TY - JOUR
T1 - Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress and extracellular vesicle release in vascular smooth muscle cells
AU - Petsophonsakul, Ploingarm
AU - Burgmaier, Mathias
AU - Willems, Brecht
AU - Heeneman, Sylvia
AU - Stadler, Nadina
AU - Gremse, Felix
AU - Reith, Sebastian
AU - Burgmaier, Kathrin
AU - Kahles, Florian
AU - Marx, Nikolaus
AU - Natour, Ehsan
AU - Bidar, Elham
AU - Jacobs, Michael
AU - Mees, Barend
AU - Reutelingsperger, Chris
AU - Furmanik, Malgorzata
AU - Schurgers, Leon
N1 - © The Author(s) 2021. Published by Oxford University Press on behalf of the European Society of Cardiology.
PY - 2022/7/20
Y1 - 2022/7/20
N2 - AIMS: Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms.METHODS AND RESULTS: We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed-tomography (µCT)-scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; p < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine induced human primary VSMC calcification, increased osteogenic gene expression (Runx2, Osx, BSP and OPN), and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels.CONCLUSION: In this study we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.
AB - AIMS: Smokers are at increased risk of cardiovascular events. However, the exact mechanisms through which smoking influences cardiovascular disease resulting in accelerated atherosclerosis and vascular calcification are unknown. The aim of this study was to investigate effects of nicotine on initiation of vascular smooth muscle cell (VSMC) calcification and to elucidate underlying mechanisms.METHODS AND RESULTS: We assessed vascular calcification of 62 carotid lesions of both smoking and non-smoking patients using ex vivo micro-computed-tomography (µCT)-scanning. Calcification was present more often in carotid plaques of smokers (n = 22 of 30, 73.3%) compared to non-smokers (n = 11 of 32, 34.3%; p < 0.001), confirming higher atherosclerotic burden. The difference was particularly profound for microcalcifications, which was 17-fold higher in smokers compared to non-smokers. In vitro, nicotine induced human primary VSMC calcification, increased osteogenic gene expression (Runx2, Osx, BSP and OPN), and extracellular vesicle (EV) secretion. The pro-calcifying effects of nicotine were mediated by Ca2+-dependent Nox5. SiRNA knock-down of Nox5 inhibited nicotine-induced EV release and calcification. Moreover, pre-treatment of hVSMCs with vitamin K2 ameliorated nicotine-induced intracellular oxidative stress, EV secretion, and calcification. Using nicotinic acetylcholine receptor (nAChR) blockers α-bungarotoxin and hexamethonium bromide we found that the effects of nicotine on intracellular Ca2+ and oxidative stress were mediated by α7 and α3 nAChR. Finally, we showed that Nox5 expression was higher in carotid arteries of smokers and correlated with calcification levels in these vessels.CONCLUSION: In this study we provide evidence that nicotine induces Nox5-mediated pro-calcific processes as novel mechanism of increased atherosclerotic calcification. We identified that activation of α7 and α3 nAChR by nicotine increases intracellular Ca2+ and initiates calcification of hVSMCs through increased Nox5 activity, leading to oxidative stress-mediated EV release. Identifying the role of Nox5-induced oxidative stress opens novel avenues for diagnosis and treatment of smoking-induced cardiovascular disease.
KW - Nicotine Vascular calcification Vascular smooth muscle cell phenotypic switching Nox5 Vitamin K2
KW - CORONARY-ARTERY CALCIFICATION
KW - NECROSIS-FACTOR-ALPHA
KW - VITAMIN-K
KW - CIGARETTE-SMOKING
KW - FETUIN-A
KW - SUBCLINICAL ATHEROSCLEROSIS
KW - CARDIOVASCULAR-DISEASE
KW - CALCIUM-PHOSPHATE
KW - AORTIC-ANEURYSMS
KW - UP-REGULATION
U2 - 10.1093/cvr/cvab244
DO - 10.1093/cvr/cvab244
M3 - Article
C2 - 34273166
SN - 0008-6363
VL - 118
SP - 2196
EP - 2210
JO - Cardiovascular Research
JF - Cardiovascular Research
IS - 9
ER -