Arterioscler Thromb Vasc Biol Nicotine Accelerates Atherosclerosis in Apolipoprotein E–Deficient Mice by Activating α7 Nicotinic Acetylcholine Receptor on Mast CellsHighlights by Chen Wang, Han Chen, Wei Zhu, Yinchuan Xu, Mingfei Liu, Lianlian Zhu, Fan Yang, Ling Zhang, Xianbao Liu, Zhiwei Zhong, Jing Zhao, Jun Jiang, Meixiang Xiang, Hong Yu, Xinyang Hu, Hong Lu, and Jian’an Wang Arterioscler Thromb Vasc Biol Volume 37(1):53-65 December 21, 2016 Copyright © American Heart Association, Inc. All rights reserved.

Chen Wang et al. Arterioscler Thromb Vasc Biol. 2017;37:53-65 Copyright © American Heart Association, Inc. All rights reserved.

Nicotine increases atherosclerotic lesion size and changes lesional compositions in apolipoprotein E–deficient (Apoe−/−) mice. 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. Chen Wang et al. Arterioscler Thromb Vasc Biol. 2017;37:53-65 Copyright © American Heart Association, Inc. All rights reserved.

Nicotine increases mast cell (MC) activation in apolipoprotein E–deficient (Apoe−/−) mice. 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. Nondegranulated MCs show smooth edges, whereas the degranulated ones are surrounded by violet-stained granules. B, Count of MC number 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. Chen Wang et al. Arterioscler Thromb Vasc Biol. 2017;37:53-65 Copyright © American Heart Association, Inc. All rights reserved.

Mast cell (MC) deficiency prevents nicotine-induced plaque formation and composition change. 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. Chen Wang et al. Arterioscler Thromb Vasc Biol. 2017;37:53-65 Copyright © American Heart Association, Inc. All rights reserved.

Nicotine activates bone marrow–derived mast cells (MCs) via α7 nicotinic acetylcholine receptor (α7nAChR). Nicotine activates bone marrow–derived mast cells (MCs) via α7 nicotinic acetylcholine receptor (α7nAChR). A and B, Supernatants 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, α7nAChR, and α9nAChR mRNA in MCs incubated with Tyrode buffer, nicotine, or nicotine+mecamylamine (pretreatment 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. Chen Wang et al. Arterioscler Thromb Vasc Biol. 2017;37:53-65 Copyright © American Heart Association, Inc. All rights reserved.

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 cassette transporter G1 (ABCG1) expression in macrophages. 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 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 real-time polymerase chain reaction. M, MMP-2 and MMP-9 expression levels in peritoneal macrophages incubated with conditional supernatants collected 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. Chen Wang et al. Arterioscler Thromb Vasc Biol. 2017;37:53-65 Copyright © American Heart Association, Inc. All rights reserved.

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. 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 administration, 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, 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. Chen Wang et al. Arterioscler Thromb Vasc Biol. 2017;37:53-65 Copyright © American Heart Association, Inc. All rights reserved.