Experimental and Molecular Pathology 96 (2014) 250–256
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Probucol via inhibition of NHE1 attenuates LPS-accelerated atherosclerosis and promotes plaque stability in vivo Li Jian-Fei a,1, Chen Song a,1, Feng Jun-Duo b, Zhang Ming-Yu a,⁎, Liu Xiao-Xia a a b
Department of Cardiology, The 4th Hospital of Harbin Medical University, Harbin 150001, China Health examination center, Hospital of Heilongjiang Armed Police Corps, 558 Xinyanglu Road, Harbin 150076, China
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Article history: Received 23 February 2014 Available online 1 March 2014 Keywords: Atherosclerosis Lipopolysaccharide Na+/H+ exchanger 1 Probucol Plaque stability
a b s t r a c t Activation of Na+/H+ exchanger 1 (NHE1) by lipopolysaccharide (LPS) via Ca2+/calpain is responsible in vascular smooth muscle cell (VSMC) apoptosis and to the process of atherosclerosis. Probucol is a lipid-lowering drug which has an anti-atherosclerosis effect. The mechanism remains poorly understood. Here we hypothesized that probucol via inhibition of NHE1 in VSMCs attenuates LPS-accelerated atherosclerosis and promotes plaque stability. Our results revealed that treatment of VSMCs with LPS increased the NHE1 activity in a timedependent manner, associated with the increased Ca2+i. Probucol inhibited the LPS-induced increase of NHE1 activity in a dose-dependent manner in VSMCs for 24-hour co-incubation, as well as the change of Ca2 +i. In addition, LPS enhanced the calpain activity. Both probucol and calcium chelation of Ca2+ abolished the LPSinduced increase of calpain activity. Treatment of VSMCs with LPS reduced the expression of Bcl-2 without altering the mRNA level. Probucol inhibited the LPS-reduced expression of Bcl-2 protein in VSMCs. Animal studies indicated administration of probucol suppressed LPS-accelerated apoptosis, atherosclerosis and plaque instability in Apoe−/− mice. In conclusion, probucol via inhibition of NHE1 attenuates atherosclerosis lesion growth and promotes plaque stability. © 2014 Elsevier Inc. All rights reserved.
Introduction Atherosclerosis is a common cardiovascular disease characterized by the depositions of fatty substances on and fibrosis of the inner lining of the arteries. Probucol is a unique cholesterol lowering drug with an anti-atherosclerosis effect (Alessio et al., 2011). Probucol trial revealed that probucol reduces coronary restenosis after percutaneous transluminal coronary angioplasty. Probucol observational study illuminating therapeutic impact on vascular events showed that probucol was useful in lowering the risk of cardiovascular events in secondary prevention in spite of causing a decrease in HDL-C levels. Although probucol decreases HDL-C levels, it shows greatly controlled progression of atherosclerosis (Bocan et al., 1992). The molecular mechanism against atherosclerosis still remains unknown. Lipopolysaccharide (LPS) is a critical glycolipid component of the outer wall of Gram-negative bacteria, and many of the cellular signals activated by Gram-negative bacteria are attributed to LPS (Raetz, 1990). Recent report has shown that LPS can induce VSMC injury by activating calpain (Liu et al., 2008) and accelerate atherosclerosis in mice (C. Wang et al., 2007). The vascular cell is a prime target of the LPS molecule, ⁎ Corresponding author at: Department of Cardiology, The 4th Hospital of Harbin Medical University, 37 Yiyuan Road, Harbin 150001, China. 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.yexmp.2014.02.010 0014-4800/© 2014 Elsevier Inc. All rights reserved.
and vascular complications of septic shock due to Gram-negative bacteria are related to vascular injury (Westhorpe et al., 2012). Indeed, LPSinduced systemic organ failure is triggered initially by vascular injury, characterized by vascular occlusion, perivascular accumulation of leukocytes and cell death. VSMC apoptosis is caused by various mechanisms, such as cytokines, free radicals, death receptors and cell interaction, which is related to atherosclerosis. The induction of apoptosis by the Na+/H+ exchanger (NHE1) activation has been extensively studied. The NHE1 is expressed ubiquitously in the plasma membrane of mammalian cells and exchanges intracellular H+ for extracellular Na+ to regulate intracellular pH (pHi) value and the concentration of Na+i (Shuang-Xi et al., 2005). The activation of NHE1 increases Na+i concentration that leads to Ca2+ overload through the Na+/Ca2+ exchanger, which is assumed to be the crucial factor in cell injury (Wang et al., 2003). Increase intracellular Ca2+ concentration automatically activates calpain, a calcium-dependent protease (Goll et al., 2003). Many proteins in cells are the natural substrates of calpain, including anti-apoptotic family member, Bcl-2 (Gil-Parrado et al., 2002). Previous studies have showed that the inhibition of NHE-1 has antiapoptotic effects and the role of Bcl-2 in LPS-induced apoptosis (Garciarena et al., 2009; Schelling and Abu Jawdeh, 2008; H.L. Wang et al., 2007). In this report we hypothesized that LPS stimulates a calpainmediated death pathway in VSMCs and probucol potentially suppresses
J.-F. Li et al. / Experimental and Molecular Pathology 96 (2014) 250–256
this pathway to attenuate atherosclerosis progression and formation of vulnerable plaque via inhibition of apoptosis. The study provides new insights to understand the effects of probucol on atherosclerosis. Materials and methods Materials Probucol was from Sigma Company. Calcium chelator (BAPTA) was obtained from Invitrogen Corporation. 2-Carboxyethyl-5(6)-carboxyfluorescein (BCECF) were purchased from Calbiochem (USA). Other chemicals were purchased from Sigma Chemical Co, USA. Primary antibodies (Bcl-2, CD68, actin, GAPDH) and secondary antibodies were obtained from Cell signaling company. All chemicals were of reagent grade. All concentrations are the final concentration. Mice Male Apo E gene knockout (Apoe−/−) mice originally purchased from Jackson Laboratories (Bar Harbor, ME), were used at 8 to 10 weeks of age and then fed a high-fat diet (HFD) with or without 0.5% (w/w) probucol and LPS infusion for 4 weeks. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by Harbin Medical University. Cell culture VSMCs were purchased from ATCC and cultured according to the method described previously (Wang et al., 2008a). VSMCs were grown in M200 (Clonetics Inc. Walkersville, MD) supplemented with 10% FBS, 12.5 mg/ml ECGF, 1 mg/ml hydrocortisone, 100 μ/ml penicillin and 100 mg/ml streptomycin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Culture medium was replaced twice a week, and cells were sub-cultured when confluent. Cultures were expanded by brief trypsinization using 0.25% trypsin in phosphate-buffered saline (PBS) containing 0.02% EDTA. Cells at passage 3–8 were used for all experiments. Western blot After treatment, VSMCs were lysated in cell-lysis buffer (Cell Signaling Company). The protein content was assayed by BCA protein assay reagent (Pierce, USA). 20 μg of protein was loaded to SDS-PAGE and then transferred to membrane. Membrane was incubated with a 1:1000 dilution of primary antibody, followed by a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized by ECL (GE Healthcare). The intensity (area × density) of the individual bands on western blots was measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). The background was subtracted from the calculated area.
