# E-cigarette and endothelial function -a critical review of preclinical studies

Mariam Bitar, Jérémie Pourchez, Claire Le Hello, Laurent Bertoletti, Valérie Forest

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Mariam Bitar, Jérémie Pourchez, Claire Le Hello, Laurent Bertoletti, Valérie Forest. E-cigarette and endothelial function -a critical review of preclinical studies. Chemical Research in Toxicology, 2025, 10.1021/acs.chemrestox.5c00357. [hal-05349537](https://hal.science/hal-05349537v1)

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Distributed under a Creative Commons Attribution 4.0 International License
E-cigarette and endothelial function - a critical review of preclinical studies

Mariam Bitar†, Jérémie Pourchez†, Claire Le Hello‡, §, ||, Laurent Bertoletti‡, §, ||, Valérie Forest†*.

† Mines Saint-Etienne, Univ Jean Monnet, INSERM, U 1059 Sainbiose, Centre CIS, F-42023 Saint-Etienne, France

‡ Service de Médecine Vasculaire et Thérapeutique, CHU de Saint-Etienne, Saint-Etienne, France

§ INSERM, UMR1059, Equipe Dysfonction Vasculaire et Hémostase, Université Jean-Monnet, F-42055, Saint-Etienne, France

|| INSERM, CIC-1408, CHU Saint-Etienne, F-42055, Saint-Etienne, France

<footnote>* Corresponding author: Valérie Forest:
Mines Saint-Etienne</footnote>

<footnote n="158">158 cours Fauriel, CS 62362</footnote>

<footnote n="42023">42023 Saint-Etienne Cedex 2. FRANCE.</footnote>

<footnote>Email address: vforest@emse.fr - Telephone number: +33 477 499 776</footnote>
Table of Contents Graphic

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**Abstract**

The electronic cigarette has been suggested as a safer alternative to the conventional tobacco cigarette. However, some vaping products have been shown to have cardiovascular effects although this remains controversial. Several clinical studies have identified a possible alteration of endothelial function due to exposure to e-cigarette aerosols. However, the underlying biological mechanisms responsible for this observation in humans are still unclear. Thus, the development of preclinical mechanistic studies seems necessary. The aim of this literature review is therefore to provide a comprehensive overview of preclinical studies addressing the question of how e-cigarettes may cause endothelial dysfunction, a predictive marker of cardiovascular events. 53 papers were included in the analysis. We analyzed these papers qualitatively and quantitatively and discussed their limitations. We found that while 30% of *in vitro* studies showed no effect of e-cigarette aerosols on endothelial cells 26% showed variable effects and 44% showed a significant adverse effect on endothelial function. *In vivo* studies were more consistent, with the vast majority (96%) reporting negative effects of e-cigarettes on endothelial function. We concluded that e-cigarettes should not be considered harmless in terms of cardiovascular effects as they may impair endothelial function through various mechanisms such as oxidative stress and inflammation. However, more studies with standardized and optimized designs are still needed to distinguish the role of nicotine, which is known to affect the cardiovascular system, from that of other components in e-cigarette aerosol.

**Keywords:** Electronic cigarette; E-cig; Vaping; Endothelial function; Preclinical studies.
# 1. Introduction

The adverse health effects of tobacco smoking are well documented, not only on the lungs but also on the cardiovascular system. A clear relationship has been established between smoking and cardiovascular diseases, including atherosclerosis and hypertension, coronary artery disease, heart failure, peripheral arterial disease, cardiac arrhythmia or arrest, and venous thromboembolism ¹.

