# Flavor-induced inflammation and cytotoxicity in human aortic smooth muscle cells: Potential implications for E-cigarette safety

Mariam Bitar, Clément Mercier, Laurent Bertoletti, Jérémie Pourchez, Valérie Forest

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## To cite this version:

Mariam Bitar, Clément Mercier, Laurent Bertoletti, Jérémie Pourchez, Valérie Forest. Flavor-induced inflammation and cytotoxicity in human aortic smooth muscle cells: Potential implications for E-cigarette safety. Toxicology and Applied Pharmacology, 2025, 500, pp.117388. 10.1016/j.taap.2025.117388. hal-05064907

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https://hal.science/hal-05064907v1

Submitted on 13 May 2025

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# Flavor-induced inflammation and cytotoxicity in human aortic smooth muscle cells: Potential implications for *E*-cigarette safety

Mariam Bitar<sup>a</sup>, Clément Mercier<sup>a</sup>, Laurent Bertoletti<sup>b,c,d</sup>, Jérémie Pourchez<sup>a</sup>, Valérie Forest<sup>a,*</sup>

## ARTICLE INFO

Editor: Lawrence Lash

**Keywords:**

E-cigarettes

Cytotoxicity

Pro-inflammatory response

Cardiovascular effect

Nicotine

Flavor

Aortic smooth muscle cells

## ABSTRACT

Electronic nicotine delivery systems (ENDS), commonly known as e-cigarettes, are considered safer alternatives to tobacco smoking, yet their long-term health effects, particularly on cardiovascular health, remain unclear. The aim of this study was to investigate the cytotoxic and pro-inflammatory effects of device power, nicotine content and flavor molecules on human aortic smooth muscle cells. AoSMCs cells were exposed to e-liquids and e-cigarette aerosol condensates containing different ratios of propylene glycol (PG) and vegetable glycerin (VG), nicotine (0, 10, 20 mg/mL), and flavors (cinnamon, menthol, tobacco), with the devices operated at different power levels (10 W, 15 W, 25 W). After a 24 h incubation, cytotoxicity was evaluated using lactate dehydrogenase (LDH) release, while pro-inflammatory effects were measured by interleukin-8 (IL-8) production. The results showed no significant cytotoxicity or inflammation in cells exposed to PG/VG base or nicotine-containing e-liquids. However, e-liquids as well as aerosol condensates containing flavors induced significant increases in IL-8 production compared to controls without flavor. Moreover, the pro-inflammatory response was more pronounced in response to aerosol condensates than to the corresponding e-liquids. Cinnamon, in particular, produced the highest inflammatory response, and the effect was enhanced at higher power settings (25 W), which also induced cytotoxicity, particularly at high concentrations. These findings demonstrate that flavors, especially cinnamon, and device power levels are key factors influencing the inflammatory potential and cytotoxicity of e-cigarette aerosols. Further studies are needed to explore the long-term cardiovascular risks associated with ENDS use and the role of flavor molecules and of their thermal degradation products.

## 1. Introduction

Electronic nicotine delivery systems (ENDS), commonly known as electronic cigarettes (e-cigs), are devices that produce aerosols that simulate traditional tobacco smoking by vaporizing nicotine-containing or nicotine-free e-liquids (Brown and Balk, 2020). Unlike traditional cigarettes, e-cigarettes do not rely on the combustion of tobacco, reducing exposure to many of the toxins produced by burning tobacco. The composition of e-cigarette aerosol therefore differs significantly from tobacco cigarette smoke, which suggests that the toxicity of e-cigarette aerosols is significantly lower than that of conventional cigarette smoke (Flouris et al., 2013). While numerous studies emphasize the health benefits of switching from tobacco smoking to e-cigarettes and their potential role in smoking cessation, other studies raise concerns,

particularly regarding the health risks they may pose (Kalkhoran and Glantz, 2016). However, the current scientific data in the literature is still insufficient to rigorously assess the actual health effects of e-cigarette use. The mechanisms by which ENDS may affect cardiovascular health are still largely unexplored, underlining the need for comprehensive toxicological studies (Basma et al., 2020).

