Biochem. J. (2008) 410, 525–534 (Printed in Great Britain)
525
doi:10.1042/BJ20071063
Unsaturated lipid peroxidation-derived aldehydes activate autophagy
in vascular smooth-muscle cells
Bradford G. HILL, Petra HABERZETTL, Yonis AHMED, Sanjay SRIVASTAVA and Aruni BHATNAGAR1
Institute of Molecular Cardiology, Department of Medicine, University of Louisville, Louisville, KY 40202, U.S.A.
Proteins modified by aldehydes generated from oxidized lipids
accumulate in cells during oxidative stress and are commonly
detected in diseased or aged tissue. The mechanisms by
which cells remove aldehyde-adducted proteins, however, remain
unclear. Here, we report that products of lipid peroxidation such
as 4-HNE (4-hydroxynonenal) and acrolein activate autophagy
in rat aortic smooth-muscle cells in culture. Exposure to 4HNE led to the modification of several proteins, as detected
by anti-protein–4-HNE antibodies or protein-bound radioactivity
in [3 H]4-HNE-treated cells. The 4-HNE-modified proteins were
gradually removed from cells. The removal of 4-HNE-modified
proteins was not affected by the oxidized protein hydrolase
inhibitor, acetyl leucine chloromethyl ketone, or lactacystin,
although it was significantly decreased by PSI (proteasome
inhibitor I), the lysosome/proteasome inhibitor MG-132
(carbobenzoxy-L-leucyl-L-leucyl-leucinal), insulin or the autophagy inhibitor 3-MA (3-methyladenine). Pre-incubation of cells
with rapamycin accelerated the removal of 4-HNE-modified
proteins. Treatment with 4-HNE, nonenal and acrolein, but
not nonanal or POVPC (1-palmitoyl-2-oxovaleroyl phosphatidyl
choline), caused a robust increase in LC3-II (microtubuleassociated protein 1 light chain 3-II) formation, which was
increased also by rapamycin, but prevented by insulin.
Electron micrographs of 4-HNE-treated cells showed extensive
vacuolization, pinocytic body formation, crescent-shaped
phagophores, and multilamellar vesicles. Treatment with 3-MA
and MG-132, but not proteasome-specific inhibitors, induced cell
death in 4-HNE-treated cells. Collectively, these results show
that lipid peroxidation-derived aldehydes stimulate autophagy,
which removes aldehyde-modified proteins, and that inhibition
of autophagy precipitates cell death in aldehyde-treated cells.
Autophagy may be an important mechanism for the survival
of arterial smooth-muscle cells under conditions associated with
excessive lipid peroxidation.
INTRODUCTION
cumulative injury or whether it is in fact reflective or causative
of ongoing pathology. Products of lipid peroxidation interfere
with signal transduction [6], energy production [7,8], ion channel
function [9] and cell death pathways [10]. In addition, aldehydes
generated from lipid oxidation could interfere with cell function
by forming covalent adducts with proteins and thereby disrupt
their function.
Proteins modified by lipid peroxidation-derived aldehydes such
as malondialdehyde, 4-HNE (4-hydroxynonenal) or POVPC (1palmitoyl-2-oxovaleroyl phosphatidylcholine) have been detected
in animal and human tissues under several pathological conditions including myocardial ischaemia [11], atherosclerosis
[12,13], restenosis [14], Parkinson’s disease [15] and Alzheimer’s
disease [16]. Sustained presence of protein–aldehyde adducts
in diseased lesions suggests that these products are generated
continuously or are associated with cells that have either died,
remain uncleared or are otherwise unable to cope with damaged
proteins.
Mechanisms responsible for removing proteins modified by
lipid peroxidation products are not well understood. As a result,
it is difficult to delineate the contribution of such a protein modification to disease severity or progression. Previous studies
report conflicting results. In lens epithelial cells, 4-HNE-modified
proteins have been shown to be preferentially ubiquitinated
Oxidative degradation of polyunsaturated lipids generates a
variety of bioactive intermediates and end-products. These
include lipid hydroperoxides and carbonyl end-products that arise
from fragmentation of peroxyl linkages and radical-elimination
reactions [1]. In the presence of oxygen, these reactions are
autocatalytic and self-sustaining and are terminated only by
radical–radical annihilation. Non-radical metastable products
such as alkanes, hydroxides or alkyl carbonyls are by-products
of such lipid oxidation reactions. The appearance of lipid
peroxidation products in live tissue is indicative of unquenched
radical reactions and their presence has been documented under
a variety of toxicological and pathological states associated
with radical injury such as carbon tetrachloride poisoning [2],
Alzheimer’s disease [3] and atherosclerosis [4].
Reactive products of oxidized lipids could amplify and prolong
oxidative stress induced by free radicals. In addition to being
footprints of radical presence, lipid peroxidation products may
be in themselves mediators of oxidative damage, propagators
of tissue injury or triggers of stress signalling [5]. Nonetheless,
the specific role of lipid peroxidation products is poorly understood, and it remains unclear whether the association of lipid
peroxidation products with diseased tissue is merely indicative of
Key words: atherosclerosis, autophagy, 4-hydroxynonenal
(4-HNE), oxidative stress, oxidized lipid, smooth-muscle cell,
unsaturated aldehyde.
