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International Journal of Cardiology 168 (2013) 1378–1385
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International Journal of Cardiology
j ourna l homepage: www.e lsev ie r .com/ locate / i j ca rd
Regulation of autophagy and apoptosis in response to ox-LDL in vascular smoothmuscle cells, and the modulatory effects of the microRNA hsa-let-7g
Zufeng Ding, Xianwei Wang, Laura Schnackenberg, Magomed Khaidakov, Shijie Liu, Sandeep Singla,Yao Dai, Jawahar L. Mehta ⁎Central Arkansas Veterans Healthcare System and the Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA
⁎ Corresponding author at: Division of Cardiovascular Mfor Medical Sciences, Little Rock, AR 72212, USA. Tel.: +1
E-mail address: [email protected] (J.L. Mehta).
0167-5273/$ – see front matter. Published by Elsevier Ihttp://dx.doi.org/10.1016/j.ijcard.2012.12.045
a b s t r a c t
a r t i c l e i n f oArticle history:
Received 23 September 2012Received in revised form 10 November 2012Accepted 6 December 2012Available online 7 January 2013Keywords:Autophagyhsa-let-7gLOX-1ROSSmooth muscle cells
Objectives: Regulation of autophagy and apoptosis during treatment of vascular smooth muscle cells (VSMCs)with pro-atherogenic stimuli, such as oxidized low density lipoprotein (ox-LDL), remains unclear.Methods and results:We examined the expression of autophagy and apoptosis upon treatment of VSMCs withox-LDL. Exposure to ox-LDL in modest amounts (10–40 μg/ml) enhanced autophagy (expression of beclin-1,LC3-II/LC3-1 ratio and Atg5) and apoptosis (expression of caspase-3, Bax, Bcl-2 and Bcl-xL); however, expo-sure to higher concentrations (≥60 μg/ml) induced high levels of apoptosis but autophagy declined.Pretreatment of VSMCs with the miRNA hsa-let-7g inhibited autophagy, as LOX-1 expression and apoptosisdeclined. Hsa-let-7g treatment also resulted in a decrease in intracellular ROS generation. Treatment withLOX-1 antibody had similar effects as hsa-let-7g. Next, we studied autophagy and apoptosis in aortic seg-ments from wild-type and LOX-1 knockout mice fed a high cholesterol diet, and observed increasedautophagy as well as apoptosis in lipid-rich sections of aortas from wild-type mice and LOX-1 knockout
mice (vs. corresponding controls); however, both autophagy and apoptosis in lipid-rich areas in aortic sec-tions of LOX-1 knockout mice were less than in WT mice. These in vivo data are in keeping with in vitrodata showing enhanced autophagy and apoptosis of VSMCs exposed to modest amount of ox-LDL.Conclusion: This study provides first set of data on the regulation of autophagy and apoptosis inox-LDL-treated VSMCs. Our observations also suggest that hsa-let-7g acts as a critical regulator of autophagyand apoptosis by modulating LOX-1.Published by Elsevier Ireland Ltd.
1. Introduction
Atherosclerosis is a chronic inflammatory disease of the arterialwall of large and medium-sized arteries, which is characterized byformation of an atherosclerotic plaque that can partially or totally oc-clude the vascular lumen [1,2]. Oxidized-low density lipoproteins(ox-LDL) play a major role in atherogenesis [1,2]. Lectin-like oxidizedlow-density lipoprotein scavenger receptor-1 (LOX-1) is one of themajor receptors responsible for binding, internalizing and degradingox-LDL [1,2]. Activation of LOX-1 has been known to be related tomany pathophysiological events, including endothelial cells andvascular smooth muscle cell (VSMC) proliferation, alteration in cellcycle signals and apoptosis [1,2].
Autophagy is an evolutionarily conserved process involved in thedegradation of long-lived proteins and excess or dysfunctional organ-elles, which becomes manifest during tissue remodeling and starvationwhen the cell needs amino acids and fatty acids from catabolism ofproteins and lipids [3]. Under normal conditions in most vascular
edicine, University of Arkansas501 296 1426.
reland Ltd.
cells, autophagy is an important house-keeping process, and may beconsidered a cell survival program [4]. Although excessive autophagicactivity leads to total collapse of all cellular functions and inductionof autophagic death, moderately enhanced autophagy promotes cellsurvival [5]. There are no data in the literature on the effects ofox-LDL on autophagic response of VSMCs.