There are numerous types of e-cigarettes and thousands of e-liquid varieties on the market. E-liquids usually consist of a base of propylene glycol (PG) and vegetable glycerin (VG), to which nicotine and flavorings can be added (Sinha et al., 2022a). When vaporized with an atomizer, the resulting aerosol produces a visual appearance similar to that of tobacco smoke. However, while the e-liquid itself may contain potentially harmful components, the heating process can lead to the formation of additional thermal degradation products that can pose a

<footnote><sup>a</sup> Mines Saint-Etienne, Univ Jean Monnet, INSERM, U 1059 Sainbiose, Centre CIS, F-42023 Saint-Etienne, France</footnote>

<footnote><sup>b</sup> Service de Médecine Vasculaire et Thérapeutique, CHU de Saint-Etienne, Saint-Etienne, France</footnote>

<footnote><sup>c</sup> INSERM, UMR1059, Equipe Dysfonction Vasculaire et Hémostase, Université Jean-Monnet, F-42055 Saint-Etienne, France</footnote>

<footnote><sup>d</sup> INSERM, CIC-1408, CHU Saint-Etienne, F-42055 Saint-Etienne, France</footnote>

<footnote>* Corresponding author at: Mines Saint-Etienne, 158 cours Fauriel, CS 62362, 42023 Saint-Etienne, Cedex 2, France.
E-mail address: vforest@emse.fr (V. Forest).</footnote>
health risk (Marques et al., 2021). Several harmful chemicals have been detected in e-cigarette aerosols, including alkaloids, volatile organic compounds (VOCs), pyridine, and carbonyl compounds such as acrolein and formaldehyde (Bekki et al., 2014). The concentration of thermal degradation products varies depending on the power setting and specific flavor additives. Furthermore, the presence of metals such as nickel, chromium, and lead in e-cigarette aerosols raises additional health concerns, as these metals are introduced by the heating coils used in the devices (Ebersole et al., 2020).

The cardiovascular effects of e-cigarette use have indeed become an important issue, as evidence of the potential risks is mounting. Research suggests that e-cigarette use may be associated with an increased risk of adverse cardiovascular events, such as chest pain (Clapp and Jaspers, 2017), coronary heart disease (Wang et al., 2018), stroke (Farsalinos et al., 2019), arrhythmias (Kaisar et al., 2017), and myocardial infarctions (Moheimani et al., 2017). In addition, several studies have reported a significant increase in heart rate and blood pressure following the use of e-cigarette (Alzahrani et al., 2018; Spindle et al., 2017; Chaumont et al., 2018; Ikonomidis et al., 2018; Franzen et al., 2018). E-cigarette use has also been associated with arterial stiffness, increased oxidative stress, inflammation, and impaired vascular endothelial function (Anderson et al., 2016; Caporale et al., 2019; Kaisar et al., 2017; Putzhammer et al., 2016; Spindle et al., 2017). These effects may contribute to an increased risk of cardiovascular disease. Preclinical in vitro and in vivo studies have also shown that exposure to e-cigarette aerosols increases the production of reactive oxygen species, causes DNA damage and cytotoxicity (Caporale et al., 2019), and is associated with impaired cardiovascular function, inflammation, and oxidative stress (Anderson et al., 2016; Olfert et al., 2018; Crotty Alexander et al., 2018; Lee et al., 2018). In particular, recent in vitro studies have shown that e-cigarette aerosol or e-liquid condensates can induce cytotoxic and pro-inflammatory effects in cardiovascular cells. Flavored e-liquids, particularly those containing cinnamon, have been associated with oxidative stress, cytotoxicity, and impaired endothelial function (Lee et al., 2019). Similar responses have been observed in human coronary endothelial cells exposed to e-cigarette aerosol condensates, indicating potential cardiovascular toxicity (Chhor et al., 2023). Regarding smooth muscle cells, an increased expression of pro-inflammatory markers was reported following exposure to condensates containing cinnamon flavor (Michon et al., 2022). Therefore, cytotoxicity and inflammation appear as important biological endpoints to consider when investigating the cardiovascular effects of e-cigarette and underlying mechanisms.