The electronic cigarette (or e-cig) has been proposed as an alternative to the traditional tobacco cigarette, especially as a smoking cessation tool. However, its safety has not been clearly demonstrated and its ability to have effects on the lungs and cardiovascular system remains controversial as conflicting results have been reported in the literature. Nevertheless, it appears that e-cigarettes are probably less harmful than traditional tobacco cigarettes. This claim is mainly based on the chemical composition of e-cigarette aerosol compared to tobacco cigarette smoke ²,³. As no tobacco is burnt in the e-cigarette, the aerosol emitted by vaping products does not contain carbon monoxide or most of the harmful thousands of combustion products found in tobacco cigarette smoke. However, e-cigarettes cannot be considered as totally harmless. The carrier solvents of e-liquid are mainly propylene glycol (PG) and vegetable glycerin (VG). E-liquid may also contain nicotine and/or flavorings ². When heated, the components of e-liquids can form thermal dehydration products, including aldehydes (acetaldehyde, formaldehyde, acrolein, methylglyoxal), organic compounds (propylene oxide, acetol, allyl alcohol, glyoxal, glycidol), heavy metals, and particulate matter ²⁻⁷. The formation of aldehydes is strongly dependent on the temperature, which in turn depends on the power supplied to the atomizer. The neoformed substances during the aerosol generation of greatest concern in terms of cardiovascular disease are acrolein and, to a lesser extent, formaldehyde and acetaldehyde, both of which have been shown to be carcinogenic to humans and are known to cause oxidative stress and inflammation ²,⁴,⁵.

Nicotine has been shown to have a variety of effects on the cardiovascular system ⁴,⁸. Indeed, nicotine causes an increase in heart rate, which can lead to acute tachycardia, and also tends to increase blood pressure, creating a breeding ground for hypertension ⁹⁻¹¹. In addition, nicotine impairs the ability of
coronary arteries to dilate properly, reducing coronary vasodilator reserve and increasing the risk of myocardial ischemia <sup>12-15</sup>. These circulatory disturbances are associated with an imbalance in vascular resistance, causing vasoconstriction of the skin vessels and vasodilation of the muscle vessels, which in turn contributes to hypertension. Nicotine also alters the composition and fluidity of the blood. It increases the viscosity of the blood and promotes platelet aggregation, which increases the risk of thrombosis <sup>16-18</sup>. Nicotine's ability to reduce the production of nitric oxide leads to endothelial dysfunction while the increase in total and LDL cholesterol levels favors the development of atherosclerosis <sup>19</sup>. Finally, by increasing insulin resistance, nicotine can promote the development of macrovascular complications (such as atherosclerosis in the large blood vessels and coronary heart disease), thus increasing its overall detrimental effect on cardiovascular health <sup>20</sup>. The differences in the effects of nicotine on the cardiovascular system between vaping and smoking are due to the different distribution kinetics of nicotine between e-cigarettes and regular tobacco cigarettes.

Therefore, further preclinical and mechanistic studies are needed to assess whether nicotine delivery from e-cigarettes produces similar toxicity to that of regular tobacco cigarettes <sup>4</sup>. Finally, the toxicity of the aerosol can vary greatly depending on the presence and type of flavors in the e-liquid <sup>2-6,21,22</sup>.

It is widely recognized that endothelial dysfunction (microscopically characterized by disrupted regeneration of endothelial cells and impaired endothelium-dependent vasodilatory mechanisms) is associated with atherogenesis, progression of atherosclerosis, and cardiovascular events <sup>3,6</sup>. It is therefore considered an early critical event in the pathogenesis of most cardiovascular diseases <sup>23,6</sup>.

For this reason, both *in vitro*, *in vivo* and epidemiological studies have focused on the impact of e-cigarettes on endothelial function. In recent years, three human studies <sup>24-26</sup> have shown a possible alteration of endothelial function by exposure to e-cigarette aerosols, although one other study <sup>27</sup> suggests a possible impairment of vascular function specifically in relation to the endothelium but without significant change in flow-mediated dilation (FMD). However, the underlying mechanisms are still unclear. The development of preclinical studies seems necessary to determine the biological mechanisms responsible for this observation in humans and to identify the key parameters involved
such as (i) the type of e-cigarette device and device-related parameters such as power level or
resistance, (ii) the presence of certain compounds in the formulation of e-liquids, such as certain
flavors, nicotine dosage or the presence of organic acids to form nicotine salts, (iii) the chemical
composition of the generated aerosol, which includes the two aforementioned points, possibly
resulting in a newly formed compounds due to thermal decomposition during aerosol generation.