Abbreviations used: ALCK, acetyl leucine chloromethyl ketone; cpm, counts per minute; DHN, dihydroxynonene; DMEM, Dulbecco’s modified Eagle’s
medium; DNP, 2,4-dinitrophenol; DNPH, 2,4-dinitrophenylhydrazine; ER, endoplasmic reticulum; FBS, foetal bovine serum; HBSS, Hanks balanced salt
solution; 4-HNA, 4-hydroxynonenoic acid; 4-HNE, 4-hydroxynonenal; LC3, microtubule-associated protein 1 light chain 3; LDL, low-density lipoprotein;
3-MA, 3-methyladenine; mTOR, mammalian target of rapamycin; NAC, N -acetylcysteine; POVPC, 1-palmitoyl-2-oxovaleroyl phosphatidylcholine; PSI,
proteasome inhibitor I; ROS, reactive oxygen species; UPR, unfolded protein response; VSMC, vascular smooth-muscle cell.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2008 Biochemical Society
526
B. G. Hill and others
and degraded by the lysosome with little or no contribution
by the proteasomal pathway of protein degradation [17]. In
the kidney of ferric nitrilotriacetate-treated animals, proteasomal degradation has been suggested to be a crucial pathway for
the removal of protein–4-HNE adducts [18]. A predominant role
for the proteasome in the removal of protein–4-HNE adducts is,
however, inconsistent with several studies showing that oxidative
stress decreases proteasome activity and that 4-HNE inhibits the
proteasome by direct covalent modification [18,19]. In addition,
in vitro studies show that 4-HNE-cross-linked proteins inhibit
proteasomal activity [20], suggesting that protein degradation
pathways other than the proteasome may be important for the
removal of protein–4-HNE adducts. It is likely that different
pathways of protein removal are engaged by different cells and
that their contribution varies with the extent of lipid peroxidation.
The present study was designed to examine the major pathways
for the degradation of protein–4-HNE adducts in VSMCs
(vascular smooth-muscle cells). These cells make up the medial
layer of adult arteries and, under pathological conditions such as
arterial restenosis [14], vasculitis [21] and atherosclerosis [4,13],
accumulate high levels of proteins modified by products of lipid
peroxidation.
EXPERIMENTAL
Materials
Reagent 4-HNE, [3 H]4-HNE and POVPC were synthesized
as described in [22,23]. All other chemicals were obtained
from Sigma (St. Louis, MO, U.S.A.) unless otherwise stated.
Electrophoresis supplies were purchased from Bio-Rad. Primary
antibodies against LC3 (microtubule-associated protein 1 light
chain 3) were obtained from MBL International (Woburn, MA,
U.S.A.). Polyclonal antibodies against KLH (keyhole-limpet
haemocyanin)–HNE (protein–4-HNE) were raised and tested as
previously described [14]. ECL® reagents were purchased from
GE Healthcare (Amersham Biosciences, Pittsburgh, PA, U.S.A.).
The Oxyblot kit was obtained from Chemicon International
(Temecula, CA, U.S.A.). ALCK (acetyl leucine chloromethyl
ketone) was synthesized as described in [24].
Cell culture experiments
Primary VSMCs obtained from Sprague–Dawley rat aortic
explants were grown in DMEM (Dulbecco’s modified Eagle’s
medium; Life Technologies–Invitrogen) supplemented with 10 %
(v/v) FBS (foetal bovine serum) and 0.1 % streptomycin/penicillin. Only passages 4–12 were used for study. At 95–100 %
confluency, the culture medium (DMEM containing 10 % FBS)
was removed and the cells were washed twice with HBSS (Hanks
balanced salt solution; pH 7.4) containing 20 mM Hepes, 135 mM
NaCl, 5.4 mM KCl, 1.0 mM MgCl2 , 2.0 mM CaCl2 ,
2.0 mM NaH2 PO4 and 5.5 mM glucose. To avoid reactions
between 4-HNE and nucleophilic constituents of the culture
medium (lysine, albumin etc.), cells were treated with 4-HNE
in HBSS. After indicated treatments, the cells were washed twice
with fresh HBSS and scraped with a rubber policeman in lysis
buffer containing 25 mM Hepes (pH 7.0), 1 mM EDTA, 1 mM
EGTA, 1 % Nonidet P40, 0.1 % SDS, 1:100 protease inhibitor
cocktail (Sigma) and 1:100 phosphatase inhibitor cocktail (Pierce,
Rockford, IL, U.S.A.). The cell suspension was further lysed
by sonication, and lysates were centrifuged at 14 000 g for
15 min at 4 ◦C. Total protein concentration was measured using a
commercially available kit (Bio-Rad).
c The Authors Journal compilation c 2008 Biochemical Society
Measurement of protein-bound 4-HNE
[3 H]4-HNE [50 µM; 2.7 × 106 cpm (counts per minute) per
well] was added to cultured VSMCs. After 30 min, the 4HNE-containing medium was removed, and the cells were
washed extensively with HBSS and were either collected or
incubated further in 4-HNE-free culture medium. At the indicated
times, cells were collected in 5 % (v/v) HClO4 and sonicated,
and the protein precipitates were sedimented by centrifugation.
Radioactivity in the supernatant was used for HPLC analysis
of acid-soluble metabolites (as described in the Supplementary
material at http://www.BiochemJ.org/bj/410/bj4100525add.htm).
The protein pellet was washed extensively with acetone and solubilized in 0.5 M Tris (pH 8.8) containing 1 mM EDTA and
1 % SDS. Protein-bound 4-HNE was measured by scintillation
counting and normalized to total protein.
Immunological detection and quantification of protein–4-HNE
adducts
SDS/PAGE, Western blotting and slot blotting were performed
as previously described [25]. For quantification of protein–
4-HNE adducts, protein from tissue homogenate (2.0 µg) was
loaded on to the wells of the Bio-Dot SF apparatus (Bio-Rad,
Hercules, CA, U.S.A.) and microfiltered through nitrocellulose
membranes under vacuum. The nitrocellulose membranes were
subjected to standard immunodetection techniques using antiprotein–4-HNE antibodies. Western and slot blots were developed
using ECL® plus reagents and a Typhoon 9400 variable mode
imager (Amersham Biosciences). Intensity of the immunoreactive
bands was quantified using ImageQuant TL software (Amersham
Biosciences).
Electron microscopy
VSMCs were grown on Arclar coverslips (Ladd Research,
Williston, VT, U.S.A.) and treated in HBSS with 50 µM 4HNE for 30 min. The medium was removed and the cells were
incubated in culture medium for an additional 2.5 h. The cells
were washed with PBS and fixed with 3 % glutaraldehyde
in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h at room
temperature (25 ◦C). The cells were then post-fixed with 1 %
osmium tetroxide, sectioned and embedded in LX112 plastic.