The aim of this study was to systematically evaluate the biological effects of different e-liquid compositions and e-cigarette settings on an in vitro model of human aortic smooth muscle cells. The novelty of this work lies in the systematic analysis of several parameters. Therefore, cells were exposed to e-liquids and condensates of e-liquids containing a variable amount of nicotine (0, 10 and 20 mg/mL), with or without flavorings (cinnamon, menthol, and tobacco), and produced at a variable power (10, 15 and 25 W). The cell response was analyzed in terms of cytotoxicity and pro-inflammatory response.

# 2. Materials and methods

## 2.1. Cell culture

Human aortic smooth muscle cells (AoSMCs) were purchased from Lonza (Basel, Switzerland) and maintained in smooth muscle cell growth medium-2 (SmGM, Lonza, Basel, Switzerland). This medium contains Fetal Bovine Serum (FBS), human fibroblast growth factors, epidermal growth factors, insulin, and gentamicin-amphotericin. Flasks were trypsinized when cells reached approximately 80 % confluence with trypsin (0.025 %)/ EDTA (Lonza, Basel, Switzerland). Cells were then differentiated for 1–2 weeks in smooth muscle cell basal medium (SmBM, Lonza, Basel, Switzerland) added with 5 % penicillin/streptomycin (VWR, Monroeville, PA, USA). Culture was carried out in an

incubator at 37 °C and 5 % CO₂ atmosphere and the culture medium was changed two or three times a week. Then, 96-well plates were coated with 3 µg/cm² fibronectin (Sigma-Merck, Saint-Quentin-Fallavier, France), reconstituted with 1 mL of phosphate buffered saline (PBS, Fisher Scientific, Illkirch, France). Plates were then air-dried for 45 min and stored for 2–4 weeks. 15,000 cells were seeded in 50 µL of SmBM medium in each well of a 96-well plate which was incubated at 37 °C in 5 % CO₂ for 24 h. The cells were then exposed to 150 µL of e-liquid or condensates for 24 h. The cells used for these experiments were at passage 7.

## 2.2. Generation of aerosol condensates

The e-liquid constituents PG/VG 70/30 and PG/VG 30/70 (Propylene glycol and vegetable glycerin) were purchased from Aromea (Aromea, Saint Aunès, France). Nicotine was purchased from A&L (Arômes et Liquides, Andrézieux-Bouthéon, France). The cinnamon, menthol and tobacco flavorings were purchased from Bio Concept (Niort, France). The clearomizer was the GS Air 2 Atomizer (Eleaf, Shenzhen, China) with the resistance GS Air Series Atomizer Head 0.75 Ω (Eleaf, Shenzhen, China) and the battery iStick TC40W (Eleaf, Shenzhen, China). Aerosols were generated using a Programmable Dual Syringe Pump (PDSP, Burghart Messtechnik GmbH, Wedel, Germany). The e-cigarette was positioned at a 30° angle to simulate the tilt during inhalation, and all vaping parameters as specified by the French Standardization Association (AFNOR standard XP D90-300-3) were adhered to: a 3-s puff, 55 mL per puff, 30 s between puffs, 20 puffs per series, 2 series, with a 300-s interval between series. As a result, 40 puffs were generated in total to collect the condensates for each condition. The aerosol was subsequently gathered in a Glass Twin Impinger (GTI, Copley Scientific Limited, Nottingham, UK) containing 30 mL of PBS in the lower chamber (Erlenmeyer), representing the respirable dose of the aerosol. The setup involved a GTI linked to a vacuum pump (Low Capacity Pump Model LCP5, with a flow rate of 60 L/min) produced by Copley Scientific. The clearomizer was weighed before and after condensation to measure the e-liquid mass in the condensate and thereby determine its concentration. The condensates were then diluted in the culture medium (SmBM, Lonza, Basel, Switzerland) to achieve the desired concentrations (2–128 µg/mL). This concentration range was selected based on the pharmacokinetic data on nicotine available in the literature (Marsot and Simon, 2016). The lowest nicotine concentration used (31.25 ng/mL, corresponding to a 2 µg/mL e-liquid concentration) reflects a physiological level (approximately the peak plasma concentration of 20–40 ng/mL). In addition to condensates, we also exposed cells to e-liquids as controls.