These preclinical works focusing on endothelial function could also allow to identify the key
parameters that play a role in relation to device parameters and liquid formulation in order to propose
sanitary management measures. The aim of this literature review is therefore to provide a
comprehensive overview of preclinical studies addressing the question of how e-cigarettes may cause
endothelial dysfunction, a predictive marker of cardiovascular events.

## 2. Review of the literature

Figure 1 schematically shows the method, the results and the analysis we carried out, which is
explained in more detail below.

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**2.1. Method**

We selected the following keywords: (e-cigarette OR e-cig OR electronic cigarette OR vaping OR electronic nicotine delivery system) AND (endothelial cells OR endothelial damage OR endothelial dysfunction OR endothelial toxicity). We searched two databases: PubMed and Web of Science. We eliminated duplicates and analyzed the remaining papers. Papers were excluded from our selection based on 5 criteria: i) if they were reviews and thus did not contain original experimental data, ii) if the studies did not involve e-cigarette vaping (e.g. studies on traditional cigarette smoking or heated tobacco products) or did not focus on endothelial cells (e.g. studies on platelets or epithelial cells...), iii) if the studies did not use samples that were representative of total e-cigarette aerosol (i.e. studies assessing the effects of isolated components such as flavors alone, nicotine alone or unheated e-liquids), iv) if the studies were clinical trials, as the topic of this review was the biological effects of e-cigarette aerosols and it is too difficult to draw firm conclusions from studies with human subjects (studies conducted on vapers or vapers and smokers or vapers who are former smokers...), due to inter-individual sensitivity and heterogeneity of experimental designs, and v) if the papers were not written in English.

**2.2. Results**

As shown in Figure 1, the search (performed on 26 March 2025) in PubMed yielded 284 papers, while the search in Web of Science yielded 233 references. After the elimination of duplicates, 381 papers remained to be further analyzed. 328 papers were excluded and the remaining 53 papers were classified as *in vitro*, *in vivo* or both *in vitro* and *in vivo* studies corresponding to 23, 23 and 7 references, respectively.
2.3. Analysis of the results

Table 1 and Table 2 summarize the studies in which the *in vitro* and *in vivo* effects of e-cigarettes on endothelial function were investigated. Table 3 details the studies in which both *in vitro* and *in vivo* studies were conducted.
<table><caption>**Table 1** – Summary of the studies assessing the *in vitro* effects of e-cigarette on endothelial function., AqE: aqueous aerosol extracts, ROS: reactive oxygen species, BBB: blood brain barrier, HUVEC: human umbilical vein endothelial cells.</caption><thead><tr><th>Authors</th><th>Year</th><th>Biological model and exposure</th><th>Biological endpoints</th><th>Main results / conclusion</th><th>Underlying mechanism(s) of toxicity*</th><th>Ref</th></tr></thead><tbody><tr><td>Mahgoup et al.</td><td>2025</td><td>Mouse vascular smooth muscle cells (VSMCs) exposed to e-cigarette extract diluted 0.0625-8% for 4, 24 and 48h.</td><td>Cell viability, NO metabolism rate.</td><td>Decreased cell viability in an e-cigarette extract concentration and time-dependent manner. Increased rate of NO metabolism.</td><td>NO</td><td>28</td></tr><tr><td>Blackham-Hayward et al.</td><td>2024</td><td>Human pulmonary microvascular endothelial cells (HPMVEC) exposed to e-cigarette extract diluted 0.5-20% for 24h.</td><td>Pulmonary endothelial barrier integrity, oxidative stress and inflammation.</td><td>Barrier disruptive effect of nicotine-free e-cigarette fluid on the pulmonary microvasculature.</td><td></td><td>29</td></tr><tr><td>Kuntic et al.</td><td>2023</td><td>Endothelial cells (EA.hy 926) exposed to condensed e-cigarette aerosol (e-cigarette condensate) and acrolein for 3 days.</td><td>Cell viability, oxidative stress (ROS formation).</td><td>Induction of a concentration and time-dependent cell death by e-cigarette condensate and acrolein. Oxidative stress induced by acrolein.</td><td></td><td>30</td></tr><tr><td>Liu et al.</td><td>2023</td><td>Patient-specific induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals and patients with pulmonary arterial hypertension exposed to e-cigarette AqE for 48h.</td><td>Cell viability, intracellular ROS production, cell migration, gene expression.</td><td>PAH iPSC-ECs showed a greater amount of damage than healthy iPSC-ECs upon e-cigarette exposure.</td><td></td><td>31</td></tr><tr><td>Mekala et al.</td><td>2022</td><td>Human brain microvascular endothelial cells (hBMVEC, D3 cell-line) exposed to e-cigarette (1.8% or 0% nicotine) conditioned medium.</td><td>Mitochondrial oxidative phosphorylation.</td><td>Reduced mitochondrial oxidative phosphorylation.</td><td></td><td>32</td></tr><tr><td>Caruso et al.</td><td>2023</td><td>HUVECs exposed to AqE (40-100%) for 6h.</td><td>Endothelial cell migration (scratch wound assay).</td><td>AqEs did not induce the inhibition of endothelial cell migration.</td><td></td><td>33</td></tr></tbody></table>

