nicotine accelerates atherosclerosis in apolipoprotein e ... · 6 arterioscler thromb vasc biol...

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Atherosclerosis is a chronic process that involves a com-plex interplay between vascular wall cells and inflam-

matory cells, of which the pathology is characterized by inflammation, lipid accumulation, cell death, and fibrosis.1,2 Cigarette smoking is a risk factor for cardiovascular diseases. It has been consistently demonstrated that smoking acceler-ates the development of atherosclerotic plaques,3,4 whereas smoking cessation results in significant decreases of cardio-vascular events among smokers.5 However, quitting tobacco usage is difficult as nicotine plays a critical role in addic-tion.6 Moreover, secondhand smoking increases the risk for developing atherosclerosis and acute plaque rupture–related cardiovascular events.7,8 There is an urgent need to prevent atherogenesis and cardiovascular events in smokers and indi-viduals exposed to secondhand smoke.

Nicotine is the major component in cigarettes that causes tobacco addiction.9 Experimental studies demonstrated that nicotine promotes the development of atherosclerosis; how-ever, the specific mechanism by which this occurs remain

largely unknown.10 Effects of mast cells (MCs) have been implicated in nicotine-induced atherosclerotic lesions. For example, mucosal MCs in the respiratory tract are activated by nicotine via cigarette smoking.11,12 MCs are important play-ers in innate immunity and exert their biological effects by releasing active components via the degranulation process. MCs play an important role in atherosclerosis, by releasing proteinases and proinflammatory cytokines, and MC-deficient Apoe−/−KitW-sh/W-sh mice are resistant to atherosclerosis. Notably, Apoe−/−KitW-sh/W-sh mice have smaller atherosclerotic lesions than their wild-type controls,13 whereas systemic MC activation augments plaque size in Apoe−/− mice.14 In humans, plaque erosion, rupture, or hemorrhage is associated with MC activation.15–17 The present study determined the role of MCs in nicotine-induced atherosclerosis and its related mechanisms.

Materials and MethodsMaterials and Methods are available in the online-only Data Supplement.

© 2016 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.116.307264

Objective—Cigarette smoking is an independent risk factor for atherosclerosis. Nicotine, the addictive component of cigarettes, induces mast cell (MC) release and contributes to atherogenesis. The purpose of this study was to determine whether nicotine accelerates atherosclerosis through MC-mediated mechanisms and whether MC stabilizer prevents this pathological process.

Approach and Results—Nicotine administration increased the size of atherosclerotic lesions in apolipoprotein E–deficient (Apoe−/−) mice fed a fat-enriched diet. This was accompanied by enhanced intraplaque macrophage content and lipid deposition but reduced collagen and smooth muscle cell contents. MC deficiency in Apoe−/− mice (Apoe−/−KitW-sh/W-sh) diminished nicotine-induced atherosclerosis. Nicotine activated bone marrow–derived MCs in vitro, which was inhibited by a MC stabilizer disodium cromoglycate or a nonselective nicotinic acetylcholine receptor blocker mecamylamine. Further investigation revealed that α7 nicotinic acetylcholine receptor was a target for nicotine activation in MCs. Nicotine did not change atherosclerotic lesion size of Apoe−/−KitW-sh/W-sh mice reconstituted with MCs from Apoe−/−α7nAChR−/− animals.

Conclusions—Activation of α7 nicotinic acetylcholine receptor on MCs is a mechanism by which nicotine enhances atherosclerosis. (Arterioscler Thromb Vasc Biol. 2017;37:00-00. DOI: 10.1161/ATVBAHA.116.307264.)

Key Words: apolipoprotein E ◼ atherosclerosis ◼ hypercholesterolemia ◼ mast cell ◼ nicotine

Received on: October 14, 2016; final version accepted on: October 23, 2016.From the Department of Cardiology, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, PR China (C.W., H.C., W.Z.,

Y.X., L-L.Z., L.Z., X,-B.L., Z..Z., J.Z., J.J., M.X., H.Y., X.H., J.W.); Cardiovascular Key Laboratory of Zhejiang Province, Hangzhou, PR China (C.W., H.C., W.Z., Y.X., M.-F.L., L.-L.Z., F.Y., L.Z., X.-B.L., Z.Z., J.Z., J.J., M.X., H.Y., X.H., J.W.); and Saha Cardiovascular Research Center, Departments of Physiology, University of Kentucky, Lexington (H.L.).

*These authors contributed equally to this article.The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.116.307264/-/DC1.Correspondence to Jian’an Wang, MD, PhD, Department of Cardiology, Provincial Key Lab of Cardiovascular Research, Second Affiliated Hospital,

Zhejiang University College of Medicine, Hangzhou 310009, China. E-mail [email protected]

Nicotine Accelerates Atherosclerosis in Apolipoprotein E–Deficient Mice by Activating α7 Nicotinic

Acetylcholine Receptor on Mast CellsChen Wang,* Han Chen,* Wei Zhu, Yinchuan Xu, Ming-Fei Liu, Lian-Lian Zhu, Fan Yang, Ling Zhang, Xian-Bao Liu, Zhiwei Zhong, Jing Zhao, Jun Jiang, Meixiang Xiang, Hong Yu,

Xinyang Hu, Hong Lu, Jian’an Wang

Original Research

mailto: [email protected]

2 Arterioscler Thromb Vasc Biol January 2017

ResultsNicotine Increases Atherosclerotic Plaque Size and Changes Lesion ContentApoe−/− mice fed a fat-enriched diet for 12 weeks exhibited typical atherosclerotic lesions not found in those fed a normal diet or wild-type mice fed the fat-enriched diet. Chronic nico-tine administration via drinking water accelerates the athero-sclerotic process.9,10 We found that Apoe−/− mice administered nicotine (delivered via drinking water) showed a significant increase in serum cotinine levels (nicotine versus water alone: 283±33 versus 5±3 ng/mL; P<0.01; Figure IA in the online-only Data Supplement). It has been reported that nicotine administered at 100 µg/mL in drinking water yields plasma levels in mice similar to those observed in moderate smokers; average serum cotinine level in moderate smokers is 266 ng/mL,9 which is similar to what we found in the present study.