Apoptosis, on the other hand, represents programmed cell deathmeant to remove cells exposed to noxious stimuli, such as ox-LDL[1,2]. The number of apoptotic cells in atherosclerotic regions is in-creased [6]. The regulation of autophagy and apoptosis in response toox-LDL might be of interest in understanding VSMC biology in athero-sclerotic regions where the concentrations of ox-LDL are high [6–9].
MiRNAs are non-coding, single stranded molecules consistingof ≈22 nucleotides that regulate the expression of genes at post-transcriptional level [7]. MiRNAs have been associated with inflamma-tion, oxidative stress and angiogenesis [8,9], and have been shown tobe critical modulators for cellular function [7]. MiRNAs let-7 familymembers were firstly observed in Canenorhabditis elegan, and ninemembers of the let-7 family have been found in humans [10]. Let-7 fam-ily expression is usually noted in tissues during embryonic stages andsignificantly increases towards maturity, which suggests that it plays apivotal role in developmental processes [11]. Let-7 family has been
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shown to inhibit cancer cell proliferation through repression of onco-genes, including RAS and HMGA2, via binding to the 3′-UTR of theirmRNAs, thus it has also been reported as a tumor suppressor family[12]. Let-7 family also plays a key role in cell proliferation and migration[14], and inflammation, steps that are critical in the evolution of athero-sclerosis [13–15]. Let-7f can modulate angiogenesis, a key regulator ofatherosclerosis and cancers, by targeting the angiogenesis inhibitorthrombospondin-1 [13]. Qin et al. [15] have suggested that let-7c con-tributes to endothelial cell apoptosis through suppression of Bcl-xl. Re-cently, Chen et al. [16]. described a negative feedback regulationbetween let-7g and LOX-1, and identified a let-7g binding site on the3′-untranslated region of LOX-1 mRNA.
In the present study we show, for the first time, the regulatoryeffects of ox-LDL on autophagy and apoptosis responses in VSMCs. Fur-ther, we show that hsa-let-7g modulates both autophagy and apoptosisby inhibiting LOX-1 expression.
2. Methods
2.1. Cell culture and miRNAs transfection
Human primary aortic VSMCs were obtained from ATCC (Manassas, VA), andmaintained in vascular cell basal medium supplemented with VSMC Growth Kit(ATCC). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. Allexperiments were conducted using VSMCs between passages 2 and 5. Cells were seededinto 6-well plates and grown to semi-confluent density (≈90%) before treatment.
Human hsa-let-7g mimic and inhibitor were synthesized by Applied Biosystems(Carlsbad, CA). VSMCs were seeded onto a 6-well plate at a density of 2×105 cells/well
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Fig. 1. A. Ox-LDL increases LOX-1 protein expression in a dose-dependent manner, while hsaexpression. Bar graphs represent data in mean±SD based on 3 experiments. B. Effect of ox-assay. Ox-LDL induces expression of p.p70S6K and p.mTOR; hsa-let-7g and LOX-1 Ab inhibC. Ox-LDL induces a dose-dependent decrease on let-7g mRNAs levels, while LOX-1 Ab blWestern blot and qPCR, respectively. Bar graphs represent data in mean±SD based on 5 ex
the day before transfection. Cells were transfected with hsa-let-7g mimic, inhibitorusing Lipofectamine 2000 (Invitrogen, Grand Island, NY). The medium was replaced6 h after transfection.
High TBAR ox-LDL (90 nmol MDA/mg protein) was purchased from BiomedicalTechnologies Inc. (Stoughton, MA). LOX-1 antibody (LOX-1 Ab) TS92 was a gift of Dr.T. Sawamura (Osaka, Japan).
2.2. Western blot
Primary and secondary antibodies were purchased from Abcam (San Francisco,CA), Santa Cruz Biotechnology (Santa Cruz, CA) and Novus Biologicals (Littleton, CO).Rabbit polyclonal antibodies against phospho-mTOR (Ser2448) and phospho-p70S6K(Thr389) were obtained from Cell Signaling (Beverley, MA, USA). Details of Westernblotting have been published elsewhere [6].