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<table><tr><td>Su et al.</td><td>2022</td><td>HUVECs exposed to e-cigarette aerosol extracts (ECA).</td><td>Cell viability, apoptosis, adhesion molecules expression, inflammatory cytokines release, intracellular reactive oxygen species (ROS) formation.</td><td>Increased expression of adhesion molecules and pro-inflammatory cytokines.</td><td></td><td>34</td></tr><tr><td>Le et al.</td><td>2021</td><td>Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) exposed to e-cig aerosol extract for 24h.</td><td>Expression profile of long noncoding RNAs by microarray analysis.</td><td>Differentially expressed long noncoding RNAs and several potential regulators and pathways in ECs exposed to e-cig.</td><td></td><td>35</td></tr><tr><td>Giebe et al.</td><td>2021</td><td>Primary HUVECs exposed to AqE from e-cigarette.</td><td>Cell viability, monocyte-endothelial cell adhesion, wound healing assay.</td><td>No reduced endothelial cell viability and wound healing capability.</td><td></td><td>36</td></tr><tr><td>Moser et al.</td><td>2021</td><td>Human umbilical vein endothelial cells (HUVEC / TERT 2 cells) exposed to aerosol condensates from e-cigarette.</td><td>Metabolic activity of cells.</td><td>Significantly decreased cellular metabolic activity by treatment with e-cigarette aerosol condensate (at a high level).</td><td></td><td>37</td></tr><tr><td>Simms et al.</td><td>2021</td><td>HUVECs, alone or with immune cells, exposed to aerosol extracts.</td><td>Biological activity for a wide range of biomarkers.</td><td>Aerosols were found to be weakly active.</td><td></td><td>38</td></tr><tr><td>De Martin et al.</td><td>2021</td><td>HUVEC/tert2 immortalized cells exposed to e-cigarette condensates obtained from different e-liquids.</td><td>Cytotoxicity and metabolic activity of cells.</td><td>Cytotoxicity of the e-cigarette condensates dependent on the nature of the e-liquid.</td><td></td><td>39</td></tr><tr><td>Makwana et al.</td><td>2021</td><td>Primary Human Aortic Endothelial cells (HAECs) exposed to e-cigarette aerosol conditioned media for 24h.</td><td>Monocyte adhesion to HAECs.</td><td>No dose-dependent increase in monocyte adhesion.</td><td></td><td>40</td></tr><tr><td>Bozhilova et al.</td><td>2020</td><td>HUVECs exposed to AqE from different e-cigarette for 24h.</td><td>Cytotoxicity.</td><td>Variable effect depending on the e-cigarette.</td><td></td><td>41</td></tr><tr><td>Breheny et al.</td><td>2020</td><td>Primary HUVECs exposed to AqEs for 24h.</td><td>Endothelial migration rate assessed by the endothelial wound repair (scratch wound assay).</td><td>Little to no effect on wound healing even at the highest concentration (100 % AqE).</td><td></td><td>42</td></tr><tr><td>Sivandzade et al.</td><td>2019</td><td>Mouse primary brain microvascular endothelial cells (mBMEC-P3) exposed to 5% e-cigarette extract solution containing 2.4% nicotine for 24h.</td><td>BBB integrity assessed by the trans-endothelial electrical resistance measurement, cell viability, ROS formation, tight junction proteins expression.</td><td>Significant increase in intracellular ROS, downregulation of the expression of the tight junction accessory protein ZO-1, decreased barrier integrity.</td><td></td><td>43</td></tr></table>