## 2.3. Conditions tested

To study the impact of the previously mentioned parameters, cells were exposed to various formulations of e-liquids and e-cigarette condensates, as summarized in Table 1.

## 2.4. Toxicological tests

### 2.4.1. Cytotoxicity - LDH test

To assess cell membrane damage from exposure to e-liquids or aerosol condensates, the release of lactate dehydrogenase (LDH) into the culture supernatant was measured after a 24-h incubation period using the CytoTox-96 assay (Promega, Madison, WI, USA) according to the manufacturer's instructions (Cervellati et al., 2014a; Effah et al., 2023; Michon et al., 2022). The optical density of the samples was then determined using a microplate reader set to 490 nm (Multiskan GO, ThermoFisher Scientific, Waltham, MA, USA) and compared with that of the control group (unexposed cells). A positive control representing the maximum LDH release (100 %) after cell lysis was used.
<table><caption>**Table 1**
Summary of the studied parameters and the associated formulations of e-liquids and e-cigarette condensates.</caption><thead><tr><th>Impact of PG/VG ratio</th><th>Impact of nicotine</th></tr></thead><tbody><tr><td>Condensate PG/VG (70/30)</td><td>E-liquid PG/VG (70/30)</td></tr><tr><td>Condensate PG/VG (30/70)</td><td>E-liquid PG/VG (70/30) + nicotine (20 mg/mL)</td></tr><tr><td></td><td>Condensate PG/VG (70/30)</td></tr><tr><td></td><td>Condensate PG/VG (70/30) + nicotine (10 mg/mL)</td></tr><tr><td></td><td>Condensate PG/VG (70/30) + nicotine (20 mg/mL)</td></tr></tbody><thead><tr><th>Impact of flavors</th><th>Impact of power</th></tr></thead><tbody><tr><td>E-liquid PG/VG (70/30)</td><td>Condensate PG/VG (70/30) + nicotine (20 mg/mL) + cinnamon flavor + power 10 W</td></tr><tr><td>E-liquid PG/VG (70/30) + menthol flavor</td><td>Condensate PG/VG (70/30) + nicotine (20 mg/mL) + cinnamon flavor + power 15 W</td></tr><tr><td>E-liquid PG/VG (70/30) + cinnamon flavor</td><td>Condensate PG/VG (70/30) + nicotine (20 mg/mL) + cinnamon flavor + power 25 W</td></tr><tr><td>E-liquid PG/VG (70/30) + tobacco flavor</td><td>Condensate PG/VG (70/30) + nicotine (20 mg/mL) + cinnamon flavor + power 25 W</td></tr><tr><td></td><td></td></tr><tr><td>Condensate PG/VG (70/30)</td><td></td></tr><tr><td>Condensate PG/VG (70/30) + menthol flavor</td><td></td></tr><tr><td>Condensate PG/VG (70/30) + cinnamon flavor</td><td></td></tr><tr><td>Condensate PG/VG (70/30) + tobacco flavor</td><td></td></tr></tbody></table>

### 2.4.2. Pro-inflammatory response - Interleukin-8 test

The production of Interleukin-8 (IL-8) was evaluated using the human IL-8 ELISA Kit (ThermoFisher Scientific, Waltham, MA, USA) following the manufacturer's guidelines. Absorbance was measured at 450 nm with a microplate reader (Multiskan GO, ThermoFisher Scientific, Waltham, MA, USA) (Michon et al., 2022; Sinha et al., 2022b; Staal et al., 2023; Vasanthi Bathrinarayanan et al., 2018). IL-8 levels were then compared to those of the control group, consisting of unexposed cells.