2.3. Quantitative real-time PCR
Total RNA was extracted using TRIZOL reagent (Invitrogen). Quantitative real-timePCR was carried out using Applied Biosystems 7500 Real-Time PCR System (AppliedBiosystems, USA). For hsa-let-7g (mature sequence: CUGUACAGGCCACUGCCUUGC)detection, the PCR master mix of TaqMan 2× Universal PCR Master Mix (No AmpEraseUNG), hsa-let-7g 10× TaqMan Assay, nuclease-free water and 1.33 μl of RT productswas prepared. PCR conditions were 95 °C for 10 min, followed by 40 cycles of 95 °Cfor 15 s and 60 °C for 60 s. To determine hsa-let-7g expression levels, U6 was usedas internal control.
2.4. Dil-ox-LDL uptake
Cultures of VSMCs in triplicate were incubated with Dil-ox-LDL 5 μg/ml for 2 h at37 °C. After incubation, cells were gently washed with PBS three times and digestedwith Trypsin-EDTA. Dil-ox-LDL uptake was measured using flow cytometry.
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-let-7g and LOX-1 antibody attenuate it. Note that n-LDL has almost no effect on LOX-1LDL, let-7g and LOX-1 Ab on cell viability. The viability of VSMCs was measured by MTTit their expression. Bar graphs represent data in mean±SD based on 5 experiments.ocks this decrease. The expression levels of LOX-1 and hsa-let-7g were measured byperiments, #Pb0.05 vs. Control, *Pb0.05 vs. ox-LDL.
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Fig. 2. Ox-LDL increases ROS generation, whereas n-LDL has no effect. Hsa-let-7g inhib-itor enhances ox-LDL-mediated ROS generation while hsa-let-7g decreases it.Pretreatment of cells with LOX-1 Ab before ox-LDL treatment also prevents ROS gener-ation. Bar graphs represent data in mean±SD based on 5 experiments, #Pb0.05 vs.Control, *Pb0.05 vs. ox-LDL.
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2.5. MTT analysis
VSMCs viability was performed byMTT Cell Viability Assay (ATCC) according to sup-plied protocols. Briefly, 10 μL MTT Reagent was added to VSMC cultures followed by in-cubation for 2 to 4 h until purple precipitate was visible, then 100 μL Detergent Reagentwas added and cultures were left at room temperature in the dark for 2 h. Absorbancewas measured at 570 nm in a microplate reader.
2.6. Measurement of intracellular reactive oxygen species
Intracellular ROS were measured with the use of the fluorescent dihydroethidium(DHE), a cell-permeable indicator for ROS generation. VSMCs were cultured in 6-wellplate, then incubated with 10 μmol/L DHE in PBS for 30 min. The ROS-mediated fluo-rescence was measured by flow cytometry (Becton Dickinson, Franklin Lakes, NJ),and the results were analyzed with the software WinMDI29.
2.7. Analysis of apoptosis in VSMCs
Apoptosis was analyzed by Western blot and Polycaspase FLICA apoptosis detec-tion kit (ImmunoChemistry Technologies, Bloomington, MN) according to suppliedprotocols. The caspase expression was assessed by flow cytometry (Becton Dickinson,Franklin Lakes, NJ), and the results were analyzed with the software WinMDI29.
2.8. Autophagy detection in VSMCs
Autophagy in VSMCs was detected by Western blot and Premo™ Autophagy sensorLC3-GFP (Invitrogen, Grand Island, NY) according to supplied protocols. Fluorescencemicroscopy and flow cytometry were used to assess autophagy activities.
2.9. Animal protocol
The generation of LOX-1 knockout (KO) mice on C57BL/6 background has been de-scribed recently [17]. The mice were housed in the breeding colony at University of Ar-kansas for Medical Sciences, Little Rock, AR, USA. Male animals were given ahigh-cholesterol diet (4% cholesterol/10% cocoa butter) for 18 weeks from the age of6 weeks. All experimental procedures were performed in accordance with protocolsapproved by the Institutional Animal Care and Usage Committee, and conformed tothe Guidelines for the Care and Use of Laboratory Animals published by the US NationalInstitutes of Health.