No differences were found in systolic blood pressure, body weight, and lipid profiles between vehicle and nicotine groups (Table I in the online-only Data Supplement). Nicotine administration resulted in significantly increased atheroscle-rotic lesions in the aorta (nicotine versus vehicle: 24.0±4.6 versus 13.2±1.5%; P<0.05; Figure 1A and 1C). To assess regional differences of the lesions, lesion areas were compared for the aortic arch (nicotine versus vehicle: 42.1±3.0 versus 27.1±2.6%; P<0.01; Figure 1A and 1D), descending thoracic region (nicotine versus vehicle: 14.7±3.0 versus 5.2±0.8%; P<0.05; Figure 1A and 1E) and abdominal region (nicotine versus vehicle: 11.2±1.8 versus 5.9±1%; P<0.05; Figure 1A and 1F). Lesion areas in the aortic root was also increased in the nicotine group (nicotine versus vehicle: 21.8±1.8×104 versus 12.6±2.2×104 μm2; P<0.05; Figure 1B and 1G).

Nicotine administration significantly altered the composi-tion of atherosclerotic lesions. Both collagen and smooth mus-cle cell (SMC) contents were reduced in the nicotine group (collagen, 23.4±2.0%; SMC, 6.6±2.1%) after a 12-week administration period, compared with the vehicle group (collagen, 34.6±3.2%, P<0.01; SMC, 11.6±1.0%, P<0.01; Figure 1B, 1H, and 1I). However, macrophage content and lipid levels increased after 12 weeks of nicotine exposure (macrophage, 45.2±8.3%; lipid, 31.0±2.8%), compared with the vehicle group (macrophage, 21.2±4.5%, P<0.01; lipid, 11.4±1.3%; P<0.01; Figure 1B, 1J, and 1K).

Cross sectioning also showed that nicotine administra-tion increased lesion areas in the aortic arch (nicotine versus

vehicle: 16.7±0.9×104 versus 11.3±0.7×104 μm2; P<0.01; Figure IIA and IIF in the online-only Data Supplement). Necrotic core ratios were altered by nicotine intervention (nicotine versus vehicle: 35.8±2.1 versus 17.0±2.1%; P<0.01; Figure IIA and IIG in the online-only Data Supplement). Collagen and SMC contents in the aortic arch were decreased in the nicotine group (collagen, 24.4±1.9%; SMC, 8.3±1.0%) compared with the vehicle group (collagen, 38.8±1.8%, SMC, 16.3±1.0%; all P<0.01; Figure IIA through IIC in the online-only Data Supplement). In contrast, macrophage and lipid contents in the aortic arch were increased after 12 weeks of nicotine administration (macrophage, 48.3±4.5%; lipid, 35.1±2.1%) compared with the vehicle group (macrophage, 23.3±2.3%; lipid, 13.8±1.2%; all P<0.01; Figure IID and IIE in the online-only Data Supplement).

Serum levels of the proinflammatory cytokines, such as interleukin (IL)-6, tumor necrosis factor-α, interferon-γ, and IL-4, were significantly increased in the nicotine group, com-pared with the vehicle counterparts, whereas the anti-inflam-matory cytokine IL-10 showed similar levels in the 2 groups (Supplementary Figure IB through IF in the online-only Data Supplement).

Nicotine Increases MC Activation in Apoe−/− MiceSignificantly increased MC accumulation (nicotine versus vehicle: 20.4±1.2 versus 13.9±0.7 cells/section; P<0.01) and degranulation ratio (nicotine versus vehicle: 37.0±4.0 versus 14.8±3.4%; P<0.01; Figure 2A through 2C) were found in the aortic adventitia of the aortic root region. MC activity was assessed via serum levels of histamine and chymase, 2 major components of MC granules, by ELISA. Serum histamine levels were higher in mice administered nicotine (P<0.05; Figure 2D). Similarly, nicotine significantly increased serum chymase amounts (P<0.01; Figure 2E). However, no signifi-cant difference was observed in serum tryptase levels between the 2 groups (Figure 2F).

MC Deficiency Attenuates Nicotine-Induced Plaque DevelopmentMC activation promotes plaque progression,14 and MC defi-ciency attenuates atherosclerosis in mice.18 To further validate the effects of MC activation on nicotine-induced atheroscle-rotic formation, we assessed whether administration of nic-otine would affect atherosclerotic lesions in MC-deficient Apoe−/−Kitw-sh/w-sh mice. After 12 weeks of the same fat-enriched diet and nicotine administration, no differences were observed in systolic blood pressure and body weight (Table I in the online-only Data Supplement), as well as lipid profile (data not shown) and serum cotinine levels (Figure IA in the online-only Data Supplement), between Apoe−/−Kitw-sh/w-sh and Apoe−/− mice. Apoe−/−Kitw-sh/w-sh mice displayed decreased ath-erosclerotic lesion size, as measured in both the entire aorta by en face method (Figure 3A and 3C) and the aortic root by cross sectioning (Figure 3B and 3G).

We analyzed lesions in different aortic regions. Lesions in the aortic arch (Apoe−/−Kitw-sh/w-sh versus Apoe−/−: 28.3±3.0 versus 42.1±3.6%; P<0.05; Figure 3A and 3D) and descend-ing thoracic aorta (Apoe−/−Kitw-sh/w-sh versus Apoe−/−: 10.1±1.5

Nonstandard Abbreviations and Acronyms

α-BTX α-bungarotoxin

α-SMA α-smooth muscle actin

BMMC bone marrow–derived mast cell

DSCG disodium cromoglycate

IL interleukin

JAK Janus kinase

MC mast cells

nAChR nicotinic acetylcholine receptor

SMC smooth muscle cell

STAT signal transducer and activator of transcription

Wang et al Nicotine Accelerates Atherosclerosis 3

Figure 1. Nicotine increases atherosclerotic lesion size and changes lesional compositions in apolipoprotein E–deficient (Apoe−/−) mice. A, Lesion area was determined in en face aorta with Oil red O staining; aortas are from vehicle- and nicotine-administered mice fed a fat-enriched diet for 12 wk. White lines divide the aortas into the aortic arch, descending thoracic region, and abdominal region. B, Cross sections of the aortic root were stained with hematoxylin and eosin (HE); Sirius Red was stained to visualize collagen and examined by polarization microscopy; α-smooth muscle actin (α-SMA) is a marker of smooth muscle cells (SMCs); Mac-3 is a marker of macrophages; Oil Red O staining revealed aortic root lipid deposit (red-stained areas indicate lipid-rich area). C–F, Quantification of Oil Red O–positive areas in the entire aorta, aortic arch, descending thoracic aorta, and abdominal aorta, respectively. G, Lesion size of the aortic root (n=7/group). H–K, Quantification of Sirius Red–, α-SMA–, Mac-3–, and Oil Red O–stained areas of lesions in the aortic root. P<0.05 and P<0.01 by Student t test. Inverted triangles represent individual data, circles are mean, and error bars are SEM.


versus 15.3±1.4%; P<0.05; Figure 3A and 3E) were decreased in Apoe−/−Kitw-sh/w-sh mice. Differences of lesion areas in the abdominal aorta were not statistically significant between the 2 genotypes (Figure 3A and 3F).

