Autophagy dysfunction and regulatory cystatin C in
macrophage death of atherosclerosis
Wei Li
a,
*,Nargis Sultana b ,Nabeel Siraj b,c ,Liam J Ward a,b ,Monika Pawlik d ,Efrat Levy e ,
Stefan Jovinge f ,Eva Bengtsson f ,Xi-Ming Yuan b
a
Division of Obstetrics and Gynaecology,Department of Clinical and Experimental Medicine,Faculty of Health Sciences,
Link €o
ping University,Link €o ping,Sweden b
Occupational and Environmental Medicine Center,Heart and Medicine Center,
County Council of €Osterg €o
tland,Link €o ping,Sweden c
Department of internal medicine,University of Alberta Edmonton,Alberta,Canada d
Nathan S.Kline Institute for Psychiatric Rearch,Orangeburg,NY,USA
e
Nathan S.Kline Institute for Psychiatric Rearch,Departments of Psychiatry and Biochemistry and Molecular Pharmacology,
New York University Langone School of Medicine,New York,NY,USA
f
Department of Clinical Sciences,Sk ane University Hospital,Lund University,Lund,Sweden
Received:December 13,2015;Accepted:February 29,2016
Abstract
Autophagy dysfunction in mou atherosclerosis models has been associated with incread lipid accumulation,apoptosis and inflammation.
Expression of cystatin C (CysC)is decread in human atheroma,and CysC deficiency enhances atherosclerosis in mice.Here,we first investi-gated the association of autophagy and CysC expression levels with atheroma plaque verity in human atherosclerotic lesions.We found that autophagy proteins Atg5and LC3b in advanced human carotid atherosclerotic lesions are decread,while markers of dysfunctional autophagy p62/SQSTM1and ubiquitin are incread together with elevated levels of lipid accumulation and apoptosis.The expressions of LC3b and Atg5were positively associated with CysC expression.Second,we investigated whether CysC expression is involved in autophagy in atherosclerotic apoE-deficient mice,demonstrating that CysC deficiency (CysC À/À)in the mice results in reduction of Atg5and LC3b levels and induction of apoptosis.Third,macrophages isolated from CysC À/Àmice displayed incread levels of p62/SQSTM1and higher nsitivity to 7-oxysterol-mediated lysosomal membrane destabilization and apoptosis.Finally,CysC treatment minimized oxysterol-mediated cellular lipid accumulation.We conclude that autophagy dysfunction is a characteristic of advanced human atherosclerotic lesions and is associated with reduced levels of CysC.The deficiency of CysC caus autophagy dysfunctio
n and apoptosis in macrophages and apoE-deficient mice.The results indicate that CysC plays an important regulatory role in combating cell death via the autophagic pathway in atherosclerosis.
Keywords:autophagy cystatin C macrophage cell death lysosomal membrane permeabilization
Introduction
Accumulation of oxidized lipids and apoptotic cells in atherosclerotic lesions contributes to plaque rupture and clinical complications,including myocardial infarction and stroke [1].In atherosclerotic dis-ea,lipids are accumulated and oxidized in the intima of the vesl wall.Oxidized lipids are taken up by macrophages that become foam cells.Oxidized lipids in lysosomes of macrophages not only cau expansion of lysosomal compartments [2]but also increa lysoso-mal pH [3]and alter lysosomal functions and enzyme activity [4–7].All the result in further accumulation of toxic materials within ather-oma lesion including oxidized lipids and un-degradable material,fol-lowed by cellular stress and macrophage cell death [8].Conquently,macrophage apoptosis results in intimal necrosis and promotes destabilization of advanced lesions.In this process,the accumulation of oxidized lipids may also lead to induction of autophagy [9–12].
Autophagy is a lysosomal degradation pathway for cytoplasmic material and is activated under stres
s conditions [8].Autophagy can be a cell survival mechanism by generating free amino acids and fatty
*Correspondence to:Wei LI,M.D.,Ph.D.E-mail:wei.li@liu.
ª2016The Authors.
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This is an open access article under the terms of the Creative Commons Attribution Licen,which permits u,
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doi:10.1111/jcmm.12859
J.Cell.Mol.Med.Vol 20,No 9,2016pp.1664-1672
acids to maintain cellular function.However,it can also promote cell death through excessive lf-dig
estion and degradation of esntial cellular constituents[13].In the autophagy process,a pathway of apoptotic cell death has been described,which involves lysosomal disruption with relea of proteolytic enzymes into the cytosol[14]. Cystatin C(CysC)is an important endogenous inhibitor of cathepsins B,H,K,L and S.Recent work suggests that CysC may play a protec-tive role in neurodegenerative dias and cerebral vasospasm by inducing autophagy[15].Increasing endogenous and exogenous CysC decread brain ischaemia in rats via activating autophagy path-ways[16,17].Expression of CysC is decread in human atheroma [18],and CysC deficiency enhances atherosclerosis[19].At prent, the role of CysC and its relation to autophagy and apoptotic cell death in atherosclerosis has not been investigated.