### 2.5. Statistical analysis

Analyses and graphs were performed on Prism software (GraphPad, San Diego, CA). Statistical significance was declared when *p* < 0.05, as calculated with two-way ANOVA tests and Tukey post-tests. Each data point represents the mean of three independent experiments each performed in quadruplicate and is presented with the arithmetic standard error of the mean (± SEM).

## 3. Results

### 3.1. Impact of the PG/VG ratio

We first investigated the effects of different PG/VG ratios (70/30 and 30/70) on cytotoxicity and pro-inflammatory responses. As shown in Fig. 1, part A, LDH release was not significantly different from that of control cells under any experimental condition. This indicates that aerosol condensates with a PG/VG ratio of 70/30 and 30/70 did not induce cytotoxicity, regardless of the concentration tested. As shown in Fig. 1, part B, IL-8 production was also not significantly different from that of control cells under any condition. These results suggest that the PG/VG ratio has no effect on cytotoxicity and the pro-inflammatory response.

As no effects of the PG/VG ratio were observed, we decided to

continue the study with the 70/30 PG/VG ratio as it is widely used in high performance devices and potentially produces higher amounts of toxic degradation products such as aldehydes. This decision is also consistent with existing studies in which a 70/30 PG/VG ratio is often used for reproducibility and comparative purposes (Mercier et al., 2024).

### 3.2. Impact of the nicotine

We then analyzed the effects of nicotine on cytotoxicity and the pro-inflammatory response. As shown in Fig. 2, part A, LDH release showed no significant difference compared to control cells, indicating that neither nicotine-containing e-liquids nor aerosol condensates induce cytotoxicity, regardless of concentration. Furthermore, no differences were observed between nicotine-free and nicotine-containing conditions. In Fig. 2, part B, IL-8 production showed no significant difference compared to the control cells. This indicates that nicotine, regardless of whether it is contained in e-liquids or aerosol condensates, does not induce a pro-inflammatory response at any of the concentrations tested.

Furthermore, no significant difference in IL-8 production was observed between the groups exposed to e-liquids and the groups exposed to e-liquids with nicotine (20 mg/mL) (*p* = 0.328). Also, when comparing the groups exposed to the condensates with those exposed to the nicotine-containing condensates (at concentrations of 10 mg/mL and 20 mg/mL), no significant differences were observed. These findings suggest that the presence of nicotine has no effect on cytotoxicity or IL-8 production.

### 3.3. Impact of the flavors

Next, we investigated the effects of the different flavors on cytotoxicity and pro-inflammatory response. Fig. 3, part A illustrates the LDH release observed following exposure of cells to e-liquids or aerosol condensates containing PG/VG (70/30), cinnamon, menthol and
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Relative cytotoxicity (A) and pro-inflammatory response (B) induced by the different experimental conditions assessed after 24 h of cell exposure through LDH release and IL-8 production. Results are expressed relative to control (unexposed) cells and represent the mean of three independent experiments, each performed in quadruplicate.</figcaption></figure>

tobacco flavors. We find that LDH release was not significantly increased compared to the unexposed control cells, indicating that the e-liquids or condensates did not induce cytotoxicity regardless of their concentration. Since no difference was observed between the groups of e-liquids with and without flavorings on the one hand and between the groups of condensates with and without flavorings on the other hand, no influence of the flavorings on cytotoxicity was observed.

In Fig. 3, part B, IL-8 production was evaluated. We observed that the cinnamon, menthol and tobacco flavored e-liquids induced a slightly significant increase in IL-8 production, but only at concentrations of 64 µg/mL and 128 µg/mL. In contrast, condensates with cinnamon, menthol and tobacco flavors caused a significant increase in IL-8 production at all concentrations tested. We also found that there was a significant difference between the groups of e-liquids without flavors and the groups with flavors but only at high concentrations of 64 µg/mL and 128 µg/mL. Additionally, there is a highly significant difference between the groups of flavorless condensates and the groups with flavors at all tested concentrations. These results suggest that the flavors, particularly cinnamon, may be responsible for the observed pro-inflammatory response.

