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

Abstract

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, and 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.

Keywords: Nicotine Vascular calcification Vascular smooth muscle cell phenotypic switching Nox5 Vitamin K2.

Graphical Abstract

Figure 1

Smoking increases the incidence and amount of calcification in human atherosclerotic plaques. (A) Representative µCT image of carotid plaque from a non-smoker. Carotid plaque including soft tissue and (B) the same plaque without soft tissue displaying compartmentation of microcalcification in green, intermediate size calcification in magenta, and macrocalcification in turquoise. (C) Number of microcalcifications, (D) intermediate size calcifications, and (E) macrocalcifications in carotid artery atherosclerotic plaques from smokers and non-smokers quantified by µCT. (F) Volume of microcalcifications, (G) intermediate size calcifications, and (H) from the same analysis. Statistical significance was tested using t-test, n = 30 plaques for smokers, n = 32 plaques for non-smokers. All graphs show mean and SEM.

Figure 2

Nicotine increases VSMC calcification in vitro. (A) Calcification was induced with elevated Ca²⁺ concentrations (5.4 mM) in DMEM with 2.5% FBS in the presence of nicotine (1 mM, 5–7 days) and quantified using an o-cresolphthalein colorimetric assay. Cells were seeded in 48-well plates at 15 000 cells/well. Data from n = 9 independent experiments. Statistical significance was tested with one-sample test. (B–F) qPCR analysis of osteogenic genes RUNX2, Osx, OCN, BSP, OPN, and (G) contractile marker SM22α in VSMCs treated with nicotine (1 µM or 1 mM, 7 days in DMEM with 20% FBS). Statistical significance was tested with one-sample test. Data from n = 3 (B, D), n = 5 (C), n = 7 (E), n = 4 (F, G) independent experiments. In case of borderline significance, more samples were added to rule in/rule out the genes that are involved in nicotine-induced changes in VSMCs. (H–K) Western blotting and quantification of contractile markers αSMA, CNN1, p-MLC in VSMCs treated with nicotine (1 mM, 7 days in DMEM with 20% FBS). Labels show protein standard (kDa). Data from n = 4 (I, J) and n = 3 (K) independent experiments. Statistical significance was tested with one-sample test. (L) VSMCs treated with nicotine (1 mM, 24 h) in DMEM with 0.5% FBS secrete more EVs than VSMCs treated with vehicle control. EVs were captured with anti-CD63-coulped beads, detected with a fluorescently labelled anti-CD81 antibody and quantified using flow cytometry. Cells were seeded in 48-well plates at 15 000 cells/well. Data from n = 3 independent experiments in triplicate or quadruplicate. Statistical significance was tested with one-sample test. (M and N) qPCR analysis of EV-related genes; CD63 and SMPD3 mRNA expression treated with nicotine (1 mM, 7 days in DMEM with 20% FBS). Data from n = 12 (M) and n = 8 (N) independent experiments. Statistical significance was tested with one-sample test.

Figure 3

Nicotine-induced ROS production leads to VSMC calcification. (A) Stimulation of nicotine (1 mM, 40 min) induces intracellular ROS of VSMCs compared to vehicle control. ROS production was measured using a fluorescent assay with DCFDA. Cells were seeded in 96-well black plates at 8000 cells/well. Data from n = 7 independent experiments in quadruplicate. Statistical significance was tested with one-sample test. Pre-treatment of VSMCs with vitamin K (10 µM, 1 h) prior to stimulation with nicotine reduces (B) intracellular ROS (1 mM nicotine, 40 min, 8000 cells/well), data from n = 3 to 4 independent experiments (C) EV secretion (1 mM nicotine in DMEM with 0.5% FBS, 24 h, 15 000 cells/well, and (D) VSMC calcification (1 mM nicotine in DMEM with 2.5% FBS and 3.6 mM Ca²⁺, 3–5 days, 15 000 cells/well). Data from n = 3 independent experiments in duplicate or triplicate (C) and n = 3 to 5 independent experiments in duplicate, triplicate, or quadruplicate (D). Data of K2 control n = 1 experiment in quadruplicate (C) and n = 2 independent experiments in duplicate and triplicate (D). Statistical significance was tested with ANOVA (B–D). (E) Pre-treatment with NAC (0.5 µM, 1 h) prior to nicotine stimulation reduces VSMC calcification induced by DMEM with 2.5% FBS and 3.6 mM Ca²⁺ (3–5 days). Cells were seeded in 48-well plates at 15 000 cells/well. Calcification was detected using Fetuin A-Alexa-546. Data from n = 5 independent experiments, each in triplicate or quadruplicate. Data of NAC control n = 1 experiment in quadruplicate. Statistical significance was tested with Kruskal–Wallis test.