Ultrathin sections were stained with uranyl acetate and lead
citrate, and electron micrographs were taken using a Philips CM10
transmission electron microscope operating at 60 kV (× 1000–
72 000 magnification).
Photomicrography and estimation of cell survival
Cells were treated as indicated, and a Nikon 990 digital camera
was used to capture × 10 inverted microscope images. Adherent
cells were counted in each photomicrograph and used for cell survival analyses. All treatments were performed at least in triplicate.
Statistical analysis
Results are reported as means +
− S.E.M. The unpaired Student’s
t test was used to compare two treatment groups. A P value of
< 0.05 was considered statistically significant.
RESULTS
4-HNE metabolism and protein–4-HNE modification in VSMCs
To examine the formation and the removal of protein–4-HNE adducts, VSMCs were exposed to 50 nmol of reagent 4-HNE in 1 ml
Unsaturated aldehydes activate autophagy
Figure 1
527
Formation and removal of protein–4-HNE adducts in VSMCs
Cells were exposed to either non-radioactive or radioactive 4-HNE (50 µM) for 30 min. The medium was removed and the cells were either collected immediately or incubated in 4-HNE-free
culture medium for the indicated times. After incubation, the cells were collected in lysis buffer for subsequent analysis. (A) Time-dependent changes of protein–4-HNE adducts were assessed
by slot immunoblotting using anti-protein–4-HNE antibodies. Slot densities were quantified by densitometry. Bars represent means, expressed as arbitrary units (n = 3 per group; ∗ P < 0.05).
(B) Representative Western blot of lysates prepared from cells treated with 4-HNE by using anti-protein–4-HNE antibodies. (C) Oxyblot analysis of 4-HNE-exposed VSMCs. Proteins from lysates
of 4-HNE-exposed cells were derivatized with DNPH and subjected to Western blotting using anti-DNP antibodies. (D) Time-dependent changes in protein-bound [3 H]4-HNE. Cells were treated
with [3 H]4-HNE and radioactivity in the protein precipitates was measured by scintillation counting and normalized to total protein. Protein-bound 4-HNE is expressed as nanomoles of 4-HNE per
∗
milligram of protein. (Values are expressed as the means +
− S.E.M. for eight replicates per group; P < 0.0001.)
of HBSS for 30 min. This treatment corresponds to ∼ 50 nmol of
4-HNE per 500 000 cells. After 30 min of incubation, the medium
was removed and replaced with 4-HNE-free culture medium. At
the indicated times, cells were lysed and protein–4-HNE adducts
were measured by immunoblot analysis using anti-protein–4HNE antibodies. As shown in Figures 1(A) and 1(B), the protein–
4-HNE adducts formed 30 min after 4-HNE exposure were almost
completely removed within 8 h (P < 0.0001). Notably, 4-HNE
did not form adducts with proteins during shorter time exposures,
indicating that at least 30 min are needed for immunologically
detectable increases in protein modification (results not shown).
No protein adducts were detected at 4-HNE concentrations
< 50 µM (Figure 3Bii).
Antibodies raised against protein–4-HNE adducts recognize
both Michael adducts and Schiff bases formed between 4-HNE
and protein cysteine, lysine and arginine residues. Therefore,
to identify whether Michael adducts of 4-HNE (in which the
carbonyl group remains free) are also removed with a similar
time course, the proteins recovered from 4-HNE-treated cells
were derivatized with DNPH (2,4-dinitrophenylhydrazine), which
derivatizes the aldehyde moiety of 4-HNE–Michael adducts to a
hydrazone. Modified proteins were detected by immunoblotting
with anti-DNP (2,4-dinitrophenol) antibodies. Treatment with 4HNE led to an increase in protein-bound DNP. Upon removal
of 4-HNE, the abundance of DNP adducts decreased with a
time course similar to that observed with anti-protein–4-HNE
antibodies (Figure 1C).
To confirm further that immunopositive bands were indeed
due to 4-HNE-bound proteins, the VSMCs were treated with
[3 H]4-HNE (2.7 × 106 cpm per well). The cells were then either
collected after 30 min of exposure to 4-HNE or incubated further
for the indicated times in 4-HNE-free culture medium. As
shown in Figure 1(D), the abundance of 4-HNE-bound proteins,
as determined by scintillation counting, decreased with time after
[3 H]4-HNE exposure (P < 0.0001). Some detectable modification persisted after 8 h as indicated by protein-bound radioactivity. The close agreement with tracer studies suggests that
immunological measurements reflect valid and quantifiable rates
of protein–4-HNE removal. Most of the 4-HNE-modified proteins
within the SDS/PAGE resolving range were removed at similar
rates (see Supplementary Figure 1 at http://www.BiochemJ.org/
bj/410/bj4100525add.htm). Proteins migrating to 250, 150, 80
and 50 kDa that were immunoreactive with anti-protein–4-HNE
antibodies had a calculated half-life of 169 +
− 16 min. Because
most 4-HNE-modified proteins were lost with similar rates,
immunological assessments of the rate of removal of protein–
HNE adducts appear to reflect the loss of most 4-HNE-modified
proteins and are not dominated by one heavily modified protein.
Our previous studies show that VSMCs rapidly metabolize 4HNE [26]. Hence, the rate of loss of protein adducts after bolus
exposure to 4-HNE could be confounded by HNE metabolites
that could persistently and recurrently induce new modifications.
To determine whether metabolites contribute to adduct formation,
VSMCs were treated with [3 H]4-HNE for 30 min and the medium
was collected for analysis of 4-HNE metabolites by HPLC (see
Supplementary Figure 2A at http://www.BiochemJ.org/bj/410/
bj4100525add.htm). Approx. 60 % of the radioactivity in the
medium was assigned to unmetabolized reagent 4-HNE. Other
metabolites found in the HBSS medium were: GS-X (glutathione conjugates of 4-HNE; ∼ 8 %), DHN (dihydroxynonene;
∼ 5 %), 4-HNA (4-hydroxynonenoic acid; ∼ 12 %) and an
unidentified metabolite (∼ 13 %) eluting with a τ R (retention
time) of 48–49 min (see Supplementary Table 1 at http://www.