In this study,wefirst examined the expression of the autophagy markers LC3b,Atg5,p62/SQSTM1and ubiquitin in human carotid plaques and their relation to CysC expression,lipid accumulation and atheroma plaque verity.Second,we investigated the role of CysC in autophagy and apoptosis in ApoE-deficient mice and in macrophages isolated from CysCÀ/Àor WT mice.Third,we studied effect of exoge-nous CysC on lipid accumulation in7-oxysterol-treated macrophages. Materials and methods
Cell culture
Human monocytic THP-1cells were maintained in RPMI-1640culture medium with10%foetal bovine rum in a humidified atmosphere(5% CO2)at37°C and subcultivated twice a week.In some experiments, THP-1cells were differentiated into macrophages after incubation with phorbol myristate acetate(300nM)for24hrs.After being washed with culture medium,the cells were further cultured for24hrs under stan-dard culture conditions before experiment initiation.
For experiments,the above cells were treated with a mixture of7b-hydroxycholesterol(7b OH)and7keto-cholesterol(7keto;Sigma-Aldrich, St.Louis,MO,USA)at a ratio of1:1.8(2mix)for12,24or48hrs(28l M) as described previously[20].Cells treated with cholesterol or ethanol were ud as controls.In some experiments,cells were expod to recombinant CysC(Sigma-Aldrich)at1–2l g/ml together with2mix for24hrs.After treatment,cells were collected and analyd as described below. Peritoneal macrophages from CysC knockout or wild-type mice
Homozygous CysC-deficient(CysCÀ/À)mice described by Huh et al.
[21]were kindly provided by Dr.Anders Grubb,Lund University Hospi-tal,Lund,Sweden[19].All animal procedures were performed following the National Institutes of Health guidelines with approval from the Insti-tutional Animal Care and U Committee at the Nathan S.Kline Institute for Psychiatric Rearch.All efforts have been made to minimize animal suffering and the numbers of mice ud.
Resident peritoneal cells were prepared from7-to9-week-old CysCÀ/Àand wild-type mice(CysC+/+)of both xes according to the basic protocol as described by Zhang et al.[22].Briefly,the mice were killed by cervical dislocation exposing them to a minimum of stress. Under sterile conditions,their peritoneal cavities werefilled with cold Dulbecco’s without magnesium and calcium(Gibco Life Technologies, New York City,NY,USA)and thefluid reaspirated with a20G1.5″nee-dle.The peritoneal exudate cells were centrifuged for10min.at 4009g,4°C,and the pellet was resuspended in complete DMEM F12. Lysosomal membrane integrity
The integrity of the lysosomal membranes was assd by the acridine orange(AO)relocation test as established previously[23].In brief,cells werefirst stained with AO and then underwent different treatments for different times,and then analyd by confocal microscopy.Cells with incread cytosolic/nuclear AO greenfluorescence were identified as cells with lysosomal membrane permeabilization(LMP). Intracellular lipid
Cellular lipid levels were assayed by Oil red O(Sigma-Aldrich)staining of cultured cells or ctions from human carotid plaques.Briefly,cells or cryoctions werefixed with2%formalin,stained for6min.with 0.15%Oil red O in76%methanol and0.2M NaOH,washed with water, counterstained with haematoxylin and visualized by light microscopy. Positive areas of oil Red O were analyd by Adob
e Photoshop(v5.5) (Adobe Systems Inc.,San Jo,California,USA).
Human carotid atheroma
The Link€o ping Carotid Study is a prospective clinical-pathology study in which atherosclerotic carotid arteries were collected from patients who underwent carotid endarterectomy at Link€o ping University Hospital.The ethics committee of Link€o ping University Hospital has approved this study as described previously[24],and informed connt was obtained from all participants.
Atherosclerotic carotid samples obtained from15patients(average-age of72)were included in this study.Ten patients with cerebrovascu-lar symptoms were classified as symptomatic,andfive patients without cerebrovascular symptoms were defined as asymptomatic.Several stroke risk factors were recorded,including hypertension(defined by hypertension history and diastolic blood pressure≥110mmHg, n=10),smoking(smoking>5years,n=4)and diabetes mellitus(dia-betes medication,n=3).Carotid artery samples were collected imme-diately after endarterectomy.All operations were performed with minimal manipulation of the specimen without opening of the arterial lumen.The samples were divided into two parts:one part was directly frozen atÀ70°C and another part prepared for histopathology analysis after decalcification.Three tofive transver gments were
taken from each specimen andfixed in4%(w/v)formaldehyde and embedded in paraffin.The gments were ctioned with an interval of2l m.