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Measurement of NHE1 activity NHE1 activity in VSMCs in situ was determined by measuring the initial rate of pHi recovery following intracellular acidification by using NH4Cl pulse method (Wang et al., 2006). Briefly, VSMCs were washed with HCO− 3 -free HBS buffer (mM: NaCl, 140; KCl, 5; CaCl2, 1; MgCl2, 1; glucose, 5; HEPES, 6; pH 7.4). After the incubation of HUVECs with HBS containing 10 μM BCECF at 37 °C for 30 min and the removal of free-BCECF by washing, 40 mM NH4Cl was added into HBS, incubated for 5 min, and washed out with Na+-free HBS buffer. Cells were acidloaded and pHi decreased. When 100 mM NaCl was added, intracellular H+ was pumped out via Na+/H+ exchange and pHi increased linearly during the initial 40 s. This initial rate of pHi recovery (dpHi/dt) was considered to reflect the Na+/H+ exchange activity. Calpain activity assay The calpain activity was measured by using the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC as a substrate following the procedure described previously with slight modification (Dong et al., 2006). Shortly, cells were cultured in 24-well plates in medium with different treatments. After being washed twice with PBS, fluorogenic peptide was added to a final concentration of 80 μM in PBS. Immediately after the addition of fluorogenic peptide, fluorescence was recorded at 2 min intervals for 20 min at an excitation of 360 nm and an emission of 460 nm using a Synergy HT Multi-Detection Microplate Reader (BIOTEK Instruments Inc.). The initial rate of peptidyl-AMC hydrolysis was used as the velocity of enzyme activity. Measurement of Ca2+i concentration The Ca2 +i concentration was measured by using a Fluo-4 NW kit from Invitrogen following kit protocol (Wang et al., 2008b). Briefly, VSMCs were treated as indicated, the cell culture medium was aspirated, washed with HEPES buffer (pH 7.4) once, and 1 ml of HEPES buffer with fluorescent dye was added to cultured cells. After 30 min incubation, fluorescence strength was measured in wavelength of excitation/ emission of 485/520 nm. Atherosclerotic lesion analysis The ascending aorta tissue from to the ideal bifurcation was removed and fixed in 4% paraformaldehyde for 16 h. The adventitia was then thoroughly cleaned under a dissecting microscope. For each mouse, four consecutive sections were stained with HE. Images of plaques were captured using an Olympus microscope connected to a QImaging Retiga CCD camera. The aortic lesion size in each animal was obtained by averaging the lesion areas in the four sections. The lesion area, from the aortic arch to 5 mm distal to the left subclavian artery, was quantified using Alpha Ease FC software (Version 4.0, Alpha Innotech). Determination of plaque vulnerable index
RT-PCR analysis for Bcl-2 mRNA Total RNA was extracted from cells after treatment by using a TRIZOL (Life Technologies, USA) reagent according to the manufacturer's protocol (Kyronlahti et al., 2008). The primer sequences were: Bcl-2 sense, 5′-GTGGATGACTGAGTACCTGAACC-3′; Bcl-2 antisense, 5′-AGCC AGGAGAAA TCAAACAGAG-3′; and GAPDH sense, 5′-TCATTTCCTGGTAT GACAACG-3′; GAPDH antisense, 5′-TTACTCCTTGGAGGCCATGT-3′. The PCR protocol for Bcl-2consisted of denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 2 min, and final extension at 72 °C for 15 min. The protocol for GAPDH was the same except for annealing at 65 °C for 1 min and amplification of 25 cycles.
The plaque vulnerable index was determined by using the ration of CD68-positive (%) plus Oil Red (%) to α-actin (%) plus collagen (%) as described previously (Dong et al., 2013). Two different lesion areas were selected to account in each segments and the mean was used in statistical analysis. Immunohistochemistry The aortic section was dissected, fixed in 4% paraformaldehyde for 16 h, and embedded in paraffin. Four micron-thick sections were deparaffinized, rehydrated, and microwaved in citrate buffer for antigen retrieval. Sections were successively incubated in endogenous
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Fig. 1. LPS increases probucol-sensitive NHE1 activity. A, VSMCs were incubated with LPS (100 ng/ml) for 1–24 h. B, VSMCs were incubated with LPS (100 ng/ml) in presence or absence of probucol (1, 5, 10 μM) for 24 h. Cells were subjected to detect NHE1 activity by NH4Cl pulse method plus BCECF dye. Data are expressed by mean ± SEM (n = 5). *P b 0.05 vs. Control, #P b 0.05 vs. LPS alone.
peroxidase and alkaline phosphatase block buffer (DAKO), protein block buffer, and primary antibodies, with the antibody incubation being performed overnight at 4 °C. After rinsing in wash buffer, sections were incubated with labeled polymer-horseradish peroxidase antimouse or anti-rabbit antibodies and DAB chromogen. Alternatively, they were incubated with polymer-alkaline phosphatase anti-mouse or anti-rabbit antibody and Permanent Red chromogen (EnVision™ G| 2 Doublestain System, DAKO). After final washes, sections were counterstained with hematoxylin. Statistical analysis Statistical analysis was performed using SPSS10.0 software. Data are expressed as means ± SEM. The statistical significance of differences was evaluated by using one-way ANOVA followed by the Student's t-test. P b 0.05 was considered statistically significant.