BiochemJ.org/bj/410/bj4100525add.htm). To examine metabolites that remained inside cells, cells were collected in HClO4
and sedimented by centrifugation. Acid-soluble metabolites in
c The Authors Journal compilation c 2008 Biochemical Society
528
B. G. Hill and others
the supernatant were separated by HPLC. The major intracellular
metabolite after 0.5 h of exposure to 4-HNE eluted with the τ R of
a glutathione conjugate (Supplementary Figure 2B). Significant
radioactivity recovered from the extracellular medium 4 and
8 h after exposure to 4-HNE was also due to the glutathione
conjugates of HNE (Supplementary Table 1). Collectively, these
results show that 4-HNE metabolites and unmetabolized 4-HNE
are rapidly extruded from VSMCs. Glutathione conjugates are
the major intracellular metabolites and, although extruded more
slowly, are quantitatively recovered in the extracellular medium.
These observations suggest that protein adducts are formed directly from 4-HNE and are the longest-lived products of 4-HNE.
Removal of protein–4-HNE adducts
In most cells, major pathways for the removal of modified
proteins are proteasomal degradation and lysosomal autophagy
[27]. In addition, oxidized proteins have also been shown to
be degraded by oxidized protein hydrolase [28]. To examine
how VSMCs remove protein–4-HNE adducts, we examined
each of these pathways. First, cells were treated with MG132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucylleucinal), which inhibits both the lysosomal/autophagic and
proteasomal pathways [17], and the formation and loss of protein–
4-HNE adducts was followed by slot-immunoblot analysis. As
shown in Figure 2(A), treatment with MG-132 decreased adduct
removal, such that 4 h after 4-HNE treatment, significantly
higher levels (38.0 +
− 6.1 %; P < 0.02) of protein–4-HNE adducts
were observed in treated compared with untreated cells. 4HNE co-treatment with MG-132 also increased adduct formation
by 29.2 +
− 4.5 % after 30 min of 4-HNE exposure (P < 0.05),
suggesting that adduct formation and removal may be concurrent
processes, initiated simultaneously upon 4-HNE exposure.
Because interpretation of the results shown in Figure 2(A) could
be confounded due to the presence of MG-132 during the adduct
formation phase, MG-132 was added after [3 H]4-HNE exposure
and protein-bound radioactivity was measured. As shown in
Figure 2(A) (inset), MG-132 added to the culture medium after 4HNE exposure modestly but significantly increased the amount of
protein-bound [3 H]4-HNE at 4 h, suggesting that MG-132 inhibits
removal of protein–4-HNE adducts.
The specific contribution of the proteasome in mediating
protein–4-HNE adduct removal in VSMCs was assessed by
treatment with the proteasome-selective inhibitors, lactacystin
[29] and Z (benzyloxycarbonyl)-Ile-Glu(OtBu)-Ala-Leu-CHO
[PSI (proteasome inhibitor I)] [30], immediately after exposure
of cells to [3 H]4-HNE. Treatment with lactacystin did not affect
protein–4-HNE removal (Figure 2B), whereas treatment with
PSI led to a small, statistically significant, increase in proteinbound radioactivity at 4 h (Figure 2B, inset), suggesting that the
proteasome plays a minor role in the degradation of protein–4HNE adducts. To determine the role of the lysosomal–autophagy
pathway, VSMCs were treated with 4-HNE for 30 min and
the medium was replaced with culture medium containing the
autophagy inhibitor 3-MA (3-methyladenine) [31]. As shown
in Figure 2(C), cells treated with 3-MA contained significantly
more protein–4-HNE adducts than non-3-MA-treated cells.
The relative contribution of autophagy was also examined by
measurement of protein-bound [3 H]4-HNE. Cells were exposed
to [3 H]4-HNE followed by treatment with 3-MA. Comparable
with immunological measurements, inhibition of autophagy
significantly decreased removal of protein–[3 H]4-HNE adducts
(Figure 2C, inset). In contrast, treatment with the oxidized protein
hydrolase inhibitor, ALCK (10 µM), did not affect the abundance of adducts 4 h after 4-HNE treatment (results not shown).
c The Authors Journal compilation c 2008 Biochemical Society
Figure 2 Removal of protein–4-HNE adducts by proteasomal and
autophagic pathways
Slot immunoblots and radioactive measurements of protein–HNE adducts in VSMCs in the
presence and absence of inhibitors of the proteasome and autophagy. Cells were left untreated
(C) or treated with 4-HNE (50 µM) in HBSS for 0.5 h in the presence or absence of (A) MG-132
(10 µM) or (B) lactacystin (10 µM). Cells were then collected immediately in lysis buffer or
were incubated for an additional 3.5 h in culture medium containing the inhibitor. After cell lysis,
protein–4-HNE adducts were measured by slot immunoblotting and quantitative densitometry
(n = 3 per group; ∗ P < 0.05). (C) Cells were treated with 4-HNE (50 µM) in HBSS for 0.5 h.
Cells were then lysed, or culture medium alone or containing 3-MA (10 mM) was added to
the cells for 3.5 h. After lysis, protein–4-HNE adducts were measured by slot immunoblotting
and quantitative densitometry (n = 3 per group; ∗ P < 0.05); insets to (A–C) show levels of
radioactive protein remaining in cells after treatment with [3 H]4-HNE (50 µM). After 30 min
of [3 H]HNE exposure, culture medium alone or medium containing MG-132 (10 µM; A), PSI
(50 µM; B) or 3-MA (10 mM; C) was added to the cells for 3.5 h. Cells were then lysed,
and protein-bound radioactivity was measured by scintillation counting and normalized to total
protein (n = 6 per group; ∗ P < 0.05).
Taken together, these results suggest that autophagy plays a
significant role in the removal of protein–4-HNE adducts in
VSMCs.