Figure 4

Nox5 mediates pro-calcific effects of nicotine. (A–D) qPCR analysis of NOX subunit mRNA expression in VSMCs treated with nicotine (1 mM, 7 days in DMEM with 20% FBS). Data from n = 3 independent experiments in single (A) and n = 4 independent experiments in duplicate or triplicate (B–D). Statistical significance was tested with t-test. (E–G) Western blotting and quantification of Nox4 and Nox5 protein in VSMCs treated with nicotine (1 mM, 7 days in DMEM with 20% FBS). Labels show protein standard (kDa). Data from n = 4 (F) independent experiments in single or duplicate and n = 4 (G) independent experiments in single or triplicate. Statistical significance was tested with t-test. (H) Pre-treatment of VSMCs with GKT136901 (1 µM, 1 h) prior nicotine stimulation (1 mM, 40 min, 8000 cells/well) reduces nicotine-induced intracellular ROS. Data from one experiment in quadruplicate, representative of three independent experiments. Statistical significance was tested with ANOVA. (I) VSMC calcification was induced with calcification medium (2.5% FBS, 3.6 mM Ca²⁺) in the presence of nicotine (1 mM), with or without pre-treatment with GKT (1 µM, 1 h), for 3–5 days. Cells were seeded in 48-well plates at 15 000 cells/well. Calcification was detected using Fetuin A-Alexa-546. Data from four independent experiments, each in triplicate or quadruplicate. Statistical significance was tested with Kruskal–Wallis test. (J) Nicotine (10 mM, 5 min) induces an increase in intracellular Ca²⁺ (measured using Fluo-4-AM). Cells were seeded in 96-well plates at 8000 cells/well. Data from one experiment in quadruplicate, representative of three independent experiments. Statistical significance was tested with t-test. (K) Pre-treatment of VSMCs with BAPTA-AM (1 µM, 1 h) reduces nicotine-induced intracellular Ca²⁺ (10 mM nicotine, 5 min, 8000 cells/well). Data from one experiment in quadruplicate, representative of three independent experiments. Statistical significance was tested with ANOVA. (L–M) Western blot showing that siRNA knock-down (24 h) decreased Nox5 protein expression. Labels show protein standard (kDa). Data from n = 4 independent experiments in single or duplicate. Statistical significance was tested with one-sample test (N) SiRNA knock-down of Nox5 (24 h) decreased VSMC intracellular ROS (data from one experiment in triplicate, representative of three independent experiments), (O) EV secretion (data from one experiment in triplicate, representative of three independent experiments), and (P) VSMC calcification (2.5% FBS, 3.6 mM Ca²⁺, 3–5 days). Data from one experiment in quadruplicate, representative of three independent experiments. Statistical significance was tested with ANOVA(N–P).

Figure 5

Nicotine mediates its effects through interaction with α3 and α7 nicotinic acetylcholine receptor. (A) Pre-treatment with α-bungarotoxin (1 µM, 1 h) and hexamethonium bromide (100 µM, 1 h) reduced nicotine-induced intracellular ROS (1 mM nicotine, 40 min, 8000 cells/well, data from one experiment in triplicate or quadruplicate, representative of three independent experiments) and (B) VSMC calcification (1 mM nicotine in DMEM with 2.5% FBS, 3.6 mM Ca²⁺, 3–5 days, 15 000 cells/well). Calcification was quantified using an o-cresolphthalein colorimetric assay. Data from n = 3 independent experiments, each in triplicate. Statistical significance was tested with ANOVA (A and B). (C) Pre-treatment with α-bungarotoxin (1 µM, 1 h) prior to nicotine stimulation (1 mM, 5 min) reduces intracellular Ca²⁺. Cells were seeded in 96-well plates at 8000 cells/well. Data from n = 3 independent experiments, each in triplicate or quadruplicate. Statistical significance was tested with ANOVA. (D) VSMCs were treated with nicotine (1 mM, 24 h) with or without pre-treatment with α-bungarotoxin (1 µM, 1 h) in DMEM with 0.5% FBS. EVs were captured with anti-CD63-coulped beads, detected with a fluorescently labelled anti-CD81 antibody and quantified using flow cytometry. Cells were seeded in 48-well plates at 15 000 cells/well. Data from n = 4 independent experiments, each in triplicate or quadruplicate. Statistical significance was tested with Kruskal–Wallis test. (E) qPCR analysis of Nox5 (data from n = 6 or 8 independent experiments) and (F–G) EV-related gene SMPD3 (data from n = 7 to 10 independent experiments) and CD63 (data from n = 6 or 7 independent experiments) expression in VSMCs treated with nicotine (1 mM, 7 days in DMEM with 20% FBS) with or without α-bungarotoxin pre-treatment (1 µM, 1 h). Statistical significance was tested with ANOVA (E) and Kruskal–Wallis test (F–G).

Figure 6

Smoking and calcification are associated with increased carotid artery Nox5 expression. (A) Immunohistochemical analysis of Nox5, CNN, and S100A4 expression in carotid artery samples from human donors. I and M indicate intima and media, respectively. Scale bars are 250 µm. Figure shows representative images from 32 carotid artery samples that were stained (15 smokers, 15 non-smokers). (B–D) Quantification of protein expression scores. Statistical significance was tested with Mann–Whitney U-test. Dots denote individual data points. Graphs (B–D) show n ≥ 15 plaques for smokers, n ≥ 15 plaques for non-smokers. (E) Nox5 expression positively correlates with calcification (P < 0.001, r = 0.8187 and P < 0.0001, r = 0.794, when outliers are removed). Nox5 expression scores were plotted against µCT total calcification scores for each patient. Graph shows individual data points (both smokers and non-smokers). Linear regression was plotted, and a two-tailed Pearson correlation test was carried out. Graph shows n = 14 plaques.

Figure 7

Mechanism of nicotine-induced VSMC calcification. Nicotine induces changes in VSMCs that initiate their calcification. First, nicotine binds to α3 and α7 nAchR on VSMCs, which rapidly raises intracellular Ca²⁺ levels. Nox5, whose activity is dependent on Ca²⁺, is then activated and subsequently increases production of intracellular ROS. Oxidative stress induces VSMC phenotypic switching and promotes transdifferentiation of VSMCs towards an osteogenic phenotype. Phenotypic switching of VMSC also results in higher secretion of EVs, which mineralize and contribute to calcification.