Lesion areas in the aortic root were also decreased in the Apoe−/−Kitw-sh/w-sh group (Apoe−/−Kitw-sh/w-sh versus Apoe−/−: 14.3±2.3×104 versus 20.7±2.7×104 μm2; P<0.05; Figure 3B and 3G). Apoe−/−Kitw-sh/w-sh mice had higher col-lagen (37.3±4.5%; Figure 3B and 3H) and SMC content (17.8±1.2%; Figure 3B and 3I) but displayed less macrophage content (25.7±2.5%; Figure 3B and 3J) and lipid deposit (20.3±1.3%; Figure 3B and 3K) compared with Apoe−/− mice (collagen, 22.9±2%, P<0.05; SMC, 9.0±1.3%, P<0.01; mac-rophage, 38.3±4.7%, P<0.05; lipid, 32.4±3.6%, P<0.01).

Similarly, MC deficiency resulted in decreased lesion areas in the aortic arch (Apoe−/−Kitw-sh/w-sh versus Apoe−/−: 13.4±0.8×104 versus 17.6±1.2×104 μm2; P<0.05; Figure IIIA and IIIF in the online-only Data Supplement). Necrotic core ratios were decreased in the Apoe−/−Kitw-sh/w-sh mice (Apoe−/−Kitw-

sh/w-sh versus Apoe−/−: 19.3±1.5 versus 32.7±1.9%; P<0.01; Supplementary Figure IIIA and IIIG in the online-only Data Supplement). Collagen levels and SMC contents in the aortic arch were better preserved in the Apoe−/−Kitw-sh/w-sh mice com-pared with the Apoe−/− mice. Collagen levels were 32.9±2.2 and 21.9±2.1% in the Apoe−/−Kitw-sh/w-sh and Apoe−/− groups, respectively (P<0.01; Figure IIIA and IIIB in the online-only Data Supplement). SMC contents in the Apoe−/−Kitw-sh/w-sh and Apoe−/− groups were 15.9±1.1 and 9.5±1.0%, respectively

Figure 2. Nicotine increases mast cell (MC) activation in apolipoprotein E–deficient (Apoe−/−) mice. A, Representative cross sections of the aortic root were stained with toluidine blue for MCs. Violet-stained cells are MCs distributed in the adventitia of the aortic root. Non-degranulated MCs show smooth edges, whereas the degranulated ones are surrounded by violet-stained granules. B, Count of MC num-ber in the adventitia. C, Quantification of MC number and degranulation ratio in the adventitia of the aortic root; degranulation ratio was calculated as (number of degranulated MCs)/(total MC number)×100%. P<0.05 by Student t test. Inverted triangles represent individual data, circles are mean, and error bars are SEM. D–F, MC granule contents of histamine, chymase, and tryptase measured by ELISA (n=8). Values are mean±SEM. P<0.05 and P<0.01 by Student t test.


Figure 3. Mast cell (MC) deficiency prevents nicotine-induced plaque formation and composition change. A, Representative en face Oil Red O–stained aortas. White lines divide the aortas into the aortic arch, descending thoracic aorta, and abdominal aorta. B, Representative cross sections of the aortic root were stained with hematoxylin and eosin (HE); Sirius Red, α-smooth muscle actin (α-SMA), Mac-3, Oil Red O, and toluidine blue staining (n=7). C–F, Quantification of Oil Red O–positive areas of the entire aorta, aortic arch, descending thoracic aorta, and abdominal aorta (n=7). G, Lesion size of the aortic root. H–K, Quantification of Sirius Red–, α-SMA–, Mac-3–, and Oil Red O–stained areas in the aortic root. P<0.05 and P<0.01 by Student t test (n=7). L and M, Quantification of MC number and degranulation ratio in the adventitia of the aorta. P<0.05 and P<0.01 by Student t test. Inverted triangles represent individual data, circles are mean, and error bars are SEM.


Figure 4. Nicotine activates bone marrow–derived mast cells (MCs) via α7 nicotinic acetylcholine receptor (α7nAChR). A and B, Super-natants containing β-hexosaminidase and histamine released from bone marrow–derived mast cells (BMMCs). Tyrode buffer was used as negative control, and C48/80 was used as positive control. Other groups included nicotine, nicotine+the nonselective nAChR blocker mecamylamine (pretreatment for 30 min), and nicotine+MC stabilizer disodium cromoglycate (DSCG; pretreatment for 30 min). At 0.5 h, 1 h, and 2 h, supernatants were collected for β-hexosaminidase and histamine analysis. C, Expression levels of α3nAChR, (Continued )


(P<0.01; Figure IIIA and IIIC in the online-only Data Supplement).

Meanwhile, macrophage and lipid contents in the aortic arch were decreased in the Apoe−/−Kitw-sh/w-sh mice compared with Apoe−/− mice. Macrophage contents were 23.0±2.1 and 46.8±2.6%, respectively, in the Apoe−/−Kitw-sh/w-sh and Apoe−/− groups (P<0.01; Supplementary Figure IIIA and IIID in the online-only Data Supplement), and lipid contents were 22.9±1.3 and 37.2±3.2%, respectively (P<0.01; Figure IIIA and IIIE in the online-only Data Supplement). Of note, Apoe−/−Kitw-sh/w-sh mice administered nicotine had no toluidine blue–positive MCs in the aortic adventitia (Figure 3B, 3L, and 3M), demonstrating that genetic deficiency of MCs decreased plaque burden and prevented plaque composition alteration. Moreover, in Apoe−/−Kitw-sh/w-sh mice, decreased serum hista-mine and chymase levels were observed compared with the Apoe−/− mice. Serum levels of proinflammatory cytokines such as IL-6, tumor necrosis factor-α, interferon-γ, and IL-4 were also significantly lower in the Apoe−/−Kitw-sh/w-sh mice compared with the Apoe−/− mice, whereas serum tryptase and IL-10 levels and cotinine (nicotine metabolite) amounts showed no significant differences between the 2 genotypes (Figure I in the online-only Data Supplement).