2.10. Measurement of plasma lipids atherosclerosis
Plasma lipids fromwild-type (WT) and LOX-1 KOmice was analyzed using nuclearmagnetic resonance (NMR) spectroscopy as described previously [17]. In brief, pro-teins were precipitated by addition of 200 μL acetonitrile. Samples were centrifugedat 13,000 rpm and 4 °C for 15 min and the supernatant collected and stored. Theremaining pellets were extracted with 800 μL 2:1 v/v CHCl3:MeOH and centrifuged.Following centrifugation, the supernatant was removed and the solvent evaporatedvia high speed vacuum. The samples were reconstituted in 200 μL of a 2:1 v/v mixtureof CHCl3-d and MeOH-d4. Samples were analyzed on a Bruker Avance spectrometerequipped with a triple resonance cryoprobe and operating at 600.133 MHz for proton.Lipophilic extract spectra were acquired using the standard Bruker proton pulse se-quence. All 1D NMR spectra were processed using ACD/Labs 1D NMR Manager (ACD/Labs, Toronto, Canada).
2.11. Analysis of atherogenesis
Extent of fatty deposits (index of atherosclerotic lesion formation) was quantified bytwo methods: first, as percent of aorta (en face staining of entire aorta), and second, byintimal thickness. Briefly, 7 mice from each group were euthanized and the aortas sepa-rated from surrounding tissues. After removal of the adventitial fat, aortas were openedlongitudinally from the aorta arch to the iliac bifurcation, and fixed in 10% formalin for24 h. The aortas were then rinsed in 70% alcohol and stained with Sudan IV for 15 min.Aortas were then mounted and photographed with a camera connected to a dissectionmicroscope. The images were analyzed by Image-Pro Plus (Media Cybernetics). 5 μmcross-sections were made at 5 predefined points (proximal ascending aorta, aortic arch,descending aorta, mid thoracic aorta, and abdominal aorta above the renal arteries).The sections were stained with H&E. In each case, the average value in each animalwas used for measurement of intima thickness.
2.12. Autophagy and apoptosis in mice aortas
Seven sections of aortic arch from 7 animals in each group were used to assessautophagy and apoptosis in normal areas as well as those with fatty deposits. Forautophagy, sections of aortic arch were incubated with primary antibody to beclin-1(Novus Biologicals, CO) for 2 h at room temperature, rinsed with PBS, and rabbit spe-cific HRP/DAB detection IHC kit (Abcam, San Francisco, CA) was applied. For apoptosisdetection, DeadEnd™ Fluorometric TUNEL System (Promega Corporation, Madison,WI) was used.
2.13. Statistical analysis
Statistical analysis was performed with SPSS 11.5 software. Data are presented asmeans and standard deviation (SD) from 3 to 7 independent experiments. Univariatecomparisons of means were evaluated using Student t-tests and/or one-way ANOVAwith Tukey's post-hoc adjustment for multiple comparisons when appropriate. Pb0.05was considered statistically significant.
3. Results
3.1. Ox-LDL, LOX-1, let-7g expression and VSMC viability
As observed previously in endothelial cells [6], LOX-1 expressionin VSMCs increased in response to increasing ox-LDL concentration(10–60 μg/ml, incubation time 24 h), whereas n-LDL had no effect(Fig. 1A). LOX-1 expression in response to ox-LDL was inhibited bytreatment of cells with LOX-1 Ab.
Treatment with ox-LDL (10 to 40 μg/ml) resulted in enhanced cellviability, and reached its highest value at 40 μg/ml. However, concen-tration beyond 60 μg/ml decreased cell viability (Fig. 1B). Pretreatmentwith LOX-1 Ab blunted the decrease in viability in response to highconcentrations of ox-LDL. It has been suggested thatmTOR/p70S6K sig-naling pathway plays a key role in proliferation of SMCs, and this path-way can be activated by ox-LDL [18]. We examined and found thathsa-let-7g inhibited ox-LDL-induced expression of p-p70S6K andp-mTOR, which had an effect similar to that of LOX-1 Ab.
Over-expression of hsa-let-7g into VSMCs inhibited ox-LDL-mediatedLOX-1 expression; this effect was similar to that of pretreatment of cells
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with LOX-1 Ab. Further, ox-LDL-mediated decrease in cell viability athigher concentration was also inhibited by over-expression of hsa-let-7g similar to the observed effect of pretreatment of cells with LOX-1Ab (Fig. 1A and B).
3.2. Ox-LDL and let-7g expression
Using quantitative PCR, we found that ox-LDL reduced the expres-sion of hsa-let-7g in a dose-dependent manner, and LOX-1 Ab reversedthe effect of ox-LDL on let-7g levels (Fig. 1C). Since 20 μg/ml concentra-tion of ox-LDL (incubation time 24 h) gave the most consistent resultson LOX-1 expression and expression of autophagy and apoptosis, weused this concentration in all subsequent experiments.