### 3.4. Impact of the power level

Finally, we investigated the effect of e-cigarettes power settings on cytotoxicity and the pro-inflammatory response. For this purpose, cells were exposed to condensates containing PG/VG (70/30), nicotine (20 mg/mL) and cinnamon flavoring at increasing power levels of 10 W, 15 W and 25 W. As shown in Fig. 4, Part A, LDH release was significantly different from that of control cells when cells were exposed to condensates prepared at 25 W power at the highest concentrations (8, 64 and 128 µg/mL) or to condensates prepared at 15 W but only at the highest concentration (128 µg/mL). These results indicate that exposure to cinnamon-flavored aerosols, especially at high concentration and high heating power, resulted in cytotoxic effects which could be due to the formation of some degradation products.

Fig. 4, part B shows IL-8 production. We found that it was strongly and significantly increased at all tested power levels and at all condensate concentrations. In addition, significant differences were found between the PG/VG + nicotine + cinnamon condensate groups heated at 10 W and the groups heated at 15 W and 25 W, especially at concentrations of 2 µg/mL and 8 µg/mL.

## 4. Discussion

The results of this study reveal significant effects on cytotoxicity and inflammatory response, particularly under specific conditions related to the flavors used and in particular the cinnamon flavor when heated at high power.

First, a pro-inflammatory response was induced in aortic smooth muscle cells after exposure to e-cigarette aerosol condensates produced from cinnamon, menthol or tobacco flavored e-liquids. In contrast, condensates containing only the components of e-liquids (PG/VG in the ratios 70/30 or 30/70), elicited neither cytotoxicity nor a pro-inflammatory response in the cells, even when nicotine was added at concentrations of 10 and 20 mg/mL. However, exposure to cinnamon-flavored aerosols, especially at a high heating power of 25 W, led to clear cytotoxic effects. This effect may be partly explained by the influence of power level on aerosol composition. Higher power increases the coil temperature, promoting thermal degradation of e-liquid constituents and leading to the formation of harmful carbonyl compounds such as formaldehyde and acrolein (Gillman et al., 2016; Uchiyama et al., 2020).

The absence of cytotoxic and pro-inflammatory responses observed in cells exposed to PG/VG base and nicotine is consistent with several recent studies showing that these components, are not inherently toxic at moderate concentrations. For example, one study demonstrated that PG/VG-based e-liquids do not induce acute toxicity in human cardiac cells even in the presence of nicotine (Farsalinos et al., 2015), suggesting that these substances do not exhibit significant cytotoxic effects under conditions similar to our experiments. Similarly, no pro-inflammatory response was observed in human bronchial epithelial cells after exposure to PG/VG-containing e-liquids, with or without nicotine (Cervellati et al., 2014b). No significant increase in inflammatory markers such as IL-8 was observed, supporting the absence of inflammation in our results. However, it is important to note that some studies have shown other effects. For example, a moderate long-term inflammatory response from nicotine was observed in animal models (Wu et al., 2014), suggesting that chronic exposure may have more harmful effects than the acute exposure studied here. In parallel, in a more recent study, mild cytotoxic and pro-inflammatory effects were observed following prolonged exposure to nicotine in certain cell models (Muthumalage et al., 2018), suggesting that the duration and conditions of exposure may influence the results.

It is known that the aerosol of e-cigarettes can contain various carbonyl compounds such as formaldehyde, acetaldehyde, and acrolein, which can be formed during the thermal decomposition of propylene glycol, glycerol and flavorings (Bekki et al., 2014; Son et al., 2020).
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Relative cytotoxicity (A) and pro-inflammatory response (B) induced by the different experimental conditions were assessed after 24 h of cell exposure through LDH release and IL-8 production. Results are expressed relative to control (unexposed) cells and represent the mean of three independent experiments, each performed in quadruplicate. Nic: nicotine.</figcaption></figure>

These neoformed compounds are associated with oxidative stress and inflammation. Some studies suggest that certain vaping conditions (e.g. high-voltage settings) strongly contribute to increasing the concentration of these harmful substances, which in turn are associated with inflammatory responses in different cell types. For example, exposure to e-cigarette aerosol has been shown to trigger pro-inflammatory effects in human bronchial epithelial cells and macrophages, leading to increased levels of inflammatory cytokines (Auschwitz et al., 2023; Buchanan et al., 2020).