Unsaturated aldehydes activate autophagy
We next hypothesized that the electrophilic stress induced
by aldehyde exposure stimulates autophagy. To test this, we
Unsaturated aldehydes activate autophagy
Figure 3
Unsaturated electrophilic aldehydes promote LC3-II formation
(A) VSMCs were treated for 30 min with HNE (50 µM), POVPC (50 µM) or acrolein (ACR;
25 µM), followed by a 2.5 h incubation in aldehyde-free culture medium. Cells were collected
and lysed, and changes in LC3-II formation were analysed by Western blotting using anti-LC3
antibodies. For loading control, the blot was stripped and reprobed with anti-actin antibodies
(n = 3 per group; ∗ P < 0.05 compared with untreated cells). (B) HNE-induced changes in
LC3-II formation. (i) Cells were either left untreated (C) or treated with 4-HNE (0–200 µM) for
0.5 h and 4-HNE-free culture medium was added for 2.5 h. Concentration-dependent effects of
4-HNE on LC3 conversion were assessed by Western blotting. Due to slight image saturation,
the actin loading control was adjusted linearly using brightness/contrast tools in Microsoft
PowerPoint. (ii) Western blots showing concentration dependence of protein–4-HNE adduct
formation. Cells were treated with 4-HNE (0–200 µM) for 0.5 h and protein–4-HNE adduct
formation was assessed by Western blotting using anti-protein–4-HNE antibodies. (C) Relative
changes in LC3-II formation by HNE analogues. Cells were left untreated (control) or treated with
nonanal (50 µM), trans -2-nonenal (nonenal; 50 µM) or 4-HNE (50 µM), and LC3-II formation
was assessed by Western blotting.
measured the processing of LC3 in aldehyde-treated VSMCs.
Formation of LC3-II (phosphatidylethanolamine-conjugated
form) is an essential step in autophagosome formation; the abundance of LC3-II correlates with the number of autophagosomes
and is therefore a practical index of autophagic activity in
mammalian cells [32,33]. Untreated cells maintained in culture
showed detectable levels of LC3-II, indicating baseline autophagy
(Figure 3A). 4-HNE treatment caused a robust and significant
529
increase in the abundance of the autophagy-indicative form of
LC3 (16 kDa; LC3-II) (Figure 3A). Similar increases in LC3-II
were also observed with other aldehydes. Stimulation with the C3
unsaturated aldehyde, acrolein, led to a more profound increase
in LC3-II formation than did 4-HNE; however, the saturated
phospholipid aldehyde, POVPC, only marginally increased LC3II formation.
Exposure to 4-HNE led to a concentration-dependent increase
in LC3-II formation in VSMCs (Figure 3Bi). Appreciable
increases in LC3-II were observed at concentrations of 4-HNE
50 µM. The concentration dependence of LC3-II formation was
similar to that of protein–4-HNE adduct formation (Figure 3Bii).
While some protein–4-HNE adducts were also detected in
untreated cells, treatment with low concentrations of 4-HNE
(< 50 µM) led to a decrease in protein–4-HNE adducts. This
may be a reflection of increased proteolysis stimulated by low
concentrations of 4-HNE. Indeed, it has been shown that, at low
concentrations, carbonyls such as 4-HNE activate the proteasome
by as much as 400 % [34–36].
Because acrolein is more electrophilic than 4-HNE or POVPC,
it appears that the ability of the aldehydes to induce autophagy
may be dependent on their electrophilicity. A proportional
relationship between electrophilicity and autophagy stimulation
was supported by a more systematic evaluation of the role
of aldehyde electrophilicity in LC3-II activation. As shown in
Figure 3(C), the electrophilic C9 aldehyde 4-HNE was more potent
in stimulating LC3-II formation than the less electrophilic C9
aldehyde nonenal, which in turn was more active than the least
reactive C9 saturated aldehyde nonanal. On the basis of these
observations, we conclude that highly electrophilic aldehydes
such as 4-HNE and acrolein are likely to be more potent in
stimulating autophagosome formation.
Previous studies show that the mTOR (mammalian target of
rapamycin) is a negative regulator of autophagy and that inhibition of mTOR by rapamycin activates autophagy [37], whereas
activation of mTOR by insulin decreases autophagy [38]. As
shown in Figure 4(A), cells pretreated with rapamycin accumulated less protein–4-HNE adducts after 30 min, and rapamycin
pretreatment decreased the abundance of protein–4-HNE adducts
after 4 h by 50 % when compared with cells treated with 4HNE alone (P < 0.05). Both rapamycin pretreatment and 4-HNE
exposure increased LC3-II formation; the combination of the two
treatments appeared to have a synergistic effect and increased
LC3-II formation by 2.5 +
− 0.3-fold (P < 0.05; Figure 4B). In
contrast, treatment with insulin resulted in a small but statistically
significant increase in the amount of adducts retained in the cells
at 4 h (P < 0.05; Figure 4C), and insulin prevented the formation
of LC3-II by 4-HNE (P < 0.0005; Figure 4D). Interestingly,
when rapamycin was added to the cells at the same time as 4HNE, no change in adduct removal was observed (results not
shown), as opposed to an increase in adduct removal in cells that
were pretreated with rapamycin 30 min before 4-HNE treatment
(Figure 4A). These results suggest that exposure to 4-HNE results
in prompt and early activation of autophagic responses.
Ultrastructural changes induced by 4-HNE
The autophagic programme involves multiple signalling pathways
that converge on the formation of autophagosomes. These collect
targeted cell constituents and subsequently fuse with lysosomes
[39]. While many biochemical markers could be used to follow
autophagosome formation, electron microscopy is considered
the gold standard for documenting autophagy. Hence, we examined ultrastructural changes in VSMCs upon 4-HNE treatment.