activity from 4 hour incubation. The increased NHE1 activity induced by LPS reached the peak at 16 to 24 h. However, probucol inhibited the LPS-induced increase of NHE1 activity in a dose-dependent manner (Fig. 1B). Probucol decreases intracellular calcium level via inactivation of NHE1 The activation of NHE1 increases Na+i that leads to Ca2+ overload through the Na+/Ca2+ exchanger, which is assumed to be the crucial factor in many diseases, such as ischemia (Toda et al., 2007), atherosclerosis (Koliakos et al., 2007), and others. Here we further investigated whether activation of NHE1 by LPS results in the increased intracellular calcium level. As shown in Fig. 2A, LPS time-dependently increased intracellular calcium level in VSMCs. In addition, the LPS-induced increase of intracellular calcium level was reversed by probucol treatment (Fig. 2B). These data suggest to us that LPS-enhanced intracellular calcium level is NHE1 dependent.
Results LPS-enhanced calpain activity is both NHE1 and calcium dependent Probucol inhibits LPS-increased NHE1 activity Early studies have shown that LPS, an inflammatory factor, induced VSMC apoptosis and vascular dysfunction, which contributes to cardiovascular disease (Ulevitch et al., 1976). We first determined whether LPS activates NHE1 in VSMCs. As shown in Fig. 1A, VSMCs were treated with LPS (100 ng/mL) for various times. LPS started to increase NHE1
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Calpain is a family of Ca2+-dependent cysteine proteases found in mammals and many lower organisms (Azuma and Shearer, 2008). In the presence of an elevated Ca2+i concentration, calpain is activated in cells and regulated cell function by degrading a lot of proteins, such as HSP90 (Stalker et al., 2003, 2005). We next detected whether LPS increased calpain activity in VSMCs. In Fig. 3A and B, the incubation of
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Fig. 2. LPS increases intracellular calcium levels via activation of NHE1. A, VSMCs were incubated with LPS (100 ng/ml) for 1–24 h. B, VSMCs were incubated with LPS (100 ng/ml) in presence or absence of probucol (10 μM). The intracellular calcium concentration was determined by Fluo-4 fluorescence. Control group was defined as 100%. Data are expressed by mean ± SEM (n = 5). *P b 0.05 vs control, #P b 0.05 vs LPS alone.
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Fig. 3. LPS-enhanced calpain activity is NHE1 or calcium dependent. A, VSMCs were incubated with LPS (100 ng/mL) for 24 h in the absence or presence of probucol (10 μM). B, VSMCs were incubated with LPS (100 ng/mL) for 24 h in the absence or presence of BAPTA (0.5 mM). Calpain activity was assayed by using the fluorogenic peptide substrate. Data are expressed as means ± SEM (n = 5), *P b 0.05 vs control, #P b 0.05 vs LPS alone.
VSMCs with LPS increased calpain activity. The inhibition of NHE1 by probucol or binding intracellular free-Ca2 + by BAPTA abolished the LPS-increased calpain activity. Taking these data together, it indicated that the activation of NHE1 resulted in the elevation of Ca2+i, causing the activation of calpain.
either. These data suggested that LPS decreased Bcl-2 protein levels via increasing the degradation but not decreasing the biosynthesis.
Inhibition of NHE1 by probucol abolishes LPS-induced down-regulation of Bcl-2 protein but not mRNA expression
We next investigated whether probucol reversed LPS-induced atherosclerosis in vivo. Apoe−/− mice were fed with high fat diet in the presence or absence of 0.5% probucol (w/w) for 4 weeks. As shown in Fig. 5A, LPS remarkably accelerated atherosclerosis lesion in HFD-fed Apoe−/− mice and probucol dramatically suppressed the atherosclerosis lesion in both basal and LPS-stimulated conditions in mice aortas. The anti-apoptotic protein of Bcl-2 was detected by IHC. In order to confirm whether the effect of probucol in atherosclerosis is due to its function of inhibiting VSMC apoptosis, we detected the cell apoptosis by TUNEL. As shown in Fig. 5B, the administration of probucol attenuated the LPS-increased cell apoptosis in aortas as well as the inhibition of NHE1 activity (Fig. 5C), indicating that probucol may suppress cell apoptosis via NHE1 inhibition to inhibit atherosclerosis.
Anti-apoptotic members of the Bcl family (Bcl-2 etc.) play important roles in regulating the cell death (Lessene et al., 2008; Yip and Reed, 2008). Next we determined whether LPS-increased calpain activity induced degradation of Bcl-2. As shown in Fig. 4A, the treatment of VSMCs with LPS reduced the protein levels of Bcl-2. The inhibition of NHE1 by probucol abolished the LPS-decreased Bcl-2 protein levels. In order to exclude the LPS-decreased Bcl-2 protein expression in VSMCs due to the decreased gene transcription by decreasing mRNA biosynthesis, we determined the mRNA of Bcl-2. As indicated in Fig. 4B, LPS alone did not change Bcl-2 mRNA level. Probucol did not alter the mRNA level of Bcl-2 in basal condition or LPS-treated VSMCs,
Probucol attenuates LPS-accelerated atherosclerosis in Apoe−/− mice
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Fig. 4. Inhibition of NHE1 or calpain abolishes LPS-induced down-regulation of Bcl-2 protein expression in HUVECs. A and B, VSMCs were incubated with LPS (100 ng/mL) for 24 h in the absence or presence of probucol (10 μM). Cells were subjected to detect (A) Bcl-2 protein level by western blot and (B) Bcl-2 mRNA level by RT-PCR. Data are expressed as means ± SEM. The picture is a representative from 3 independent experiments. *P b 0.05 vs control, #P b 0.05 vs LPS alone, NS is indicated as no significant difference.