Nicotine Activates MCs in the Aortic Adventitia via α7 Nicotinic Acetylcholine Receptor to Promote AtherogenesisIgE is considered the most effective activator of MCs. However, non–IgE-mediated MC activation has been doc-umented, with nicotine representing one critical factor in MC activation.19 In addition, nicotine-induced activation of MCs was demonstrated in guttate psoriasis lesions and stenotic aortic valves in humans.20,21 Administration of a nonselective nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine inhibits nicotine-induced MC activation in vitro.20–22 To assess mechanisms by which nicotine stimulates MCs, bone marrow–derived MCs (BMMCs) were isolated from Apoe−/− mice and differ-entiated into mature MCs. The purity of BMMCs was 93.8±0.01% as assessed by toluidine blue staining and 94.6±0.03% by tryptase immunostaining (Figure IJ and IK in the online-only Data Supplement). β-Hexosaminidase release and histamine release assays were performed to assess degranulation ratios.23,24 Supernatants of cultured BMMCs were collected after nicotine stimulation for 0.5, 1, and 2 hours, respectively. BMMCs released ≤48.1±0.5% of total β-hexosaminidase content (Figure 4A) and 50.3±0.5% of total histamine content (Figure 4B) in a time-dependent manner. Pretreatment of BMMCs with mecamylamine and a MC stabilizer disodium cromoglycate (DSCG) for 30 minutes significantly attenuated nicotine-induced β-hexosaminidase (30.0±0.7%, P<0.01; 31.1±0.7%, P<0.01,

respectively) and histamine (29.6±0.3%, P<0.01; 35.0±1.1%, P<0.01, respectively) release compared with nicotine-incu-bated MCs (Figure 4A and 4B).

To identify the nAChR subunits involved in nicotine-induced MC activation, RT-PCR was performed to quantify the gene expression levels of α3nAChR, α7nAChR, and α9nAChR. α7nAChR mRNA were increased by >100-fold after nicotine stimulation compared with the control group (P<0.01), whereas no significant differences were observed in α3nAChR and α9nAChR mRNA levels (Figure 4C). These findings suggest that α7nAChR is an important mediator in nicotine-induced MC activation.

Preincubation with a selective α7nAChR blocker α-bungarotoxin (α-BTX) or genetic suppression of α7nAChR prevented nicotine-induced MC activation, as validated by β-hexosaminidase and histamine release assays (Figure 4D and 4E). Western blot demonstrated that α7nAChR protein levels were significantly higher after 12 hours of nicotine stimulation and attenuated by the nAChR nonselective blocker mecamylamine and the α7nAChR-selective blocker α-BTX. Taken together, these results indicate that nicotine activates BMMCs through α7nAChR (Figure 4F and 4G).

Nicotine Activates MCs via the Janus Kinase/Signal Transducer and Activator of Transcription Pathway and Influences Foam Cell Formation In VitroThe Janus kinase/signal transducer and activator of tran-scription (JAK-STAT) pathway mediates important responses in immune cells. Although there are 4 members in the mammalian JAK family, JAK2 is best studied in vas-cular and neuronal tissues. Previous studies have demon-strated that α7nAChR-JAK2–mediated signaling plays a central role in nicotine-induced neuroprotection.25 BMMCs exposed to nicotine for 2 hours showed phosphorylation of JAK2, STAT3, and Akt, all of which could be restored by preincubation with the nAChR nonselective blocker mecamylamine or the α7nAChR-selective blocker α-BTX (Figure 5A through 5D). These results demonstrated that nicotine-induced MC activation was mediated through JAK2 phosphorylation and subsequent activation of STAT3 and Akt, leading to MC degranulation and enhanced cyto-kine production.19,26,27

To assess the effects of nicotine-induced MC activation on macrophages, peritoneal macrophages were incubated with conditional supernatants from MCs. RT-PCR demonstrated that stimulation with supernatants from nicotine-activated MCs resulted in decreased expression levels of ATP-binding membrane cassette transporter A1 and ATP-binding mem-brane cassette transporter G1 (Figure 5E and 5F). Meanwhile, CD36 levels (Figure 5G) and the amounts of proinflammatory cytokines IL-6, monocyte chemotactic protein-1, interferon-γ,

Figure 4 Continued. α7nAChR, and α9nAChR mRNA in MCs incubated with Tyrode buffer, nicotine, or nicotine+mecamylamine (pre-treatment for 30 min) for 2 h. D and E, To confirm whether α7nAChR is a target of nicotine for MC activation, supernatants containing β-hexosaminidase and histamine released from BMMCs were incubated with Tyrode buffer, nicotine, or nicotine+α-bungarotoxin (α-BTX; pretreatment for 30 min), or α7nAChR−/−BMMCs were incubated with nicotine for 0.5 h, 1 h, and 2 h, respectively. Then, supernatants were collected for β-hexosaminidase and histamine assessments. F and G, α7nAChR protein expression levels in BMMCs incubated with Tyrode buffer, nicotine, nicotine+mecamylamine (pretreatment for 30 min), or nicotine+α-BTX (pretreatment for 30 min) incubated for 12 h. Protein levels were assessed by Western blotting; quantitative analysis of α7nAChR expression was performed by 1-way ANOVA.


Figure 5. Nicotine induces mast cell (MC) degranulation via Janus kinase (JAK)/signal transducer and activator of transcription (STAT) cell signaling in vitro, and supernatants from degranulated MCs enhance the expression levels of inflammatory cytokines, CD36, and matrix metalloproteinases (MMPs), decreasing ATP-binding membrane cassette transporter A1 (ABCA1) and ATP-binding membrane (Continued )


and tumor necrosis factor-α (Figure 5H through 5K) were increased in macrophages. IL-10 levels did not change (Figure 5L). Western blotting demonstrated that matrix metal-loproteinase-2 and matrix metalloproteinase-9, which cause collagen degradation in atherosclerotic lesions, were also increased by incubation with supernatants from nicotine-acti-vated MCs (Figure 5M through 5O).

Similarly, in vitro foam cell formation assay demonstrated that MC supernatants strongly promoted foam cell forma-tion (63.3±6.3%) compared with the control (22.2±4.3%, P<0.01), nicotine (40.1±4.3%, P<0.05), and nicotine with DSCG (25.3±5.3%, P<0.01) groups (Figure VA and VB in the online-only Data Supplement). Cholesterol efflux assay showed that supernatants from nicotine-triggered MCs sig-nificantly impaired the cholesterol efflux capability of mac-rophages (Supplementary Figure VC in the online-only Data Supplement).