As for flavors, a study analyzed several flavors commonly used in e-liquids and showed that each of these chemical compounds can trigger inflammatory and oxidative reactions in human lung cells that increase cellular and inflammatory stress, processes that are closely linked to respiratory and cardiovascular diseases. For example, cinnamon flavoring has been associated with particularly high toxicity (Muthumalage et al., 2018). Indeed, although cinnamon is a safe and approved food additive, its effects can be different when inhaled, and can even lead to toxicity. This is partly due to the thermal degradation of cinnamaldehyde during the vaporization process, producing reactive and potentially harmful compounds not typically generated during food or medicinal use. Another study confirmed these observations by showing that flavors such as mint and vanilla used in electronic nicotine delivery systems (ENDS) have toxic effects on the heart both in vitro and in vivo. These flavors impaired endothelial cell function and increased the risk of atherosclerosis and cardiovascular disease (Abouassali et al., 2021). Flavors, even without nicotine, have also been reported to cause marked cytotoxicity and inflammatory responses in human bronchial cells. Popular flavors such as tobacco, apple and tropical fruits were

tested, and some fruit flavors showed a high inflammatory potential and contributed to significant cell damage (Sinha et al., 2022a). Finally, a study focusing on tobacco and menthol flavors in nicotine-free e-cigarettes found that these specific flavors caused significant inflammation in lung epithelial cells and fibroblasts while interfering with cellular repair mechanisms (Wang et al., 2024). We chose to work with human aortic smooth muscle cells because these cells play a key role in the regulation of actomyosin, which is involved in contraction, and contribute to the stiffening of arteries, a clinical effect associated with e-cigarette use (Lacolley et al., 2017; Zhuge et al., 2020). These cells represent a relevant model for investigating the cardiovascular toxicity of e-liquids and aerosol condensates from these devices. Although smooth muscle cells are less frequently studied, partly because they are potentially exposed to lower concentrations of these substances, their importance lies in their direct involvement in arterial contraction mechanisms.

In contrast, endothelial cells have been studied more intensively as they are directly exposed to the condensate components that enter the bloodstream. Several studies have shown the effects of e-liquid condensates on endothelial cells. For example, Chhor et al. have shown that exposure of human coronary endothelial cells to aerosol condensates, even without nicotine, causes a significant reduction in cell viability, an increase in oxidative stress (due to increased production of reactive oxygen species), and an increase in inflammatory markers (Chhor et al., 2023). Another study by Anderson et al. showed that exposure to e-liquid aerosols causes an increase in reactive oxygen species (ROS), DNA damage, and cell death in endothelial cells, indicating a potential risk to vascular health (Anderson et al., 2016). In response to biostress,
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Relative cytotoxicity (A) and pro-inflammatory response (B) induced by the different experimental conditions assessed after 24 h of cell exposure through LDH release and IL-8 production. Results are expressed relative to control (unexposed) cells and are means of three independent experiments, each performed in quadruplicate. Statistical significance is also indicated: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 as determined with a two-ways ANOVA followed by Tukey's post-hoc test between control and experimental groups. For inter-group comparisons, a two-ways ANOVA followed by Tukey's post-hoc was also performed and indicated by: \#p < 0.05.</figcaption></figure>

endothelial cells activate a paracrine metabolic pathway that induces a reaction in smooth muscle cells. As a result, assessing the vascular response must also include evaluating its impact on smooth muscle cells. However, to date, only one toxicological study has been conducted on cardiovascular smooth muscle cells, which showed pro-inflammatory effects when aortic smooth muscle cells were exposed to e-liquid condensates containing cinnamon flavor and produced at high potency (Michon et al., 2022). These results emphasize the urgent need to deepen our understanding of the impact of electronic cigarettes on vascular smooth muscle cells in order to better assess their cardiovascular health risks.