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Fig. 5. Probucol administration suppresses LPS-accelerated atherosclerosis in Apoe−/−− mice. LPS was injected into Apoe−/− mice fed with high fat diet with or without 0.5% probucol. A, Representative photographs of Oil Red O staining in aortic root Sections. B, Cell apoptosis by TUNEL in situ. C, NHE1 activity in rat aortas. Data are expressed by mean ± SEM. N is 7 in each group. *P b 0.05 vs Control, #P b 0.05 vs LPS.
Probucol promotes the stability of atherosclerosis plaque in LPS-treated mice The rupture of vulnerable atherosclerotic plaque is one of the common risk factors of acute coronary syndrome and stroke (Hadamitzky et al., 2013; Kwan et al., 2013; Pelisek et al., 2012). Apoptosis in VSMCs is an important factor contributing to the formation of vulnerable plaque in atherosclerosis. Thus, we investigated the effect of probucol on plaque stability by analyzing the lipid content by Oil Red, collagen content by picrosirius red, number of macrophage cells by staining with CD68 antibody, and number of vascular smooth muscle cells by staining with α-actin antibody as described previously (Guo et al., 2013). As shown in Fig. 6A, the lipid content and number of CD68 positive macrophages were significantly increased in the plaques of LPS-treated mice. Probucol decreased macrophage infiltration and lipid content while increasing collagen content and number of vascular smooth muscle cells compared to control mice. To assess plaque stability, we determined the vulnerability index (VI) of atherosclerotic plaque as: VI = (macrophages stained % + lipids stained %) / (smooth muscle cells stained % + collagen stained %) (Torzewski et al., 1998). Plaque rupture was defined as discontinuity of the fibrous cap with luminal thrombosis or a buried fibrous cap within a plaque (Williams et al., 2002). The VI was 0 and 1.63 in control mice and LPS-treated mice, respectively. The administration of probucol dramatically decreased the VI to 0.82, which is significantly lower than that in mice treated with LPS. In addition, probucol also increased Bcl-2 protein expression assayed by IHC (Fig. 6B) and Western blot (Fig. 6C). Together, these data indicate that probucol promotes the plaque stability in atherosclerotic mice.
Discussion The current study demonstrates that probucol attenuates LPSaccelerated atherosclerosis formation and promotes plaque stability by inhibiting NHE1-dependent calpain-mediated Bcl-2 degradation in VSMCs. The inhibition of NHE1 by probucol reverses LPS-induced increase of calcium concentration and calpain activity. In addition, the inhibition of NHE1 by probucol blocked the decrease of Bcl-2 degradation caused by LPS. These results strongly suggest that NHE1 is required for LPS-induced cell apoptosis and the anti-atherosclerotic effects of probucol. The injury of vascular cells, such as endothelial cells and VSMCs, is a common finding in the pathogenesis of several diseases including atherosclerosis (Chen and Keaney, 2004), hypertension (Chen et al., 2004), and congestive heart failure (Cines et al., 1998). During bacterial
sepsis, cell death is the final step of a process beginning when quiescent cells are induced to express procoagulant, proadhesive, and vasoconstrictive factors. Results from this study demonstrate that LPS induces apoptosis in VSMCs. These findings are in agreement with those obtained in other species both in vitro and in vivo (Frey and Finlay, 1998; McCuskey et al., 1996). Our data clearly implicate that LPS-induced apoptosis is mediated by NHE1 via calcium/calpain pathway. The calpain is a family of calciumdependent protease that acts independently of the proteasome pathway and cleaves a number of cellular substrates, including kinases, phosphatases, transcription factors, and cytoskeletal protein. In this study, we found that LPS increased calpain activity in VSMCs as well as increased intracellular calcium. In addition, the chelation of intracellular free-calcium by BAPTA inhibited the activation of calpain. So we speculated that LPS-induced calpain activation is calcium dependent because it has been reported that calpain is activated in response to large calcium fluxes, such as during apoptosis (Lu et al., 2013). To determine whether LPS produced a probucol-sensitive activation of NHE1 in VSMCs, we measured NHE1 activity in this study. Our data showed that LPS produced a probucol-sensitive activation of NHE1 in VSMCs. A potential mechanism of LPS-induced increase of NHE1 activity is possibly a phosphorylation-dependent increase in the activity of existing exchangers or the activation of dormant membraneassociated exchangers. In fact, Sardet et al. (1990). have demonstrated that NHE1 is rapidly phosphorylated in response to various mitogens and concluded that this phosphorylation of NHE1 is temporally correlated with its activation. The anti-apoptotic Bcl-2 protein is localized on outer mitochondrial membrane, and functions to prevent cytochrome C release from mitochondria (Green and Reed, 1998). In our results, LPS decreased the level of Bcl-2 protein but not mRNA in VSMCs; however, probucol reversed the effects of LPS. All the results indicate that calpain may play some roles in LPS-induced degradation of Bcl-2, however which calpain isoform is involved in the degradation of Bcl-2 and whether Bcl-2 is the direct substrate of calpain in LPS-treated VSMCs need further investigations. In conclusion, the inhibition of NHE1 by probucol completely blocked LPS-induced atherosclerosis in mice, mediated by calcium and calpain dependent Bcl-2 degradation in VSMCs (Fig. 6D). The inhibition of NHE1 could be a promising novel approach to halt or even reverse atherosclerosis in patients.
Conflict of interest The authors declare no competing financial interests.
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Fig. 6. Probucol promotes plaque stability in LPS-injected in Apoe−/−− mice. LPS was injected into Apoe−/− mice fed with high fat diet containing 0.5% probucol or not. A, The lipid content by Oil Red staining, collagen by picrosirius red, numbers of vascular smooth muscle cells by α-actin staining, and numbers of macrophages by CD68 staining were determined in left carotid artery. B and C, Bcl-2 protein expression by IHC or Western blot. Data are expressed by mean ± SEM. 5–10 mice in each group. *P b 0.05 vs control, #P b 0.05 vs LPS alone. D, Proposed mechanism by which probucol produces the anti-atherosclerotic effects.