ApoEÀ/ÀCysC+/+mice and apoEÀ/ÀCysCÀ/Àmice
Cryoctions from the aortic sinus of atherosclerotic apoE-deficient and CysC-competent(apoEÀ/ÀCysC+/+)mice or apoE-and CysC-deficient
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(apoEÀ/ÀCysCÀ/À)mice[19],which were fed a high fat diet,21%cocoa
fat,0.15%cholesterol without sodium cholate(AnalyCen Nordic,if公式
Link€o ping,Sweden)for25weeks,were ud for immunohistochemistry or TUNEL technique.The hearts and proximal aortas from the mice
werefixed in Histochoice embedded in Tissue-Tek(Sakura,Torrance,
CA,USA)and stained as described below.All animal experiments were
approved by the local animal care ethical committee.
Apoptosis
In human carotid plaques or lesions from apoEÀ/ÀCysC+/+and apoEÀ/ÀCysCÀ/Àmice,apoptotic cells were assayed by the TUNEL technique using TUNEL in situ cell death detection kit(Roche Molecular Biochemi-cal,Mannheim,Germany).Cryoctions werefixed in2%PFA and per-meabilized with0.1%Triton x-100in0.1%sodium citrate.The slides were incubated with TUNEL reaction mixture for60min.at37°C in the dark followed by incubation with converter peroxida(POD)/hor rad-ish peroxida(HRP)or with covert alkaline phosphata(AP)for 30min.at37°C.Immunoreaction was visualized by3,30-diaminobenzi-dine(DAB)for POD system or by fast red for AP system and counter-stained with haematoxylin.Endogenous peroxida was blocked with 3%H2O2in methanol and unspecific background was blocked with3% bovine rum albumin and20%normal bovine rum in0.1M Tris-HCl (pH7.5).Control ctions without TUNEL reaction mixture were run for each protocol,resulting in consistently negative results.
无线热点
In cultured cells,apoptotic cells were detected by immunocytochem-
istry using an antibody recognizing activated caspa-3(BD PharMin-
gen,Franklin Lakes,NJ,USA).
Immunohistochemistry
Cryoctions and formalin-fixed paraffin embedded ctions of human
羊肉萝卜
carotid plaques or cryoctions from lesions of apoEÀ/ÀCysC+/+and
apoEÀ/ÀCysCÀ/Àmice werefixed in2%PFA,permeabilized and incu-
bated with rabbit primary antibodies:anti-LC3b(4°C overnight),anti-
Atg5(2hrs at22°C),anti-CysC(4°C,overnight),anti-p62/SQSTM1
(4°C,overnight),or anti-ubiquitin(4°C,overnight).The ctions were
incubated with AP-conjugated goat anti-rabbit condary antibody at
22°C for1hr and the immunoreaction was visualized by fast red.Other
ctions were incubated with HRP-conjugated goat anti-rabbit
condary antibody at22°C for1hr,and the immunoreaction was visu-
alized by DAB.Control ctions without primary antibodies or with
non-immune IgG were run for each protocol,resulting in consistently
negative results.The slides were counterstained with haematoxylin and
适合小孩讲的简短故事visualized by light microscopy.
Image analysis and classification of the plaques
All histological ctions were examined under a light microscope,and the images were digitalized to a Macintosh computer with Image Grab-ber program(Toronto,ON,Canada).The microscope was t on the same parameters ud to scan all samples.The plaques were classified into early and advanced plaques.Early lesions have an intact plaque with afibrous cap>100l m and have no lipid po
ol formation often with intact internal elastic membrane.Advanced lesions have intact or ruptured plaques with lipid pool formation,infiltration of leucocytes and afibrous cap<100l m.
The randomly digitalized images were analyd with Adobe Photo-shop(v5.5)as described previously[24].Positively stained areas for each ction were prented as immunopositive areas(%),which were calculated as the average immunostained area per pixel value divided by the total pixel value.
Statistics
姊妹饭For statistical analysis,one-way ANOVA followed by a post hoc Newman–Keuls test were ud for multiple comparisons.Correlation between CysC and LC3b or CysC and Atg5in human carotid plaques was anal-yd by the Spearman correlation test and prented as the Spearman correlation coefficient(r).Results are given as meanÆS.E.M. P<0.05was considered statistically significant.