Genetic Deficiency of α7nAChR in MCs or Pharmacological Stabilization of MCs Attenuates Nicotine-Induced AtherosclerosisTo test the hypothesis that nicotine activates MCs via α7nAChR, thereby contributing to atherosclerotic development, we per-formed experiments in Apoe−/− mice with α7nAChR knockout MCs. MC-deficient Apoe−/−KitW-sh/W-sh mice were reconstituted with 1×107 cultured BMMCs from Apoe−/−α7nAChR−/− mice and Apoe−/− mice.28 Because nicotine-induced MC activation could be alleviated by treatment with the MC stabilizer DSCG in vitro, we assessed whether the MC stabilizer and specific depletion of α7nAChR in MCs of Apoe−/− mice could alleviate nicotine-enhanced atherosclerosis in vivo.

After 12 weeks of fat-enriched diet feeding and nico-tine administration, no differences were observed in sys-tolic blood pressure and body weight (Table I in the online-only Data Supplement) and lipid profile (data not shown) and serum cotinine levels (Figure IA in the online-only Data Supplement) among the 4 groups. Apoe−/−KitW-

sh/W-sh+α7nAChR−/−MC+nicotine and Apoe−/−+nicotine+DSCG groups showed protection against nicotine-induced atherogen-esis. In these groups, atherosclerotic lesions in the aorta were significantly decreased compared with Apoe−/−+nicotine and Apoe−/−KitW-sh/W-sh+WT MC+nicotine groups (Figure 6A and 6C). Regional analysis of lesions in the aortic arch, thoracic aorta, and abdominal aorta revealed similar results (Figure 6A and 6C through 6F).

Similarly, the Apoe−/−KitW-sh/W-sh+α7nAChR−/−MC+nicotine and Apoe−/− + nicotine +DSCG groups showed attenuated lesions in the aortic root compared with the Apoe−/−+nicotine and Apoe−/−KitW-sh/W-sh+WT MC+nicotine groups (Figure 6B and 6G). Collagen and SMC contents were better preserved in the Apoe−/−KitW-sh/W-sh+α7nAChR−/−BMMCs+nicotine mice

and the Apoe−/−+nicotine+DSCG mice, whereas lipid and macrophage contents were significantly decreased (Figure 6B and 6H through 6K). Lesions in the aortic arch showed simi-lar changes. Apoe−/−+nicotine and Apoe−/−KitW-sh/W-sh+WT MC+nicotine groups showed larger lesion areas, enhanced necrotic area ratios, and increased macrophage and lipid contents but decreased SMC and collagen contents, com-pared with Apoe−/−KitW-sh/W-sh+α7nAChR−/−MC+nicotine and Apoe−/−+nicotine+DSCG groups (Figure IVA through IVG in the online-only Data Supplement).

The number of MCs accumulated in the aortic adventitia was increased in the Apoe−/−+nicotine group compared with the other groups. MC degranulation ratios in the Apoe−/−KitW-

sh/W-sh+α7nAChR−/−MC+nicotine (20.9±2.1%, P<0.05) and Apoe−/−KitW-sh/W-sh+nicotine+DSCG (14.7±3.4%, P<0.05) groups were significantly lower than the values obtained for the Apoe−/−+nicotine (37.0±4.0%) and Apoe−/−KitW-sh/W-

sh+WTMC+nicotine (32.8±1.8%) groups (Figure 6B, 6L, and 6M).

DiscussionNicotine enhances atherogenesis in Apoe−/− mice fed a fat-enriched diet. Nicotine increases MC number and activates MCs in the adventitia of the atherosclerosis-prone regions by releasing proinflammatory cytokines and proteinases.19,29–31 Nicotine increases macrophage content and lipid deposition but diminishes SMC and collagen contents in atherosclerotic lesions.

Although the association of nicotine with atheroscle-rosis has been recognized for decades, no approach effec-tively protects individuals exposed to cigarette smoking from developing atherosclerosis. More than 4000 chemicals have been found in cigarettes, but nicotine is considered the major contributor to cigarette addiction and adverse cardiovascular events. Nicotine in cigarettes can be rapidly absorbed through the alveoli and small airways, entering the circulation through the pulmonary fluid. Similarly, nicotine could be absorbed through the oral mucosa and small intestine. Nicotine was found to promote atherosclerosis, nonrheumatic aortic steno-sis, and psoriasis, and increased activation and accumulation of MCs are found in lesions of these diseases.10,20,21

The role of MCs in atherosclerosis has been identified recently. Both increased quantity and activation of MCs are observed in atherosclerotic plaques and the adventitia of human aortas and coronary arteries. MC progenitors originate from hematopoietic stem cells, which circulate throughout the body, eventually homing in tissues; maturing human MCs also exhibit heterogeneity and are classified by serine prote-ase content as tryptase-only MC, chymase-only MC, and both tryptase- and chymase-positive MC. However, no studies have assessed the effects of MC subgroups on atherosclerosis.32

Figure 5 Continued. cassette transporter G1 (ABCG1) expression in macrophages. A, Bone marrow–derived mast cells (BMMCs) were exposed to 100 μg/mL nicotine for 2 h. Phosphorylation levels of JAK2, STAT3, and Akt were determined by Western blotting, and β-actin was used as a loading control. B–D, Quantitative analysis of JAK2, STAT3, and Akt phosphorylation levels were analyzed with 1-way ANOVA. E–L, mRNA expression levels of ABCA1 and ABCG1, CD36, interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-10 in peritoneal macrophages incubated with conditional supernatants were assessed by RT-PCR. M, MMP-2 and MMP-9 expression levels in peritoneal macrophages incubated with conditional supernatants col-lected from the MC activation assay were assessed by Western blotting. N and O, Quantitative analysis of MMP-2 and MMP-9 expression levels of peritoneal macrophages. Values are mean±SEM.


Figure 6 α7nAChR−/− bone marrow–derived mast cell (BMMC) reconstitution or administration of a mast cell (MC) stabilizer disodium cromoglycate (DSCG) alleviates nicotine-induced atherogenesis and composition change. A, Representative en face Oil Red O–stained aortas from Apoe−/− mice, Apoe−/−Kitw-sh/w-sh mice with α7nAChR−/−MC reconstitution, Apoe−/− mice with a MC stabilizer DSCG administra-tion, and Apoe−/−Kitw-sh/w-sh mice with WT MC reconstitution. White lines divide the aortas into the aortic arch, descending thoracic aorta, and abdominal aorta. B, Cross sections of the aortic root stained with hematoxylin and eosin (HE), Sirius Red, Oil Red O, toluidine blue, α-smooth muscle actin (α-SMA), Mac-3. C–F, Quantification of Oil Red O–positive areas of the entire aorta, aortic arch, descending thoracic aorta, and abdominal aorta. P<0.05 and P<0.01 obtained by 1-way ANOVA (n=7). G, Quantification of lesion size in the aortic root; P<0.05 and P<0.01 as assessed by 1-way ANOVA (n=7). H–K, Quantification of Sirius Red, α-SMA, Mac-3, (Continued )


Mature MCs achieve their biological activity via degranula-tion, a process whereby cells release their intragranular con-tents into the extracellular space. In the lungs and skin of smokers, MC number and activation are increased.11,12 In this study, we focused on aortic adventitial MCs for a morphologi-cal evaluation of degranulation. It should be pointed out that the serum degranulation effect comes not only from MCs in the arteries but also from those in other locations such as the respiratory epithelium or skin.