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Probucol via inhibition of NHE1 attenuates LPS-accelerated atherosclerosis and promotes plaque stability in vivo Li Jian-Fei a,1, Chen Song a,1, Feng Jun-Duo b, Zhang Ming-Yu a,⁎, Liu Xiao-Xia a a b
Department of Cardiology, The 4th Hospital of Harbin Medical University, Harbin 150001, China Health examination center, Hospital of Heilongjiang Armed Police Corps, 558 Xinyanglu Road, Harbin 150076, China
a r t i c l e
i n f o
Article history: Received 23 February 2014 Available online 1 March 2014 Keywords: Atherosclerosis Lipopolysaccharide Na+/H+ exchanger 1 Probucol Plaque stability
a b s t r a c t Activation of Na+/H+ exchanger 1 (NHE1) by lipopolysaccharide (LPS) via Ca2+/calpain is responsible in vascular smooth muscle cell (VSMC) apoptosis and to the process of atherosclerosis. Probucol is a lipid-lowering drug which has an anti-atherosclerosis effect. The mechanism remains poorly understood. Here we hypothesized that probucol via inhibition of NHE1 in VSMCs attenuates LPS-accelerated atherosclerosis and promotes plaque stability. Our results revealed that treatment of VSMCs with LPS increased the NHE1 activity in a timedependent manner, associated with the increased Ca2+i. Probucol inhibited the LPS-induced increase of NHE1 activity in a dose-dependent manner in VSMCs for 24-hour co-incubation, as well as the change of Ca2 +i. In addition, LPS enhanced the calpain activity. Both probucol and calcium chelation of Ca2+ abolished the LPSinduced increase of calpain activity. Treatment of VSMCs with LPS reduced the expression of Bcl-2 without altering the mRNA level. Probucol inhibited the LPS-reduced expression of Bcl-2 protein in VSMCs. Animal studies indicated administration of probucol suppressed LPS-accelerated apoptosis, atherosclerosis and plaque instability in Apoe−/− mice. In conclusion, probucol via inhibition of NHE1 attenuates atherosclerosis lesion growth and promotes plaque stability. © 2014 Elsevier Inc. All rights reserved.
Introduction Atherosclerosis is a common cardiovascular disease characterized by the depositions of fatty substances on and fibrosis of the inner lining of the arteries. Probucol is a unique cholesterol lowering drug with an anti-atherosclerosis effect (Alessio et al., 2011). Probucol trial revealed that probucol reduces coronary restenosis after percutaneous transluminal coronary angioplasty. Probucol observational study illuminating therapeutic impact on vascular events showed that probucol was useful in lowering the risk of cardiovascular events in secondary prevention in spite of causing a decrease in HDL-C levels. Although probucol decreases HDL-C levels, it shows greatly controlled progression of atherosclerosis (Bocan et al., 1992). The molecular mechanism against atherosclerosis still remains unknown. Lipopolysaccharide (LPS) is a critical glycolipid component of the outer wall of Gram-negative bacteria, and many of the cellular signals activated by Gram-negative bacteria are attributed to LPS (Raetz, 1990). Recent report has shown that LPS can induce VSMC injury by activating calpain (Liu et al., 2008) and accelerate atherosclerosis in mice (C. Wang et al., 2007). The vascular cell is a prime target of the LPS molecule, ⁎ Corresponding author at: Department of Cardiology, The 4th Hospital of Harbin Medical University, 37 Yiyuan Road, Harbin 150001, China. 1 Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.yexmp.2014.02.010 0014-4800/© 2014 Elsevier Inc. All rights reserved.
and vascular complications of septic shock due to Gram-negative bacteria are related to vascular injury (Westhorpe et al., 2012). Indeed, LPSinduced systemic organ failure is triggered initially by vascular injury, characterized by vascular occlusion, perivascular accumulation of leukocytes and cell death. VSMC apoptosis is caused by various mechanisms, such as cytokines, free radicals, death receptors and cell interaction, which is related to atherosclerosis. The induction of apoptosis by the Na+/H+ exchanger (NHE1) activation has been extensively studied. The NHE1 is expressed ubiquitously in the plasma membrane of mammalian cells and exchanges intracellular H+ for extracellular Na+ to regulate intracellular pH (pHi) value and the concentration of Na+i (Shuang-Xi et al., 2005). The activation of NHE1 increases Na+i concentration that leads to Ca2+ overload through the Na+/Ca2+ exchanger, which is assumed to be the crucial factor in cell injury (Wang et al., 2003). Increase intracellular Ca2+ concentration automatically activates calpain, a calcium-dependent protease (Goll et al., 2003). Many proteins in cells are the natural substrates of calpain, including anti-apoptotic family member, Bcl-2 (Gil-Parrado et al., 2002). Previous studies have showed that the inhibition of NHE-1 has antiapoptotic effects and the role of Bcl-2 in LPS-induced apoptosis (Garciarena et al., 2009; Schelling and Abu Jawdeh, 2008; H.L. Wang et al., 2007). In this report we hypothesized that LPS stimulates a calpainmediated death pathway in VSMCs and probucol potentially suppresses
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this pathway to attenuate atherosclerosis progression and formation of vulnerable plaque via inhibition of apoptosis. The study provides new insights to understand the effects of probucol on atherosclerosis. Materials and methods Materials Probucol was from Sigma Company. Calcium chelator (BAPTA) was obtained from Invitrogen Corporation. 2-Carboxyethyl-5(6)-carboxyfluorescein (BCECF) were purchased from Calbiochem (USA). Other chemicals were purchased from Sigma Chemical Co, USA. Primary antibodies (Bcl-2, CD68, actin, GAPDH) and secondary antibodies were obtained from Cell signaling company. All chemicals were of reagent grade. All concentrations are the final concentration. Mice Male Apo E gene knockout (Apoe−/−) mice originally purchased from Jackson Laboratories (Bar Harbor, ME), were used at 8 to 10 weeks of age and then fed a high-fat diet (HFD) with or without 0.5% (w/w) probucol and LPS infusion for 4 weeks. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by Harbin Medical University. Cell culture VSMCs were purchased from ATCC and cultured according to the method described previously (Wang et al., 2008a). VSMCs were grown in M200 (Clonetics Inc. Walkersville, MD) supplemented with 10% FBS, 12.5 mg/ml ECGF, 1 mg/ml hydrocortisone, 100 μ/ml penicillin and 100 mg/ml streptomycin. The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Culture medium was replaced twice a week, and cells were sub-cultured when confluent. Cultures were expanded by brief trypsinization using 0.25% trypsin in phosphate-buffered saline (PBS) containing 0.02% EDTA. Cells at passage 3–8 were used for all experiments. Western blot After treatment, VSMCs were lysated in cell-lysis buffer (Cell Signaling Company). The protein content was assayed by BCA protein assay reagent (Pierce, USA). 20 μg of protein was loaded to SDS-PAGE and then transferred to membrane. Membrane was incubated with a 1:1000 dilution of primary antibody, followed by a 1:2000 dilution of horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized by ECL (GE Healthcare). The intensity (area × density) of the individual bands on western blots was measured by densitometry (model GS-700, Imaging Densitometer; Bio-Rad). The background was subtracted from the calculated area.