3.3. Ox-LDL, hsa-let-7g and reactive oxygen species generation
Ox-LDL is a potent inducer of ROS; this was confirmed in the presentstudy (Fig. 2). To investigate whether hsa-let-7g can regulate ROS gen-eration, VSMCs were transfected with hsa-let-7g mimic or its inhibitor.Hsa-let-7g inhibitor over-expression enhanced ROS generation beyondthat caused by ox-LDL alone. On the other hand, pretreatment ofVSMCs with LOX-1 Ab significantly inhibited ROS generation inresponse to ox-LDL (Fig. 2).
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Fig. 3. A, B and C. Ox-LDL enhances autophagy (measured by beclin-1, LC3 and Atg5 expressmicrographs and flow cytometry analysis of autophagy; the results are similar to those omean±SD based on 5 experiments, # Pb0.05 vs. control, * Pb0.05 vs. ox-LDL.
3.4. Ox-LDL, hsa-let-7g and autophagy
We assessed autophagy in response to ox-LDL by measuringbeclin-1, LC3-II/LC3-1 ratio, and Atg5. The conversion of LC3-I intoLC3-II is an essential step in autophagosome formation, and the abun-dance of LC3-II correlates with the number of autophagosomes [19].
As shown in Fig. 3A and C, ox-LDL treatment increased beclin-1and Atg5, and hsa-let-7g inhibited ox-LDL-induced beclin-1 andAtg5. LOX-1 Ab had an effect similar to that of hsa-let-7g.
It is of note that VSMCs maintained in culture showed barely de-tectable levels of LC3-II, indicating minimal baseline autophagy(Fig. 3B). As with measurement of beclin-1, the LC3-II:LC3-I ratio in-creased in response to ox-LDL; hsa-let-7g over-expression inhibitedthis effect. Notably LOX-1 Ab markedly inhibited, but did not elimi-nate, the effect of ox-LDL.
As noted previously [20], some of the LC3 dissociates from themembrane after fusion with the lysosome during development ofautophagy. LC3-GFP can serve as an autolysosome and as a specificmarker for autophagy. As shown in Fig. 3D, LC3B-GFP was distributeddiffusely in the cell with almost no accumulation in the lysosomes inuntreated VSMCs. After incubating VSMCs with ox-LDL for 24 h, alarge number of cells showed LC3B-GFP accumulation, suggesting ac-tivation of autophagic response. Hsa-let-7g markedly decreased LC3Baccumulation induced by ox-LDL. LOX-Ab treatment also reducedLC3B accumulation.
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ion). Hsa-let-7g and LOX-1 Ab inhibit autophagy in response to ox-LDL. D. Fluorescencebtained by Western blotting for beclin-1, LC3 and Atg5. Bar graphs represent data in
1382 Z. Ding et al. / International Journal of Cardiology 168 (2013) 1378–1385
3.5. Ox-LDL uptake, apoptosis and hsa-let-7g
Previous studies have shown that ox-LDL induces apoptosis inVSMCs via LOX-1 activation [1,2]. Further, apoptosis is regulated byvarious pro-apoptosis-related proteins, such as caspase-3 and Bax,and anti-apoptosis proteins, such as Bcl-2 and Bcl-xL [21,22]. In keep-ing with these studies, ox-LDL treatment increased caspase-3 and Baxexpression and reduced Bcl-2 and Bcl-xL expression (Fig. 4). Over-expression of hsa-let-7g in the VSMCs markedly blunted the effectof ox-LDL. We confirmed these data with flow cytometry usingPolycaspase FLICA apoptosis detection kit. As shown in Fig. 4E, apo-ptosis rate of VSMCs increased in cells treated with ox-LDL, and theover-expression of hsa-let-7g mimic or treatment of cells withLOX-1 Ab significantly attenuated ox-LDL-induced apoptosis.
Next, we examined ox-LDL uptake by VSMCs. As shown in Fig. 4F,ox-LDL enhanced Dil-ox-LDL uptake, and hsa-let-7g mimic as well asLOX-1 Ab blocked it. These changes paralleled the changes in LOX-1expression (Fig. 1A).