<table><tr><td>Kaisar et al.</td><td>2018</td><td>Mouse brain endothelial cells (bEnd.3 cell line) and mouse primary brain microvascular endothelial cells (mBMEC) exposed to 5% diluted e-cigarette extracts for 72h.</td><td>Mitochondrial membrane potential.</td><td>Disruption of the mitochondrial membrane potential in bEnd.3 cells but not in the mBMEC.</td><td></td><td>44</td></tr><tr><td>Bengalli et al.</td><td>2017</td><td>Co-culture of alveolar and lung microvascular endothelial cells (NCI-H441 epithelial lung cells/ HPMEC-ST1.6R endothelial cells) exposed to condensed aerosols from e-liquids with different favours, with or without nicotine.</td><td>Cell viability, pro-inflammatory cytokines release and alveolar-blood barrier (ABB) integrity.</td><td>Toxicity influenced by the flavor composition but not by the presence of nicotine. Cell viability significantly decreased after exposure to the mints and cinnamon condensed aerosols. No increase of the inflammatory cytokines release.</td><td></td><td>45</td></tr><tr><td>Taylor et al.</td><td>2017</td><td>Primary HUVECs exposed to AqE (40-100%) for 6h.</td><td>Endothelial migration (scratch wound assay).</td><td>No significant reduction in HUVEC migration rate.</td><td></td><td>46</td></tr><tr><td>Anderson et al.</td><td>2016</td><td>HUVECs exposed to e-cigarette aerosol extract (EAE) for 4-72h.</td><td>ROS formation, DNA damage, cell viability, and markers of programmed cell death pathways.</td><td>Concentration-dependent induction of ROS, DNA damage, and reduced cell viability.</td><td></td><td>47</td></tr><tr><td>Barber et al.</td><td>2016</td><td>HUVECs exposed to extracts produced from e-cigarette for 48h.</td><td>Endothelial cell inflammatory processes, viability, density and metabolic activity.</td><td>Significant increase in complement deposition, expression of the receptors for C1q, significant decrease in endothelial cell viability, cell proliferation and metabolic activity.</td><td></td><td>48</td></tr><tr><td>Putzhammer et al.</td><td>2016</td><td>HUVECs exposed to aerosols produced by 4 different brands of e-cigarettes and to aerosols generated from 6 different e-liquids (using the same device) for 1, 24 or 48h.</td><td>Cell death induction, proliferation rates, intracellular ROS, and cell morphology.</td><td>High variability in the acute cytotoxicity depending on the liquid and e-cigarette used. Some e-cigarette aerosol extracts showed high cytotoxicity (5/11), inhibition of cell proliferation (5/11), induction of oxidative stress (1/11) and alterations in cell morphology.</td><td></td><td>49</td></tr></table>
<table><tr><td>Teasdale et al.</td><td>2016</td><td>Human coronary artery endothelial cells (HCAEC) obtained from 3 to 6 different donors exposed to aqueous filtered extracts of e-cigarette aerosol.</td><td>Analysis of gene expression changes.</td><td>E-cigarette aerosol extract did not activate the oxidative stress pathway.</td><td></td><td>50</td></tr></table>