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Measurement of NHE1 activity NHE1 activity in VSMCs in situ was determined by measuring the initial rate of pHi recovery following intracellular acidification by using NH4Cl pulse method (Wang et al., 2006). Briefly, VSMCs were washed with HCO− 3 -free HBS buffer (mM: NaCl, 140; KCl, 5; CaCl2, 1; MgCl2, 1; glucose, 5; HEPES, 6; pH 7.4). After the incubation of HUVECs with HBS containing 10 μM BCECF at 37 °C for 30 min and the removal of free-BCECF by washing, 40 mM NH4Cl was added into HBS, incubated for 5 min, and washed out with Na+-free HBS buffer. Cells were acidloaded and pHi decreased. When 100 mM NaCl was added, intracellular H+ was pumped out via Na+/H+ exchange and pHi increased linearly during the initial 40 s. This initial rate of pHi recovery (dpHi/dt) was considered to reflect the Na+/H+ exchange activity. Calpain activity assay The calpain activity was measured by using the fluorogenic peptide Suc-Leu-Leu-Val-Tyr-AMC as a substrate following the procedure described previously with slight modification (Dong et al., 2006). Shortly, cells were cultured in 24-well plates in medium with different treatments. After being washed twice with PBS, fluorogenic peptide was added to a final concentration of 80 μM in PBS. Immediately after the addition of fluorogenic peptide, fluorescence was recorded at 2 min intervals for 20 min at an excitation of 360 nm and an emission of 460 nm using a Synergy HT Multi-Detection Microplate Reader (BIOTEK Instruments Inc.). The initial rate of peptidyl-AMC hydrolysis was used as the velocity of enzyme activity. Measurement of Ca2+i concentration The Ca2 +i concentration was measured by using a Fluo-4 NW kit from Invitrogen following kit protocol (Wang et al., 2008b). Briefly, VSMCs were treated as indicated, the cell culture medium was aspirated, washed with HEPES buffer (pH 7.4) once, and 1 ml of HEPES buffer with fluorescent dye was added to cultured cells. After 30 min incubation, fluorescence strength was measured in wavelength of excitation/ emission of 485/520 nm. Atherosclerotic lesion analysis The ascending aorta tissue from to the ideal bifurcation was removed and fixed in 4% paraformaldehyde for 16 h. The adventitia was then thoroughly cleaned under a dissecting microscope. For each mouse, four consecutive sections were stained with HE. Images of plaques were captured using an Olympus microscope connected to a QImaging Retiga CCD camera. The aortic lesion size in each animal was obtained by averaging the lesion areas in the four sections. The lesion area, from the aortic arch to 5 mm distal to the left subclavian artery, was quantified using Alpha Ease FC software (Version 4.0, Alpha Innotech). Determination of plaque vulnerable index
RT-PCR analysis for Bcl-2 mRNA Total RNA was extracted from cells after treatment by using a TRIZOL (Life Technologies, USA) reagent according to the manufacturer's protocol (Kyronlahti et al., 2008). The primer sequences were: Bcl-2 sense, 5′-GTGGATGACTGAGTACCTGAACC-3′; Bcl-2 antisense, 5′-AGCC AGGAGAAA TCAAACAGAG-3′; and GAPDH sense, 5′-TCATTTCCTGGTAT GACAACG-3′; GAPDH antisense, 5′-TTACTCCTTGGAGGCCATGT-3′. The PCR protocol for Bcl-2consisted of denaturation at 94 °C for 5 min, 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 2 min, and final extension at 72 °C for 15 min. The protocol for GAPDH was the same except for annealing at 65 °C for 1 min and amplification of 25 cycles.
The plaque vulnerable index was determined by using the ration of CD68-positive (%) plus Oil Red (%) to α-actin (%) plus collagen (%) as described previously (Dong et al., 2013). Two different lesion areas were selected to account in each segments and the mean was used in statistical analysis. Immunohistochemistry The aortic section was dissected, fixed in 4% paraformaldehyde for 16 h, and embedded in paraffin. Four micron-thick sections were deparaffinized, rehydrated, and microwaved in citrate buffer for antigen retrieval. Sections were successively incubated in endogenous
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Fig. 1. LPS increases probucol-sensitive NHE1 activity. A, VSMCs were incubated with LPS (100 ng/ml) for 1–24 h. B, VSMCs were incubated with LPS (100 ng/ml) in presence or absence of probucol (1, 5, 10 μM) for 24 h. Cells were subjected to detect NHE1 activity by NH4Cl pulse method plus BCECF dye. Data are expressed by mean ± SEM (n = 5). *P b 0.05 vs. Control, #P b 0.05 vs. LPS alone.
peroxidase and alkaline phosphatase block buffer (DAKO), protein block buffer, and primary antibodies, with the antibody incubation being performed overnight at 4 °C. After rinsing in wash buffer, sections were incubated with labeled polymer-horseradish peroxidase antimouse or anti-rabbit antibodies and DAB chromogen. Alternatively, they were incubated with polymer-alkaline phosphatase anti-mouse or anti-rabbit antibody and Permanent Red chromogen (EnVision™ G| 2 Doublestain System, DAKO). After final washes, sections were counterstained with hematoxylin. Statistical analysis Statistical analysis was performed using SPSS10.0 software. Data are expressed as means ± SEM. The statistical significance of differences was evaluated by using one-way ANOVA followed by the Student's t-test. P b 0.05 was considered statistically significant.