Newly released clinical data from the Multi-Ethnic Study of Atherosclerosis showed that persistent asthmatics have a higher incidence of cardiovascular diseases com-pared with nonasthmatics, suggesting the possibility that MCs act as a mediator for respiratory and cardiovascular diseases.33 Herein, we assessed the role of MCs in nicotine-induced atherosclerosis and observed the effects of MC defi-ciency or pharmacological stabilization on nicotine-induced atherosclerosis.

Supernatants from conditioned MC cultures promoted foam cell formation of macrophages. The underlying mecha-nism may involve decreased mRNA expression of macrophage ATP-binding cassette transporters, ATP-binding membrane cassette transporter A1 and ATP-binding membrane cassette transporter G1, and increased mRNA expression of scaven-ger receptor CD36, which suggests both increased cholesterol uptake and impaired cholesterol efflux of the macrophages.34,35 The expression levels of a variety of proinflammatory cyto-kines, including interferon-γ, IL-6, monocyte chemotactic protein-1, and tumor necrosis factor-α, were increased in activated peritoneal macrophages incubated with supernatants from conditioned MCs. Matrix metalloproteinase-2 and matrix metalloproteinase-9, the most potent proteinases for collagen degradation, also showed increased amounts in macrophages incubated with conditional MC supernatants. Increased lipid accumulation, matrix degradation, and inflammation all con-tribute to accelerated atherogenesis.36

nAChRs are mainly expressed in the central nervous sys-tem. However, recent studies reported their presence and bio-logical activities in a variety of non-neuronal cells, including MCs.37 Given that nicotine can dramatically increase α7nAChR expression in MCs, the relationship between α7nAChR and nicotine-induced MC activation was assessed. Apoe−/−KitW-

sh/W-sh mice do not develop mature MCs because of an inversion mutation in the Kit gene promoter region. As a consequence, bone marrow hematopoietic stem cells in these mice are not able to differentiate into mature MCs.38 MC-deficient Apoe−/−KitW-sh/

W-sh mice were reconstituted with cultured BMMCs from Apoe−

/−α7nAChR−/− mice to generate Apoe−/− mice with MC-specific deficiency of α7nAChR.13 α7nAChR-deficient MCs showed protection from nicotine-induced atherosclerosis. Similarly, MCs incubated with either a nonselective nAChRs blocker mecamylamine or a α7nAChR-selective blocker α-BTX had no nicotine-induced MC activation. Similarly, a MC stabilizer DSCG attenuated nicotine-induced MC activation. Our in vivo

study showed protective effects of DSCG, as evidenced by decreased plaque burden and improved preservation of plaque components. These data strongly suggest a potential therapeu-tic application of DSCG in individuals exposed to cigarette smoke. Although protection from nicotine-enhanced athero-sclerosis by DSCG treatment is suggestive, it is not known whether DSCG in this model affects atherosclerosis indepen-dent of nicotine-induced MC activation.

Phosphorylation levels of JAK2, STAT3, and Akt were increased in MCs after a 2-hour nicotine stimulation but inhibited by the nonselective nAChR blocker mecamylamine and the α7nAChR-specific blocker α-BTX. These results suggest that nicotine activates α7nAChR and exerts its effects through the downstream JAK2/STAT3 and PI3K/Akt signaling pathways. However, Barua et al39 have reported that the histamine–TLR–COX-2 axis is also involved in MC activation by cigarette smoke. Therefore, the histamine–TLR–COX-2 axis may also be a potential mechanism, which needs further studies.

The present study showed that, in vitro and in vivo, deple-tion and stabilization of MCs could not fully reverse nicotine-induced foam cell formation and atherosclerosis. Therefore, activation of MCs may represent only one of the mechanisms by which nicotine accelerates atherosclerosis. According to previous studies, other mechanisms may include (1) direct enhancement of CD36 expression on macrophages; (2) induc-tion of angiogenesis and increased neovascularization; and (3) increased oxidative stress.9,40,41 In this case, MC stabilization therapy may not be enough in smokers experiencing athero-sclerotic diseases.

In summary, we have demonstrated that MCs play an important role in nicotine-enhanced atherogenesis in Apoe−/− mice. Nicotine activated MCs in the aortic adventitia via an α7nAChR-mediated mechanism. Nicotine induces JAK2 phosphorylation, triggers STAT3 phosphorylation and PI3K-mediated activation of Akt, which contribute to MC degranula-tion and proinflammatory cytokine production and release.19,42 MC stabilization or nAChR depletion attenuates nicotine-induced atherosclerosis. These findings provide a potential therapeutic strategy for reducing atherosclerotic plaque pro-gression and preventing cardiovascular events in individuals exposed to cigarette smoke.

Sources of FundingThis work was supported by the National Basic Research Program of China (973 Program, 2014CB965103), the National High-tech R&D 863 Program (no. 2013AA020101 for X. Hu, no. 2015AA020922 for X. Liu), grants from National Natural Science Foundation of China (no. 81320108003 and 31371498 for J. Wang, no. 81370247 for X. Hu, no. 81570233 for X. Liu, no. 81500334 for H. Chen, no. 81470384 and 81270179 for M. Xiang, no. 81573641 for L. Zhu, no. 81100141 for J. Jiang), the Qianjiang Talents Project of Science and Technology Department of Zhejiang Province (no. 2013R10032 for H. Chen and no. LY16H280003 for L. Zhu), the Natural Science Foundation of Zhejiang Province (no. LQ16H020004 for H. Chen),

Figure 6 Continued. and Oil Red O staining of lesions in the aortic root; P<0.05 and P<0.01 as assessed by 1-way ANOVA (n=7). L and M, Quantification of MC number and degranulation ratio in the adventitia of the aortic root; P<0.05 and P<0.01 as assessed by 1-way ANOVA (n=7). Inverted triangles represent individual data, circles are mean, and error bars are SEM. α7nAChR indicates α7 nicotinic acetylcholine receptor; and WT, wild type.


and the Fundamental Research Funds for the Central Universities (no. 2016XZZX002-03 for X. Hu).