3.6. Ox-LDL concentration, apoptosis and autophagy
Next, we studied the development of autophagy and apoptosis inrelation to the concentration of ox-LDL in parallel experiments. We
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Fig. 4. Ox-LDL increases pro-apoptotic proteins caspase 3 (A) and Bax (B) both hsa-let-7g anBcl-2 (C) and Bcl-Xl (D) are congruent with those shown in panels A and B. Flow cytometFlow cytometry shows that ox-LDL enhances Dil-ox-LDL uptake by SMCs and hsa-let-7experiments, # Pb0.05 vs. control, * Pb0.05 vs. ox-LDL.
observed that the number of cells showing apoptosis increased sharp-ly in response to treatment with 10 to 60 μg/ml of ox-LDL, reaching aplateau upon exposure to >60 μg/ml concentration of ox-LDL (Fig. 5).On the other hand, the number of VSMCs showing autophagy in-creased rapidly in response to 20 to 40 μg/ml concentration, decliningrapidly as the concentration of ox-LDL was increased.
3.7. Plasma lipids and extent of atherosclerosis
NMR proton spectral analysis (Fig. 6A) showed that the averageLDL-cholesterol concentration was lower in LOX-1 KOmice comparedwith WT mice, while HDL and VLDL concentrations were similar inthe two groups. En face staining of aortas from WT mice revealedareas of sudanophilia, which were much fewer in the LOX-1 KOmice (Pb0.05, Fig. 6B). The intimal thickening was also less in theLOX-1 KO mice compared with that in the WT mice (Fig. 6B).
3.8. Autophagy and apoptosis in vivo
We analyzed autophagy and apoptosis in multiple sections ofaortas of WT and LOX-1 KO mice when both groups were fed highcholesterol diet (Fig. 6C and D). Immunohistochemistry showedthat beclin-1 expression was markedly increased in lipid-rich areas
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d LOX-1 Ab block the effect of ox-LDL. Data on the expression of anti-apoptotic proteinsry results also confirmed that hsa-let-7g and LOX-1 Ab inhibit caspase expression (E).g and LOX-1 Ab block it (F). Bar graphs represent data in mean±SD based on 5
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Fig. 5. The crosstalk between ox-LDL-mediated apoptosis and autophagy in VSMCs. A. Representative flow cytometry data on LC3B and caspase expression. B. Ox-LDL increases ap-optosis rate in a dose-dependent manner up to 60 μg/ml, and then the apoptosis rate stabilizes. Ox-LDL (10–40 μg/ml) increases autophagy, but at higher concentrations autophagysharply falls. Data in mean±SD based on 5 experiments.
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of aortas from WT mice compared with normal regions of the samemice or other WT mice not given high cholesterol diet (and henceno areas of lipid deposit). Though beclin-1 expression was increasedin the lipid-rich areas of aortas from LOX-1 KO mice as well, it wasmarkedly less than in the similar areas from WT mice. Beclin-1 ex-pression was minimal in the aortas of LOX-1 KO mice and was similarto WT mice, when both were fed regular chow.
We observed a similar pattern of presence of apoptotic cells. Alarge number of VSMCs exhibited apoptosis in the lipid-rich areas ofaortas from WT mice, but apoptosis was minimal in normal areas ofaortas, and was similar to that in WT mice not given high cholesteroldiet. Similarly to beclin-1 expression, enhanced apoptosis was foundin the aortas of lipid-rich areas from LOX-1 KO mice, but was alwaysless than that in similar lipid-rich areas in aortas from WT mice(Fig. 6D).
4. Discussion
Autophagy is a well conserved intracellular degradation process bywhich cytoplasmic material, including soluble macromolecules andorganelles, is delivered to lysosomes for degradation [23]. Hence,autophagy is considered a stress response that allows unicellulareukaryotic organisms to survive during harsh conditions, probably byregulating energy homeostasis and/or by protein and organelle
degradation [19,20]. Apoptosis, on the other hand, is a response to ox-idant stress, and is mediated via LOX-1 expression/activation in humanendothelial cells and VSMCs [1,2].
Atherosclerotic regions are characterized by accumulation of largeamounts of ox-LDL in macrophages, VSMCs and endothelial cells[1,2]. In addition, there is intense LOX-1 expression in the intimaand media [6]. Since VSMCs form an important constituent of athero-sclerotic lesion, we focused on this cell type to study the relative ef-fects of ox-LDL in terms of development of autophagy and apoptosisin vitro.