As shown in Figures 5(A) and 5(B), 4-HNE-exposed cells
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530
Figure 4
B. G. Hill and others
Regulation of protein–HNE adduct removal by rapamycin and insulin
Immunoblots of lysates from 4-HNE-treated VSMCs. Cells were pretreated with culture medium containing rapamycin (Rapa; 0.2 µM) or insulin (Ins; 1 m-units/ml) for 30 min followed by HNE
treatment (50 µM). Control cells (C) received the vehicle only. (A) Slot blots of protein–4-HNE adducts in 4-HNE-exposed cells pretreated with rapamycin (n = 3 per group; ∗ P < 0.05 compared
with 4-HNE treatment alone at 4 h). (B) LC3-II formation in cells exposed to rapamycin and 4-HNE (n = 3 per group; ∗ P < 0.05 compared with cells treated with HBSS alone). (C) Quantification of
protein–4-HNE adducts in 4-HNE-exposed cells pretreated with insulin (n = 3 per group; ∗ P < 0.05 compared with 4-HNE treatment alone at 4 h). (D) LC3-II formation in cells exposed to insulin
and 4-HNE (n = 3 per group; ∗ P < 0.0005 compared with cells treated with 4-HNE alone).
showed mild to extensive autophagic vacuolization, phagocytotic/
pinocytic body formation and loss or thinning of the glycocalyx.
Such changes were not apparent in untreated cells (see
Supplementary Figure 4 at http://www.BiochemJ.org/bj/410/
bj4100525add.htm). Several 4-HNE-treated cells showed
isolation membranes, representing immature phagophores, that
were observed near mitochondria (Figure 5C), and mature
phagophores and autophagosomes were observed surrounding
bulk cytoplasm or organelles such as the mitochondria and
rough ER (endoplasmic reticulum) (Figures 5D and 5E). All
stages of autophagosome formation were observed. These ranged
from early double-membraned vacuoles (Figure 5D), to crescentshaped phagophores (Figures 5D and 5E) and single-membrane
vacuoles containing electron-dense ingested material and late
multilamellar membranes (Figure 5D). Cells exposed to 4HNE also demonstrated mild to extensive membrane ruffling
(Figure 5F), indicative of processes leading to the internalization
of extracellular cargoes via vesicular intermediates.
Inhibition of autophagy causes 4-HNE-induced cell death
The results obtained so far indicated to us that exposure to 4HNE results in the accumulation of several proteins modified
by 4-HNE and that these proteins are gradually and systemically
removed by proteasomal proteolysis and autophagy. To determine
whether removal of modified proteins is essential for protection
against 4-HNE toxicity, we investigated whether inhibiting adduct
removal would affect cell viability. As shown in Figure 6(A),
cells treated with MG-132 (Figure 6Aii) or 4-HNE (Figure 6Aiii)
alone showed no signs of cell death 4 h after treatment; however,
treatment with MG-132 triggered significant cell death in 4-HNEexposed cells (Figure 6Aiv), with only 57.2 +
− 7.1 % (Figure 6B)
of the cells remaining viable after the treatment (P < 0.005).
c The Authors Journal compilation c 2008 Biochemical Society
In contrast, treatment of the cells with PSI did not induce cell
death in 4-HNE-treated cells (see Supplementary Figure 5 at
http://www.BiochemJ.org/bj/410/bj4100525add.htm). Treatment
of 4-HNE-exposed cells with 3-MA also induced cell death. As
shown in Figure 6(C), cells treated with 3-MA (Figure 6Cii) or
4-HNE (Figure 6Ciii) alone showed little to no cell death 4 h
after treatment; however, significant cell death occurred in 3-MAtreated cells that were exposed to 4-HNE, with only 63.6 +
− 2.5 %
(Figure 6D) of the cells remaining viable (P < 0.005) after
treatment.
DISCUSSION
The present study demonstrates that proteins modified by
electrophilic products of lipid peroxidation such as 4-HNE are
efficiently removed from VSMCs. We find that unsaturated
aldehydes stimulate autophagic responses and that inhibition of
autophagy inhibits the removal of protein–4-HNE adducts and
induces cell death. These observations indicate that proteins
modified by aldehydes, if not removed, are likely to be cytotoxic.
To the best of our knowledge, this is the first demonstration of
autophagy stimulated by lipid peroxidation products.
Aldehydes generated from oxidized lipids are highly reactive
[5]. They induce a variety of stress responses and interfere with
cell signalling [6], metabolism [8] and function [9]. To protect
against their potential cytotoxicity, these aldehydes are rapidly
metabolized by several biochemical pathways that include reduction, oxidation and the formation of glutathione conjugates [26].
Nonetheless, when metabolic detoxification is overwhelmed,
these aldehydes form covalent adducts with nucleophilic side
chains of proteins, particularly cysteine, lysine and histidine
residues [5]. As shown by the present study, covalent protein
Unsaturated aldehydes activate autophagy
Figure 5
531
Ultrastructure of HNE-treated VSMCs
Representative electron micrographs of cells treated with 4-HNE (50 µM) in HBSS for 0.5 h. Following treatment, 4-HNE-free medium was added, and the cells were incubated for an
additional 2.5 h. The cells were then fixed and stained, and electron micrographs were taken at × 1000–72 000 magnification. (A, B) 4-HNE-treated cells showing autophagic vacuolization
and phagocytotic/pinocytic membrane processes (A; small arrows). Original magnification = × 2750–4800. (C) Formation of isolation membrane (immature phagophore; open arrow) near
mitochondria in 4-HNE-treated VSMCs. Original magnification = × 14 000. (D) Mature phagophore (open arrow) engulfing cytosolic constituents. A double-membraned autophagosome is
also present (closed arrow). Original magnification = × 18 000. Inset to (D): multilamellar body present in 4-HNE-exposed VSMCs. Original magnification = × 27 500. (E) Mature phagophore
(open arrow) engulfing portions of the rough ER (indicated by ∗ ). Original magnification = × 35 000. (F) Extensive membrane ruffling, endocytosis and pinocytic body formation in 4-HNE-treated
VSMCs. Original magnification = × 14 000.
adducts are the most sustained outcome of 4-HNE exposure.