activity from 4 hour incubation. The increased NHE1 activity induced by LPS reached the peak at 16 to 24 h. However, probucol inhibited the LPS-induced increase of NHE1 activity in a dose-dependent manner (Fig. 1B). Probucol decreases intracellular calcium level via inactivation of NHE1 The activation of NHE1 increases Na+i that leads to Ca2+ overload through the Na+/Ca2+ exchanger, which is assumed to be the crucial factor in many diseases, such as ischemia (Toda et al., 2007), atherosclerosis (Koliakos et al., 2007), and others. Here we further investigated whether activation of NHE1 by LPS results in the increased intracellular calcium level. As shown in Fig. 2A, LPS time-dependently increased intracellular calcium level in VSMCs. In addition, the LPS-induced increase of intracellular calcium level was reversed by probucol treatment (Fig. 2B). These data suggest to us that LPS-enhanced intracellular calcium level is NHE1 dependent.
Results LPS-enhanced calpain activity is both NHE1 and calcium dependent Probucol inhibits LPS-increased NHE1 activity Early studies have shown that LPS, an inflammatory factor, induced VSMC apoptosis and vascular dysfunction, which contributes to cardiovascular disease (Ulevitch et al., 1976). We first determined whether LPS activates NHE1 in VSMCs. As shown in Fig. 1A, VSMCs were treated with LPS (100 ng/mL) for various times. LPS started to increase NHE1
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Calpain is a family of Ca2+-dependent cysteine proteases found in mammals and many lower organisms (Azuma and Shearer, 2008). In the presence of an elevated Ca2+i concentration, calpain is activated in cells and regulated cell function by degrading a lot of proteins, such as HSP90 (Stalker et al., 2003, 2005). We next detected whether LPS increased calpain activity in VSMCs. In Fig. 3A and B, the incubation of
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Fig. 2. LPS increases intracellular calcium levels via activation of NHE1. A, VSMCs were incubated with LPS (100 ng/ml) for 1–24 h. B, VSMCs were incubated with LPS (100 ng/ml) in presence or absence of probucol (10 μM). The intracellular calcium concentration was determined by Fluo-4 fluorescence. Control group was defined as 100%. Data are expressed by mean ± SEM (n = 5). *P b 0.05 vs control, #P b 0.05 vs LPS alone.
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Fig. 3. LPS-enhanced calpain activity is NHE1 or calcium dependent. A, VSMCs were incubated with LPS (100 ng/mL) for 24 h in the absence or presence of probucol (10 μM). B, VSMCs were incubated with LPS (100 ng/mL) for 24 h in the absence or presence of BAPTA (0.5 mM). Calpain activity was assayed by using the fluorogenic peptide substrate. Data are expressed as means ± SEM (n = 5), *P b 0.05 vs control, #P b 0.05 vs LPS alone.
VSMCs with LPS increased calpain activity. The inhibition of NHE1 by probucol or binding intracellular free-Ca2 + by BAPTA abolished the LPS-increased calpain activity. Taking these data together, it indicated that the activation of NHE1 resulted in the elevation of Ca2+i, causing the activation of calpain.
either. These data suggested that LPS decreased Bcl-2 protein levels via increasing the degradation but not decreasing the biosynthesis.
Inhibition of NHE1 by probucol abolishes LPS-induced down-regulation of Bcl-2 protein but not mRNA expression
We next investigated whether probucol reversed LPS-induced atherosclerosis in vivo. Apoe−/− mice were fed with high fat diet in the presence or absence of 0.5% probucol (w/w) for 4 weeks. As shown in Fig. 5A, LPS remarkably accelerated atherosclerosis lesion in HFD-fed Apoe−/− mice and probucol dramatically suppressed the atherosclerosis lesion in both basal and LPS-stimulated conditions in mice aortas. The anti-apoptotic protein of Bcl-2 was detected by IHC. In order to confirm whether the effect of probucol in atherosclerosis is due to its function of inhibiting VSMC apoptosis, we detected the cell apoptosis by TUNEL. As shown in Fig. 5B, the administration of probucol attenuated the LPS-increased cell apoptosis in aortas as well as the inhibition of NHE1 activity (Fig. 5C), indicating that probucol may suppress cell apoptosis via NHE1 inhibition to inhibit atherosclerosis.
Anti-apoptotic members of the Bcl family (Bcl-2 etc.) play important roles in regulating the cell death (Lessene et al., 2008; Yip and Reed, 2008). Next we determined whether LPS-increased calpain activity induced degradation of Bcl-2. As shown in Fig. 4A, the treatment of VSMCs with LPS reduced the protein levels of Bcl-2. The inhibition of NHE1 by probucol abolished the LPS-decreased Bcl-2 protein levels. In order to exclude the LPS-decreased Bcl-2 protein expression in VSMCs due to the decreased gene transcription by decreasing mRNA biosynthesis, we determined the mRNA of Bcl-2. As indicated in Fig. 4B, LPS alone did not change Bcl-2 mRNA level. Probucol did not alter the mRNA level of Bcl-2 in basal condition or LPS-treated VSMCs,
Probucol attenuates LPS-accelerated atherosclerosis in Apoe−/− mice
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Fig. 4. Inhibition of NHE1 or calpain abolishes LPS-induced down-regulation of Bcl-2 protein expression in HUVECs. A and B, VSMCs were incubated with LPS (100 ng/mL) for 24 h in the absence or presence of probucol (10 μM). Cells were subjected to detect (A) Bcl-2 protein level by western blot and (B) Bcl-2 mRNA level by RT-PCR. Data are expressed as means ± SEM. The picture is a representative from 3 independent experiments. *P b 0.05 vs control, #P b 0.05 vs LPS alone, NS is indicated as no significant difference.