We show that ox-LDL in concentrations of 20–40 μg/ml inducessignificant increases in LOX-1 expression and autophagy in humanVSMCs. The number of cells showing autophagy begins to decline asthe concentration of ox-LDL is increased to ≥60 μg/ml. Although theprecise concentration of ox-LDL in atherosclerotic regions is notknown, the circulating levels of ox-LDL are in the 20–40 μg/mlrange in patients with moderate to severe atherosclerosis [1,2]. Itmay be speculated that autophagy in response to circulating concen-trations of ox-LDL reflects a stress response that allows VSMCs to sur-vive during harsh conditions. As the stress increases, as reflected by≥40 μg/ml concentration of ox-LDL, the cellular defenses are lostand the cell undergoes apoptosis or necrosis. This concept issupported by our observation that VSMC apoptosis increased in re-sponse to increasing concentration of ox-LDL up to 60 μg/ml and
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Fig. 6. A. LDL-cholesterol concentrations in plasma are lower in LOX-1 knockout (LOX-1 KO) mice (Pb0.05 vs WT mice), while HDL and VLDL-cholesterol concentrations are similarin both two groups. B. LOX-1 KO mice have less fatty deposits than WT mice. Upper panel shows representative aortas from each group and the bar graphs show summary data ineach group. Intimal thickness is also reduced in the LOX-1 KO mice (Pb0.05 vs. wild-type mice). C and D. Immunohistochemistry and immunofluorescence staining for autophagyand apoptosis in aortic sections fromWT and LOX-1 KO mice. Autophagy is more prominent in lipid-rich areas of WT mice given high cholesterol diet, and less so in LOX-1 KO mice,but almost completely absent in normal areas. Apoptosis is also more prominent in lipid-rich areas of WTmice, and less in the LOX-1 KOmice given high cholesterol diet, but almostcompletely absent in aortic sections from WT and LOX-1 KO mice given regular chow. Data are representative of study of 7 sections of 7 mice in each group. Bar graphs showsummary (±SD) data in each group, *Pb0.05.
1384 Z. Ding et al. / International Journal of Cardiology 168 (2013) 1378–1385
then the rate of apoptosis stabilized indicating death of a very largepercentage of cells. These observations are confirmed by cell viabilitydata shown in Fig. 1B. These observations on the rate of apoptosis inresponse to ox-LDL are supported by results of several previousstudies [6,21].
Our data showing autophagy and apoptosis in the aortas of WTmice fed a high cholesterol diet are confirmatory of in vitro observa-tions in cultured VSMCs. It is of note that the aortas of WT mice fed ahigh cholesterol diet reveal oxidant stress and inflammatory reactionas well as LOX-1 expression [24].
Our in vitro studies show that autophagy as well as apoptosis in re-sponse to ox-LDL was caused by LOX-1 activation since a specific LOX-1antibody blocked the effects of ox-LDL. The intracellular signaling ofox-LDL/LOX-1 involves NADPH oxidase expression and generation ofROS followed by activation of MAPKs and transcription factors suchas NF-kB [1,2]. The inhibition of LOX-1 by specific antibody dramatical-ly reduced ROS generation (Fig. 2) and almost completely preventedVSMC autophagy aswell as apoptosis, suggesting that LOX-1 activation,ROS generation, autophagy and apoptosis are inter-related.
Though ROS are essential mediators of normal cell physiology,there is increasing evidence to suggest that ROS overproductionand/or alterations of the antioxidant system are key pathological
triggers of several cardiovascular disorders [1,2]. As the amount ofROS generation increases, massive apoptosis and necrosis ensue[25–27]. Release of small amounts of ROS, on the other hand, hasbeen shown to induce autophagy, which in turn serves to reduce ox-idative damage [28]. However, release of ROS in large amounts wouldbe expected to halt the cell survival mechanism and result in exten-sive injury as shown in Fig. 1.