Thermal degradation of flavors, particularly cinnamon, is a crucial aspect to consider in understanding the cytotoxic and pro-inflammatory effects observed in this study. Under high temperatures, compounds present in cinnamon flavor, such as cinnamaldehyde, undergo chemical transformations that generate potentially toxic degradation products. Among these products, we find aldehydes such as acetaldehyde and formaldehyde that can alter vascular regulatory mechanisms, promoting inflammation and oxidative damage (Jin et al., 2021). Cinnamaldehyde itself, although naturally present in cinnamon, has shown cytotoxicity and a pro-inflammatory effect when exposed to intense heating conditions, as occurs in high-power devices (Behar et al., 2016; Bengalli et al., 2017; Muthumalage et al., 2018). These degradation products can also interact with other components of e-liquids, amplifying their deleterious

effects (Kerber et al., 2022). It would therefore be relevant for future research to focus specifically on the identification of cinnamaldehyde degradation products and the in-depth assessment of their toxicity. In addition to the inflammatory and cytotoxic effects explored in this study, other important biological responses such as oxidative stress or DNA damage remain to be investigated to fully characterize the toxicity of e-cigarette aerosols. Moreover, the chemical analysis of e-cigarette aerosol by gas chromatography mass spectrometry (GC-MS) could be helpful to identify the specific volatile and semi-volatile compounds generated during vaporization and more precisely determine the compounds responsible for the observed toxicity. These data will help better understand the link between aerosol composition and cellular responses.

## 5. Conclusion

Our results highlight the significant cytotoxic and pro-inflammatory effects of e-cigarette aerosol condensates on aortic smooth muscle cells, in the presence of certain flavors such as cinnamon and at certain vaping conditions (mainly high-power settings of the device). It is noteworthy that the use of e-liquid containing the same flavors also produced effects, but they were less pronounced compared to the aerosol condensates. This suggests that the thermal degradation of flavors may enhance their cytotoxic and pro-inflammatory effects. The results emphasize the need for further research into the long-term effects of exposure to e-cigarettes
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Relative cytotoxicity (A) and pro-inflammatory response (B) induced by the different experimental conditions was evaluated after 24 h of cell exposure via LDH release and IL-8 production. The results are expressed relative to control (unexposed) cells and represent the mean of three independent experiments, each performed in quadruplicate. Statistical significance is also indicated: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001 as determined with a two-ways ANOVA followed by Tukey's post-hoc test between control and experimental groups. For inter-group comparisons, a two-ways ANOVA followed by Tukey's post-hoc was also performed and indicated by: \#p < 0.05. The nicotine used was 20 mg/mL and the PG/VG was 70/30. Nic: nicotine.</figcaption></figure>

on vascular smooth muscle cells, as these cells are crucial for maintaining vascular function and integrity. Future research should also investigate the effects of different e-liquid formulations (e.g., different flavors) and exposure durations to establish clearer links between e-cigarette use and cardiovascular effects. It would also be valuable to conduct a chemical analysis of the thermal degradation products contained in the condensates to better identify the compounds responsible for the increased toxicity observed at higher power levels.

### CRediT authorship contribution statement

**Mariam Bitar:** Writing – original draft, Visualization, Investigation, Formal analysis, Conceptualization.
**Clément Mercier:** Writing – review & editing, Methodology.
**Laurent Bertoletti:** Writing – review & editing, Supervision.
**Jérémie Pourchez:** Writing – review & editing, Supervision, Conceptualization.
**Valérie Forest:** Writing – review & editing, Supervision, Conceptualization.

### Funding

This research did not receive any specific grant from funding

agencies in the public, commercial, or not-for-profit sectors.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### References

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