Results
Defective autophagy,reduced CysC and lipid oxidation in human atheroma
Intracellular lipid accumulation is an important step in the progres-sion of atherosclerotic lesions.Co
mpared with early lesion of atherosclerosis lipid derived ceroid-like material due to lipid oxidation is particularly abundant in areas rich in apoptotic macrophage foam cells in advanced lesions(Fig.1,yellow auto-fluorescence,white arrow).
To examine the relation between autophagy function and CysC expression in atheroma progression,Atg5,LC3b,SQSTM1,ubiquitin and CysC were examined by immunostaining in human carotid pla-ques from15patients.Atg5expression was en in all ctions of both early and advanced lesions.In early lesions,Atg5was mainly expresd in endothelial cells and intimal areas of the vesl wall (Fig.1).In advanced lesions,the expression of Atg5was significantly reduced,and in the areas clo to the lipid rich areas,there were nearly no detectable levels of Atg5(Fig.1).As shown in Figure S1, Atg5was mainly expresd in thefibrous cap and the expression of Atg5appeared in cytoplasmic granules of macrophage foam cells (empty arrows)and in some endothelial cells(arrow with dotted line) and smooth muscle cells(arrow with solid line).
When autophagyflux is defective,p62/SQSTM1and ubiquitin accumulate in lysosomes[25].Here,we found that the levels of p62/ SQSTM1and ubiquitin were significantly lower in early stages of human atherosclerotic plaques(Fig.1).In contrast,massive and sig-nificant accumulation of p62/SQSTM1and ubiquitin was obrved in CD68-positive macrophages in advanced lesions,especially near lipid-rich
areas(Fig.1)suggesting deficiency of autophagyflux.
Expression of CysC,an inhibitor of lysosomal cathepsins,is decread in human atheroma[18].To examine the relationship between CysC and autophagy in human atherosclerotic lesions,the expression of CysC was determined in human carotid samples.In
1666ª2016The Authors.
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early lesions of atherosclerosis,CysC expression was mainly en in endothelial cells and in intimal areas (Fig.1),which were positive for the autophagy marker Atg5,to some extent for p62/SQSTM1and for ubiquitin (Fig.1).In rial ctions of advanced lesions,CysC expres-sion was remarkably diminished together with massive accumulation of p62/SQSTM1and ubiquitin (Fig.1).Moreover,the expression of CysC was positively associated with the expression of both Atg5(r =0.76,P <0.0001)and LC3b (r =0.75,P <0.0001).
CysC deficiency is correlated with reduced
autophagy and incread apoptotic cell death in atherosclerotic lesions of mice
满的反义词
Lack of CysC has been found previously to promote formation of large atheroma plaques with incread levels of lipid deposition in atherosclerotic apoE À/Àmice [19].Deficiency of CysC in apoE À/À
Fig.1Decread autophagy is associated with reduced CysC levels in progression of human atheroma.Reprentative photographs of autofluores-cence of ceroid,Atg5(brown),double immunohistochemistry of p62/SQSTM1(brown)and CD68(red),ubiquitin (brown)and CysC (brown)in situ in early lesions (left panels)and advanced human carotid lesions (middle panels);bars =50l m.No
世界杯美国te :In early lesion areas,there are very low levels of ceroid,significantly higher levels of Atg5and significantly lower levels of p62/SQSTM1in CD68-positive macrophages and low levels of ubiquitin,while advanced lesions show pronounced ceroid accumulation,low Atg5and massive accumulation of CD68-positive macrophages with significant incread levels of p62/SQSTM1and ubiquitin and lower levels of CysC.Histograph show quantification of immunopositive areas of Atg5,p62/SQSTM1,ubiquitin and CysC.*P <0.05,**P <0.01.
ApoE −/−CysC +/+ApoE −/−CysC −/−
电视背景墙怎么做ApoE −/−CysC −/−
ApoE −/−CysC −/−
ApoE −/−CysC +/+ApoE −/−CysC +/+
B
C
A
D
Fig.2CysC deficiency in apoE À/Àmice results in reduced autophagy and incread apoptotic cell death.Lesions from apoE À/ÀCysC +/+(n =6)and apoE À/ÀCysC À/Àmice (n =4)were immunostained with Atg5or LC3b .Apoptosis was determined by the TUNEL technique,and images were anal-yd as described in the Methods ction.The stained ctions were counterstained with haematoxylin.(A )Reprentative photographs of Atg5,LC3b and TUNEL (stained in red)from apoE À/ÀCysC +/+mice and apoE À/ÀCysC À/À,bars =25l m.(B –D )Quantification of Atg5,LC3b and TUNEL in the two groups of mice.*P <0.05versus apoE À/ÀCysC +/+mice.1668
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