Greater than 80 % of the radioactivity remaining in [3 H]4HNE-treated cells after 4 h was protein-bound (Supplementary
Figure 2). Abundant protein adducts of 4-HNE and related aldehydes have been detected in several tissues under conditions of
high oxidative stress associated with cardiovascular [4,11–14,21]
and neurodegenerative [15,16] diseases. Several toxicological
states are also associated with the accumulation of protein–
aldehyde adducts [2,40]. Moreover, as shown in Figure 1,
detectable levels of protein–4-HNE adducts are also present in
untreated cells, indicating that basal levels of lipid peroxidation
in otherwise healthy cells are sufficient to induce protein modifications. Nevertheless, the metabolic fate of aldehyde-modified
proteins remains obscure, and the cytotoxic potential of such protein modification reactions has not been assessed. Results of the
present study suggest that healthy cells progressively remove 4HNE-modified proteins and that their accumulation in diseased
tissue may be a reflection of metabolic failure resulting from
either uncontrolled lipid peroxidation or sustained inhibition of
cell processes that remove modified proteins.
We investigated several mechanisms by which protein–
4-HNE adducts are removed in VSMCs. Results of these
investigations show that ALCK does not prevent protein–4-HNE
adduct removal, indicating that degradation via oxidized protein
hydrolase is not a significant fate of protein–4-HNE adducts
in these cells. Similarly, no inhibition of protein–4-HNE
c The Authors Journal compilation c 2008 Biochemical Society
532
Figure 6
B. G. Hill and others
Inhibition of proteolysis triggers cell death in HNE-treated VSMCs
Photomicrographs of VSMCs treated with 4-HNE and with MG-132 or 3-MA. (A) Cells were treated for 0.5 h with 4-HNE (50 µM) alone (iii) or containing MG-132 (iv; 50 µM) in HBSS. The cells
were then incubated in 4-HNE-free medium in the absence or presence of MG-132 for an additional 3.5 h. Control cells (Con) were incubated with 4-HNE-free HBSS for 0.5 h and allowed to recover
in medium containing no other additives (i) or MG-132 (ii). The cells were photographed after 4 h. (B) Adherent cells were counted to estimate the entire cell population and normalized to cell
density from untreated dishes (n = 3 per group; ∗ P < 0.005 compared with other treatment groups). (C) VSMCs were treated with 4-HNE (50 µM) for 0.5 h, and medium alone (iii) or containing
3-MA (iv; 10 mM) was added for an additional 3.5 h. Control cells (Con) were incubated with 4-HNE-free HBSS for 0.5 h and allowed to recover in the medium with no extra additive (i) or 3-MA
(ii). Photomicrographs were acquired as described. (D) Cell survival was estimated as indicated above (n = 3 per group; ∗ P < 0.005 compared with other treatment groups).
removal was observed with lactacystin, although slight inhibition
was observed with PSI, suggesting that proteasome-mediated
degradation may be a minor pathway for the removal of proteins
modified by 4-HNE. This observation is consistent with previous
work showing that 4-HNE inhibits the proteasome and that
the proteasome is unable to degrade proteins heavily modified
by 4-HNE. Nevertheless, some of the proteins modified by 4HNE may be removed initially by the proteasome before it is
inhibited. Further experiments are required to delineate fully
the role of the proteasome in the removal of proteins modified
by 4-HNE and related aldehydes. In contrast, inhibition of the
lysosomal–autophagy pathway either by MG-132 or by 3-MA led
to significantly greater accumulation of protein–4-HNE adducts
(Figure 2), indicating that protein–aldehyde adducts may be
degraded as part of the autophagic response. In addition to
inhibitor data, several other lines of evidence suggest that autophagy is an important mechanism for the removal of protein–
4-HNE adducts. These include the observations that several unsaturated aldehydes (4-HNE, nonenal and acrolein) led to robust
stimulation of LC3-II formation, which is the first committed
step in autophagosome formation and autophagy (Figure 3).
Moreover, the removal of protein–4-HNE adducts was accelerated
by the autophagy stimulator rapamycin [37] and decreased by
the inhibitor of autophagy insulin [38] (Figure 4). In 4HNE-exposed cells, treatment with rapamycin also led to an
increase in LC3-II levels and insulin prevented LC3-II formation. Thus inhibition or activation of autophagy was found
to exert corresponding effects on protein–4-HNE removal and
LC3-II formation. Significantly, 4-HNE-treated cells displayed
extensive vacuole formation, double-membrane vacuoles,
multilamellar vesicles, crescent-shaped phagophores, membrane
blebs, invaginations and ruffling, features that were not observed
in untreated cells and are ultrastructural signatures of autophagy
c The Authors Journal compilation c 2008 Biochemical Society
(Figure 5). Thus, taken together, this evidence supports the
notion that degradation by autophagy is a significant fate of 4HNE-modified proteins in VSMCs. Removal of 4-HNE-modified
proteins may also be due to a general increase in protein
turnover. While this possibility could not be excluded by the
current data, basal mechanisms of proteolysis are unlikely to be
sensitive to 3-MA or insulin or associated with increased LC3-II
formation.
Mechanisms by which 4-HNE or protein–4-HNE adducts
trigger autophagy remain unclear. It has been suggested that one
mechanism by which nutrient starvation induces autophagy is
by increasing ROS (reactive oxygen species) generation [41].
In support of this idea, it was demonstrated that lipid-soluble
antioxidants such as resveratrol and vitamin E are more potent
inhibitors of autophagy than soluble ROS antioxidants such as
Tiron and NAC (N-acetylcysteine) [42]. However, neither NAC
nor Tiron was able to prevent LC3-II formation in 4-HNEtreated cells (results not shown). Hence, stimulation of LC3-II
formation by proteins modified by lipid peroxidation products
may be a downstream event in ROS-mediated autophagy. Our
data show that several products of lipid peroxidation stimulate
LC3-II formation. On a mole-per-mole basis, acrolein, the
most reactive member of the α,β-unsaturated aldehyde series,
was the most effective, followed by 4-HNE and nonenal. In
contrast, the saturated aldehyde nonanal was inactive, whereas
the phospholipid aldehyde POVPC was only marginally effective.
