

[80] A. Degterev, J. Yuan, Expansion and evolution of cell death programmes, Nat. Rev. Mol. Cell Biol. 9 (2008) 378–390.

[81] M.C. Maiuri, E. Zalckvar, A. Kimchi, G. Kroemer, Self-eating and self-killing: crosstalk between autophagy and apoptosis, Nat. Rev. Mol. Cell Biol. 8 (2007) 741–752.

[82] K. Moreau, S. Luo, D.C. Rubinsztein, Cytoprotective roles for autophagy, Curr. Opin. Cell Biol. 22 (2010) 206–211.

[83] D. Gozuacik, A. Kimchi, Autophagy as a cell death and tumor suppressor mecha-nism, Oncogene 23 (2004) 2891–2906.

[84] A. Liao, R. Hu, Q. Zhao, J. Li, Y. Li, K. Yao, R. Zhang, H. Wang, W. Yang, Z. Liu, Autophagy induced by FTY720 promotes apoptosis in U266 cells, Eur. J. Pharm. Sci. 45 (2012) 600–605.

[85] Y. Grishchuk, V. Ginet, A.C. Truttmann, P.G. Clarke, J. Puyal, Beclin 1-independent autophagy contributes to apoptosis in cortical neurons, Autophagy 7 (2011) 1115–1131.

[86] S.H. Park, G.Y. Chi, H.S. Eom, G.Y. Kim, J.W. Hyun, W.J. Kim, S.J. Lee, Y.H. Yoo, Y.H. Choi, Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI-H460 human lung carcinoma cells, Int. J. Oncol. 40 (2012) 1929–1940.

[87] V. Nogueira, Y. Park, C.C. Chen, P.Z. Xu, M.L. Chen, I. Tonic, T. Unterman, N. Hay, Akt determines replicative senescence and oxidative or oncogenic premature senescence and sensitizes cells to oxidative apoptosis, Cancer Cell 14 (2008) 458–470.

[88] M. Los, S. Maddika, B. Erb, K. Schulze-Osthoff, Switching Akt: from survival signal-ing to deadly response, Bioessays 31 (2009) 492–495.