Nicotine promotes vascular calcification via intracellular Ca2+-mediated, Nox5-induced oxidative stress and extracellular vesicle release in vascular smooth muscle cells

Ploingarm Petsophonsakul, Mathias Burgmaier, Brecht Willems, Sylvia Heeneman, Nadina Stadler, Felix Gremse, Sebastian Reith, Kathrin Burgmaier, Florian Kahles, Nikolaus Marx, Ehsan Natour, Elham Bidar, Michael Jacobs, Barend Mees, Chris Reutelingsperger, Malgorzata Furmanik, Leon Schurgers*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review


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.

Original languageEnglish
Pages (from-to)2196-2210
Number of pages15
JournalCardiovascular Research
Issue number9
Early online date17 Jul 2021
Publication statusPublished - 20 Jul 2022


  • Nicotine Vascular calcification Vascular smooth muscle cell phenotypic switching Nox5 Vitamin K2

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