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Fig. 5. Probucol administration suppresses LPS-accelerated atherosclerosis in Apoe−/−− mice. LPS was injected into Apoe−/− mice fed with high fat diet with or without 0.5% probucol. A, Representative photographs of Oil Red O staining in aortic root Sections. B, Cell apoptosis by TUNEL in situ. C, NHE1 activity in rat aortas. Data are expressed by mean ± SEM. N is 7 in each group. *P b 0.05 vs Control, #P b 0.05 vs LPS.
Probucol promotes the stability of atherosclerosis plaque in LPS-treated mice The rupture of vulnerable atherosclerotic plaque is one of the common risk factors of acute coronary syndrome and stroke (Hadamitzky et al., 2013; Kwan et al., 2013; Pelisek et al., 2012). Apoptosis in VSMCs is an important factor contributing to the formation of vulnerable plaque in atherosclerosis. Thus, we investigated the effect of probucol on plaque stability by analyzing the lipid content by Oil Red, collagen content by picrosirius red, number of macrophage cells by staining with CD68 antibody, and number of vascular smooth muscle cells by staining with α-actin antibody as described previously (Guo et al., 2013). As shown in Fig. 6A, the lipid content and number of CD68 positive macrophages were significantly increased in the plaques of LPS-treated mice. Probucol decreased macrophage infiltration and lipid content while increasing collagen content and number of vascular smooth muscle cells compared to control mice. To assess plaque stability, we determined the vulnerability index (VI) of atherosclerotic plaque as: VI = (macrophages stained % + lipids stained %) / (smooth muscle cells stained % + collagen stained %) (Torzewski et al., 1998). Plaque rupture was defined as discontinuity of the fibrous cap with luminal thrombosis or a buried fibrous cap within a plaque (Williams et al., 2002). The VI was 0 and 1.63 in control mice and LPS-treated mice, respectively. The administration of probucol dramatically decreased the VI to 0.82, which is significantly lower than that in mice treated with LPS. In addition, probucol also increased Bcl-2 protein expression assayed by IHC (Fig. 6B) and Western blot (Fig. 6C). Together, these data indicate that probucol promotes the plaque stability in atherosclerotic mice.
Discussion The current study demonstrates that probucol attenuates LPSaccelerated atherosclerosis formation and promotes plaque stability by inhibiting NHE1-dependent calpain-mediated Bcl-2 degradation in VSMCs. The inhibition of NHE1 by probucol reverses LPS-induced increase of calcium concentration and calpain activity. In addition, the inhibition of NHE1 by probucol blocked the decrease of Bcl-2 degradation caused by LPS. These results strongly suggest that NHE1 is required for LPS-induced cell apoptosis and the anti-atherosclerotic effects of probucol. The injury of vascular cells, such as endothelial cells and VSMCs, is a common finding in the pathogenesis of several diseases including atherosclerosis (Chen and Keaney, 2004), hypertension (Chen et al., 2004), and congestive heart failure (Cines et al., 1998). During bacterial
sepsis, cell death is the final step of a process beginning when quiescent cells are induced to express procoagulant, proadhesive, and vasoconstrictive factors. Results from this study demonstrate that LPS induces apoptosis in VSMCs. These findings are in agreement with those obtained in other species both in vitro and in vivo (Frey and Finlay, 1998; McCuskey et al., 1996). Our data clearly implicate that LPS-induced apoptosis is mediated by NHE1 via calcium/calpain pathway. The calpain is a family of calciumdependent protease that acts independently of the proteasome pathway and cleaves a number of cellular substrates, including kinases, phosphatases, transcription factors, and cytoskeletal protein. In this study, we found that LPS increased calpain activity in VSMCs as well as increased intracellular calcium. In addition, the chelation of intracellular free-calcium by BAPTA inhibited the activation of calpain. So we speculated that LPS-induced calpain activation is calcium dependent because it has been reported that calpain is activated in response to large calcium fluxes, such as during apoptosis (Lu et al., 2013). To determine whether LPS produced a probucol-sensitive activation of NHE1 in VSMCs, we measured NHE1 activity in this study. Our data showed that LPS produced a probucol-sensitive activation of NHE1 in VSMCs. A potential mechanism of LPS-induced increase of NHE1 activity is possibly a phosphorylation-dependent increase in the activity of existing exchangers or the activation of dormant membraneassociated exchangers. In fact, Sardet et al. (1990). have demonstrated that NHE1 is rapidly phosphorylated in response to various mitogens and concluded that this phosphorylation of NHE1 is temporally correlated with its activation. The anti-apoptotic Bcl-2 protein is localized on outer mitochondrial membrane, and functions to prevent cytochrome C release from mitochondria (Green and Reed, 1998). In our results, LPS decreased the level of Bcl-2 protein but not mRNA in VSMCs; however, probucol reversed the effects of LPS. All the results indicate that calpain may play some roles in LPS-induced degradation of Bcl-2, however which calpain isoform is involved in the degradation of Bcl-2 and whether Bcl-2 is the direct substrate of calpain in LPS-treated VSMCs need further investigations. In conclusion, the inhibition of NHE1 by probucol completely blocked LPS-induced atherosclerosis in mice, mediated by calcium and calpain dependent Bcl-2 degradation in VSMCs (Fig. 6D). The inhibition of NHE1 could be a promising novel approach to halt or even reverse atherosclerosis in patients.
Conflict of interest The authors declare no competing financial interests.
J.-F. Li et al. / Experimental and Molecular Pathology 96 (2014) 250–256
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Fig. 6. Probucol promotes plaque stability in LPS-injected in Apoe−/−− mice. LPS was injected into Apoe−/− mice fed with high fat diet containing 0.5% probucol or not. A, The lipid content by Oil Red staining, collagen by picrosirius red, numbers of vascular smooth muscle cells by α-actin staining, and numbers of macrophages by CD68 staining were determined in left carotid artery. B and C, Bcl-2 protein expression by IHC or Western blot. Data are expressed by mean ± SEM. 5–10 mice in each group. *P b 0.05 vs control, #P b 0.05 vs LPS alone. D, Proposed mechanism by which probucol produces the anti-atherosclerotic effects.
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