DisclosuresNone.

References 1. Weber C, Zernecke A, Libby P. The multifaceted contributions of leu-

kocyte subsets to atherosclerosis: lessons from mouse models. Nat Rev Immunol. 2008;8:802–815. doi: 10.1038/nri2415.

2. Libby P. Inflammation in atherosclerosis. Nature. 2002;420:868–874. doi: 10.1038/nature01323.

3. Ambrose JA, Barua RS. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J Am Coll Cardiol. 2004;43:1731–1737. doi: 10.1016/j.jacc.2003.12.047.

4. Ezzati M, Henley SJ, Thun MJ, Lopez AD. Role of smoking in global and regional cardiovascular mortality. Circulation. 2005;112:489–497. doi: 10.1161/CIRCULATIONAHA.104.521708.

5. Barnoya J, Glantz S. Association of the California tobacco control pro-gram with declines in lung cancer incidence. Cancer Causes Control. 2004;15:689–695. doi: 10.1023/B:CACO.0000036187.13805.30.

6. Crooks PA, Dwoskin LP. Contribution of CNS nicotine metabolites to the neuropharmacological effects of nicotine and tobacco smoking. Biochem Pharmacol. 1997;54:743–753.

7. Glantz SA, Parmley WW. Passive smoking and heart disease. Mechanisms and risk. JAMA. 1995;273:1047–1053.

8. He J, Vupputuri S, Allen K, Prerost MR, Hughes J, Whelton PK. Passive smoking and the risk of coronary heart disease–a meta-analysis of epi-demiologic studies. N Engl J Med. 1999;340:920–926. doi: 10.1056/NEJM199903253401204.

9. Heeschen C, Jang JJ, Weis M, Pathak A, Kaji S, Hu RS, Tsao PS, Johnson FL, Cooke JP. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nat Med. 2001;7:833–839. doi: 10.1038/89961.

10. Santanam N, Thornhill BA, Lau JK, Crabtree CM, Cook CR, Brown KC, Dasgupta P. Nicotinic acetylcholine receptor signaling in ath-erogenesis. Atherosclerosis. 2012;225:264–273. doi: 10.1016/j.atherosclerosis.2012.07.041.

11. Erbagci Z, Erkiliç S. Can smoking and/or occupational UV exposure have any role in the development of the morpheaform basal cell car-cinoma? A critical role for peritumoral mast cells. Int J Dermatol. 2002;41:275–278.

12. Pesci A, Rossi GA, Bertorelli G, Aufiero A, Zanon P, Olivieri D. Mast cells in the airway lumen and bronchial mucosa of patients with chronic bron-chitis. Am J Respir Crit Care Med. 1994;149:1311–1316. doi: 10.1164/ajrccm.149.5.8173772.

13. Sun J, Sukhova GK, Wolters PJ, Yang M, Kitamoto S, Libby P, MacFarlane LA, Mallen-St Clair J, Shi GP. Mast cells promote atherosclerosis by releasing proinflammatory cytokines. Nat Med. 2007;13:719–724. doi: 10.1038/nm1601.

14. Bot I, de Jager SC, Zernecke A, Lindstedt KA, van Berkel TJ, Weber C, Biessen EA. Perivascular mast cells promote atherogenesis and induce plaque destabilization in apolipoprotein E-deficient mice. Circulation. 2007;115:2516–2525. doi: 10.1161/CIRCULATIONAHA.106.660472.

15. Kovanen PT. Mast cells: multipotent local effector cells in athero-thrombosis. Immunol Rev. 2007;217:105–122. doi: 10.1111/j.1600- 065X.2007.00515.x.

16. Kovanen PT, Kaartinen M, Paavonen T. Infiltrates of activated mast cells at the site of coronary atheromatous erosion or rupture in myocardial infarc-tion. Circulation. 1995;92:1084–1088.

17. Virmani R, Burke AP, Farb A, Kolodgie FD. Pathology of the vulnerable plaque. J Am Coll Cardiol. 2006;47(suppl 8):C13–C18. doi: 10.1016/j.jacc.2005.10.065.

18. Shi GP, Bot I, Kovanen PT. Mast cells in human and experimental car-diometabolic diseases. Nat Rev Cardiol. 2015;12:643–658. doi: 10.1038/nrcardio.2015.117.

19. Gilfillan AM, Tkaczyk C. Integrated signalling pathways for mast-cell activation. Nat Rev Immunol. 2006;6:218–230. doi: 10.1038/nri1782.

20. Radosa J, Dyck W, Goerdt S, Kurzen H. The cholinergic system in guttate psoriasis with special reference to mast cells. Exp Dermatol. 2011;20:677–679. doi: 10.1111/j.1600-0625.2011.01283.x.

21. Helske S, Syväranta S, Kupari M, Lappalainen J, Laine M, Lommi J, Turto H, Mäyränpää M, Werkkala K, Kovanen PT, Lindstedt KA. Possible role for mast cell-derived cathepsin G in the adverse remodelling of stenotic aortic valves. Eur Heart J. 2006;27:1495–1504. doi: 10.1093/eurheartj/ehi706.

22. Wong D, Ogle CW. Chronic parenterally administered nicotine and stress- or ethanol-induced gastric mucosal damage in rats. Eur J Pharmacol. 1995;292:157–162.

23. Zhang J, Alcaide P, Liu L, Sun J, He A, Luscinskas FW, Shi GP. Regulation of endothelial cell adhesion molecule expression by mast cells, macro-phages, and neutrophils. PLoS One. 2011;6:e14525. doi: 10.1371/journal.pone.0014525.

24. Nagai K, Fukushima T, Oike H, Kobori M. High glucose increases the expression of proinflammatory cytokines and secretion of TNFα and β-hexosaminidase in human mast cells. Eur J Pharmacol. 2012;687:39–45. doi: 10.1016/j.ejphar.2012.04.038.

25. Marrero MB, Bencherif M. Convergence of alpha 7 nicotinic acetylcho-line receptor-activated pathways for anti-apoptosis and anti-inflammation: central role for JAK2 activation of STAT3 and NF-kappaB. Brain Res. 2009;1256:1–7. doi: 10.1016/j.brainres.2008.11.053.

26. Lee JH, Kim TH, Kim HS, Kim AR, Kim DK, Nam ST, Kim HW, Park YH, Her E, Park YM, Kim HS, Kim YM, Choi WS. An indoxyl com-pound 5-bromo-4-chloro-3-indolyl 1,3-diacetate, CAC-0982, suppresses activation of Fyn kinase in mast cells and IgE-mediated allergic responses in mice. Toxicol Appl Pharmacol. 2015;285:179–186. doi: 10.1016/j.taap.2015.04.009.