We used themicroRNA hsa-let-7g, a critical regulator of LOX-1 [16],to further study the role of LOX-1 in VSMC autophagy and apoptosis.We assessed autophagy by measuring three different markers,beclin-1, LC3 and Atg5, all found in endothelial cells and VSMCs [3].LC3 exists in two forms, LC3-I and LC3-II; LC3-I is localized in the cyto-sol while its proteolytic derivative LC3-II is present in autophagosomalmembranes. LC3-II accumulation is often used to estimate theautophagosomes [19,20]. The results of all three measurements weresimilar. Transfection of VSMCs with hsa-let-7g mimic reduced LOX-1expression and ROS generation and improved cell viability; in contrast,hsa-let-7g inhibitor had opposite effects. Importantly, hsa-let-7gover-expression inhibited autophagy marker (beclin-1, LC3 and Atg5)expression, which presumably would result in improved survival ofVSMCs. We showed that hsa-let-7g influences apoptosis by regulatingcaspase 3, Bax, Bcl-2 and Bcl-xL. Again, the over-expression of
1385Z. Ding et al. / International Journal of Cardiology 168 (2013) 1378–1385
hsa-let-7g inhibitor had the opposite effect on autophagy and apopto-sis. The effects of hsa-let-7g were largely similar to those of LOX-1antibody.
The Bcl-2 family of proteins, Bcl-2, Bcl-xL and Mcl-1 that maintainthe integrity of the mitochondrial outer membrane in normal cells,are well-known anti-apoptotic mediators; while other Bcl-2 familymembers, such as Bax, Bak and Bid are able to promote apoptosis,so that the ratio of expression of anti-apoptotic and pro-apoptoticproteins might determine the apoptotic potential of cells [29–31].Bcl-2 and Bcl-xL are also well known for their anti-autophagy abilitiesby binding to beclin-1, which is required for the initiation ofautophagasome formation in autophagy [29,31]. In our study, Bcl-2and Bcl-xL were inhibited by ox-LDL and enhanced by hsa-let-7g aswell as LOX-1 Ab (Fig. 4C and D). On the contrary, ox-LDL enhancedbeclin-1 expression while LOX-1 Ab decreased beclin-1 expression.Interestingly, hsa-let-7g inhibited beclin-1 expression, indicatingthat hsa-let-7g may have a variable effect on LOX-1 mediatedautophagy and apoptosis resulting in a salutary role in cell survival.
Based on the observations in cultured VSMCs presented here, webelieve that autophagy in VSMCs represents a stress adaptation thatprevents cell death and serves to eliminate superfluous, damagedcells and organelles that would include apoptotic cells when thestress is modest (equivalent to 10 to 40 μg/ml concentrations ofox-LDL). At higher concentrations (60 to 100 μg/ml), ox-LDL via gen-eration of large amounts of ROS leads to a significant increase innonviable cells and a high apoptosis rate that is beyond autophagy'sprotective ability.
Next, we studied autophagy and apoptosis in aortic sections of WTand LOX-1 KO mice fed a high fat diet. Our studies showed significantautophagy and apoptosis in lipid-rich areas of aortic sections of WTmice. Notably, aortic sections of LOX-1 KO mice had much lessautophagy and apoptosis than the WT mice. The average LDL-cholesterol concentrations were lower in the LOX-1 KO mice than theWT mice as shown earlier (Fig. 6A). It is likely that the ox-LDL concen-tration in the lipid-rich regions is in the range of 10–40 μg/ml. aconcentration that resulted in a comparable increase in autophagy aswell as apoptosis. Further, compared with lipid-rich areas, bothautophagy and apoptosis were much less in the normal areas of aortasfrom LOX-1 KO mice and WT mice fed a high cholesterol diet. Thesedata support the in vitro observations in cultured VSMCs presented inFig. 5B.
Based on these in vitro and in vivo observations, we suggest thatLOX-1 is a key mediator of both autophagy and apoptosis in thelipid-rich lesions. The use of LOX-1 KO mice provides direct evidencein support of this hypothesis.
Lastly, the present study provides first evidence that hsa-let-7gcan inhibit apoptosis and promote cell survival by inhibiting LOX-1expression. Since LOX-1 activation and autophagy are involved in anumber of disease states, e.g. tissue ischemia, certain cancers, andatherosclerosis, it is possible that hsa-let-7g might become a potentialtherapeutic target for further investigation in these disease states.
Disclaimer
The opinions expressed in this manuscript do not necessarilyrepresent those of the U.S. Food and Drug Administration.
Acknowledgments
This study was supported in part by funds from the Department ofVeterans Affairs, Veterans Health Administration, Office of Researchand Development, Biomedical Laboratory Research and Development,Washington, DC.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ijcard.2012.12.045.
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