These observations suggest that the processes that activate the
autophagic programme may be dependent on Michael additiontype reactions and that strongly electron-deficient aldehydes
that modify and cross-link proteins are more likely to trigger
autophagy. Alternatively, products of lipid peroxidation could
directly trigger autophagic signalling. However, in our studies,
we found little or no free 4-HNE (or its metabolites DHN or
Unsaturated aldehydes activate autophagy
4-HNA) in cells after HNE exposure (Supplementary Figure 2).
The glutathione conjugates of 4-HNE were found to persist and
could trigger LC3-II formation; however, the concentration or the
activity of 4-HNE, acrolein or nonenal conjugates is unlikely to
be different enough to cause different levels of LC3-II formation.
Finally, 3-MA prevented LC3-II formation after HNE exposure
(see Supplementary Figure 3 at http://www.BiochemJ.org/bj/410/
bj4100525add.htm), indicating that significant accumulation of
protein adducts was required to trigger autophagy. Hence,
it appears that electrophile-modified or cross-linked products
rather than oxidative damage caused by cytosolic ROS promote
autophagy. Our observations are consistent with the idea that
protein cross-linking (rather than simple protein modification or
free aldehydes) may be one signal common to the activation of
autophagy by different products of lipid peroxidation. Nevertheless, further experiments are required to determine whether
the accumulation of aldehyde-modified proteins also triggers
chaperone-mediated autophagy or whether processes related to
macrophagy of entire organelles are activated specifically.
Autophagy is a carefully orchestrated pathway for bulk
degradation of protein aggregates or damaged organelles [27].
During starvation, autophagy is employed to recycle proteins
to generate energy and cell constituents. Such a response may
also be an attempt to establish homoeostasis after potentially
lethal cytotoxic insults. In addition, autophagy may be a cellular
defence mechanism to remove damaged organelles or to protect
against the toxic effects of protein aggregates. That autophagy
is a pro-survival mechanism is consistent with several studies
suggesting a protective effect of autophagy during atherosclerosis,
myocardial ischaemia and diabetes mellitus [27,43]. Furthermore,
loss of autophagy has been shown to lead to the accumulation
of ubiquitin-positive inclusions in the nervous system, which
are pathological indicators of neurodegenerative disease [44].
Interestingly, age-related increases in protein carbonyls in rats
correlate with the age-related decline in lysosomal proteolysis
[45], indicating that the process of autophagy may be a primary
route for the removal of protein carbonyls that accumulate in
aged tissues. The results of the present study are consistent with
the idea that autophagy is an attempt to restore homoeostasis
and to support survival. Our data show that inhibition of the
autophagic pathway induces cell death in 4-HNE-treated cells
at concentrations of 4-HNE that were by themselves not lethal
(Figure 6). These observations suggest that 4-HNE cytotoxicity
can be attributed in part to protein modifications.
Modification of proteins by 4-HNE may be harmful not only
because it disrupts the function of the protein but also because
it leads to the accumulation of inactive or cross-linked proteins,
which must be removed to prevent further toxicity. Hence, the
removal of protein or organelle detritus to prevent further spread
of injury may be a protective function of autophagy. Yet it appears
that autophagic signalling may have additional survival benefits.
In our experiments, inhibition of the proteasome by PSI did
not induce cell death, even though it prevented the removal of
4-HNE-modified proteins, whereas inhibition of the lysosome–
autophagy pathway by 3-MA or MG-132 precipitated cell death.
While this may be due to the variable efficacy of the inhibitors
used to prevent proteolysis, our data show that only those compounds that could inhibit autophagy proved lethal in cells
exposed to 4-HNE. In addition to preventing the removal of
damaged proteins, inhibition of autophagy may be attenuating
the stimulation of additional pathways required for cell survival.
What these pathways may be remains unclear, but possibilities
include the modulation of downstream effectors of the PI3K
(phosphoinositide 3-kinase)–PKB (protein kinase B; also called
Akt) pathway and the dissociation of Beclin-1 from Bcl-2 [46].
533
Additionally, the accumulation of modified proteins could activate
the UPR (unfolded protein response) and trigger cell death
pathways. In this regard, it has been recently demonstrated that ER
stress and UPR are linked to autophagy [47,48]; hence, the UPR
may be triggered in an attempt to inhibit protein synthesis and to
activate adaptive stress signalling to remove modified proteins by
autophagy.
Stimulation of autophagic responses may be a significant component of the stress response in VSMCs and may be a particularly
important determinant of intimal proliferation or the stabilization of atherosclerotic plaques. The formation of myelin figures
and severe vacuolization typical of autophagy have been detected
by electron microscopy in smooth-muscle cells in the fibrous
cap of atherosclerotic plaques [49]. Autophagy has also been
shown to be triggered by oxidized LDL (low-density lipoprotein)
[50]: 7-ketocholesterol, an oxysterol found in oxidized LDL,
promotes protein–4-HNE modification, vacuolization, protein
ubiquitination and LC3 conversion in human VSMCs [51],
indicating that the local environment surrounding smooth-muscle
cells in atherosclerotic plaques could promote the intracellular
generation of protein–aldehyde adducts that may be cleared by
autophagy.
In summary, the results of the present study provide evidence
that electrophilic products generated by the oxidation of lipids
activate autophagic responses in VSMCs and that proteins
modified by these aldehydes are removed by autophagy. The
removal of protein adducts by autophagy appears to be a
protective mechanism, because inhibition of autophagy triggered
cell death. On the basis of these observations, we speculate that
autophagic responses stimulated by lipid peroxidation products
may be a common feature of several conditions where such
adducts accumulate, e.g. aging, diabetes and several etiologically
unrelated cardiovascular and neurological diseases.
We acknowledge Daniel Riggs for technical assistance with HPLC, and Cathie Caple for
assistance with EM. This work was supported in part by the NIH (National Institutes of
Health) grants HL55477, HL59378, HL65618, ES11594 and ES11860, Philip Morris USA
and an American Heart Association predoctoral fellowship to B. G. H.
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