27. Lu Y, Yang JH, Li X, Hwangbo K, Hwang SL, Taketomi Y, Murakami M, Chang YC, Kim CH, Son JK, Chang HW. Emodin, a naturally occurring anthraquinone derivative, suppresses IgE-mediated anaphylactic reaction and mast cell activation. Biochem Pharmacol. 2011;82:1700–1708. doi: 10.1016/j.bcp.2011.08.022.

28. Sun J, Zhang J, Lindholt JS, Sukhova GK, Liu J, He A, Abrink M, Pejler G, Stevens RL, Thompson RW, Ennis TL, Gurish MF, Libby P, Shi GP. Critical role of mast cell chymase in mouse abdominal aor-tic aneurysm formation. Circulation. 2009;120:973–982. doi: 10.1161/CIRCULATIONAHA.109.849679.

29. Li JM, Cui TX, Shiuchi T, Liu HW, Min LJ, Okumura M, Jinno T, Wu L, Iwai M, Horiuchi M. Nicotine enhances angiotensin II-induced mitogenic response in vascular smooth muscle cells and fibroblasts. Arterioscler Thromb Vasc Biol. 2004;24:80–84. doi: 10.1161/01.ATV.0000104007.17365.1c.

30. Wada T, Naito M, Kenmochi H, Tsuneki H, Sasaoka T. Chronic nicotine exposure enhances insulin-induced mitogenic signaling via up-regulation of alpha7 nicotinic receptors in isolated rat aortic smooth muscle cells. Endocrinology. 2007;148:790–799. doi: 10.1210/en.2006-0907.

31. Shaw S, Bencherif M, Marrero MB. Janus kinase 2, an early target of alpha 7 nicotinic acetylcholine receptor-mediated neuroprotection against Abeta-(1-42) amyloid. J Biol Chem. 2002;277:44920–44924. doi: 10.1074/jbc.M204610200.

32. Moon TC, St Laurent CD, Morris KE, Marcet C, Yoshimura T, Sekar Y, Befus AD. Advances in mast cell biology: new understanding of heterogeneity and function. Mucosal Immunol. 2010;3:111–128. doi: 10.1038/mi.2009.136.

33. Tattersall MC, Guo M, Korcarz CE, Gepner AD, Kaufman JD, Liu KJ, Barr RG, Donohue KM, McClelland RL, Delaney JA, Stein JH. Asthma predicts cardiovascular disease events: the multi-ethnic study of ath-erosclerosis. Arterioscler Thromb Vasc Biol. 2015;35:1520–1525. doi: 10.1161/ATVBAHA.115.305452.

34. Lee-Rueckert M, Silvennoinen R, Rotllan N, Judström I, Blanco-Vaca F, Metso J, Jauhiainen M, Kovanen PT, Escola-Gil JC. Mast cell activation in vivo impairs the macrophage reverse cholesterol transport pathway in the mouse. Arterioscler Thromb Vasc Biol. 2011;31:520–527. doi: 10.1161/ATVBAHA.110.221069.

35. Wang X, Collins HL, Ranalletta M, Fuki IV, Billheimer JT, Rothblat GH, Tall AR, Rader DJ. Macrophage ABCA1 and ABCG1, but not SR-BI, promote macrophage reverse cholesterol transport in vivo. J Clin Invest. 2007;117:2216–2224. doi: 10.1172/JCI32057.

36. Gough PJ, Gomez IG, Wille PT, Raines EW. Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. J Clin Invest. 2006;116:59–69. doi: 10.1172/JCI25074.

37. Wessler I, Kirkpatrick CJ. Acetylcholine beyond neurons: the non-neuro-nal cholinergic system in humans. Br J Pharmacol. 2008;154:1558–1571. doi: 10.1038/bjp.2008.185.

38. Duttlinger R, Manova K, Berrozpe G, Chu TY, DeLeon V, Timokhina I, Chaganti RS, Zelenetz AD, Bachvarova RF, Besmer P. The wsh and ph mutations affect the c-kit expression profile: C-kit misexpression in embryogenesis impairs melanogenesis in wsh and ph mutant mice. Proc Natl Acad Sci USA. 1995;92:3754–3758.


39. Barua RS, Sharma M, Dileepan KN. Cigarette smoke amplifies inflam-matory response and atherosclerosis progression through activation of the H1R-TLR2/4-COX2 axis. Front Immunol. 2015;6:572. doi: 10.3389/fimmu.2015.00572.

40. Zhou MS, Chadipiralla K, Mendez AJ, Jaimes EA, Silverstein RL, Webster K, Raij L. Nicotine potentiates proatherogenic effects of oxLDL by stimu-lating and upregulating macrophage CD36 signaling. Am J Physiol Heart Circ Physiol. 2013;305:H563–H574. doi: 10.1152/ajpheart.00042.2013.

41. Catanzaro DF, Zhou Y, Chen R, Yu F, Catanzaro SE, De Lorenzo MS, Subbaramaiah K, Zhou XK, Pratico D, Dannenberg AJ, Weksler BB. Potentially reduced exposure cigarettes accelerate atherosclerosis: evi-dence for the role of nicotine. Cardiovasc Toxicol. 2007;7:192–201. doi: 10.1007/s12012-007-0027-z.

42. Kopeć A, Panaszek B, Fal AM. Intracellular signaling pathways in IgE-dependent mast cell activation. Arch Immunol Ther Exp (Warsz). 2006;54:393–401. doi: 10.1007/s00005-006-0049-4.

Highlights• Asthekeycomponentofcigarettesmoke,nicotineworsensatherosclerosisinapolipoproteinE–deficient(Apoe−/−)mice,accompaniedbyen-

hancedintraplaquemacrophagecontent,lipiddeposit,anddecreasedcollagenandsmoothmusclecellcontentsintheatheroscleroticlesions.Moreover,wedemonstratedthatnicotineaugmentshypercholesterolemia-inducedatherosclerosisthroughactivatingmastcells.

• α7Nicotinicacetylcholinereceptoristhekeymediatorbywhichnicotineactivatesmastcells.• Mastcellstabilizationorα7nicotinicacetylcholinereceptorinhibitionattenuatednicotine-inducedatherosclerosisinamousemodel.• These findingsprovideanovelandpromising therapeuticstrategy for reducingatheroscleroticprogressionandpreventingcardiovascular

eventsinindividualsexposedtocigarettesmoke.

nicotine accelerates atherosclerosis in apolipoprotein e ... · 6 arterioscler thromb vasc biol...

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