* Underlying mechanism(s) of toxicity:

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Change in gene/protein expression

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Cell death/cytotoxicity

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Barrier disruptive effect

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Oxidative stress

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Pro-inflammatory effect

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Metabolic/biological activity

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Mitochondrial dysfunction

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DNA damage

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Nitric Oxide production

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No significant toxic effect observed
<table><caption>**Table 2** – Summary of the studies assessing the *in vivo* effects of e-cigarette on endothelial function., ROS: reactive oxygen species, BBB: blood brain barrier, FMD: flow-mediated dilation, MCA: middle cerebral artery.</caption><thead><tr><th>Authors</th><th>Year</th><th>Biological model and exposure</th><th>Biological endpoints</th><th>Main results / conclusion</th><th>Underlying mechanism(s) of toxicity*</th><th>Ref</th></tr></thead><tbody><tr><td>Mills et al.</td><td>2025</td><td>Pregnant Sprague-Dawley rats exposed to e-cigarette (60 puffs/day, 5 days/week for 3 weeks).</td><td>Vascular function in the offspring at 1-, 3-, 6- and 12-months of age.</td><td>Impaired middle cerebral artery reactivity.</td><td></td><td>51</td></tr><tr><td>Mills et al.</td><td>2025</td><td>Pregnant Sprague-Dawley rats exposed to e-cigarette (60 puffs/day, 5 days/week for 3 weeks).</td><td>Maternal vascular function.</td><td>Maternal vascular dysfunction, significant impairment of middle cerebral artery reactivity.</td><td></td><td>52</td></tr><tr><td>Mills et al.</td><td>2024</td><td>Pregnant Sprague-Dawley rats exposed to e-cigarette during 1st, 2nd, 3rd trimester or full-term gestation (60 puffs/day, 5 days/week).</td><td>Vascular function in the offspring.</td><td>Maternal exposure to e-cigarette in the second and third trimesters results in a decreased vascular reactivity of the middle cerebral artery (MCA) reactivity.</td><td></td><td>53</td></tr><tr><td>Mills et al.</td><td>2024</td><td>Pregnant Sprague-Dawley rats exposed to e-cigarette (60 puffs/day, 5 days/week for 3 weeks) + 2- or 5-months offspring exposed to e-cig (20 puffs/day, 5 days/week for 4 weeks).</td><td>Vascular function in the offspring.</td><td>Increased vascular dysfunction.</td><td></td><td>54</td></tr><tr><td>Jin et al.</td><td>2024</td><td>Male and female C57BL/6J wild-type (WT) and TRPA1-null mice (TRPA-1: transient receptor potential ankyrin-1) exposed to e-cigarette (20 sessions of 18 puffs per session daily for 4 days).</td><td>Vascular function.</td><td>Short-term exposures to e-cigarette aerosol induced endothelial dysfunction in female WT but not in female TRPA1-null mice.</td><td></td><td>55</td></tr><tr><td>Han et al.</td><td>2023</td><td>Sprague-Dawley rats exposed to aerosols from e-cigarette (10 cycles of 5 sec exposure over 5 min).</td><td>Flow-mediated dilation (FMD).</td><td>FMD significantly impaired.</td><td></td><td>56</td></tr><tr><td>Mills et al.</td><td>2023</td><td>Male and female C57BL/6 mice exposed to e-cigarette aerosol generated from pure</td><td>Vascular outcomes in a conducting vessel (thoracic aorta) and a resistance artery (middle cerebral artery, MCA).</td><td>Aortic endothelial-dependent dilation (EDD) impaired with PG exposure. In the MCA, EDD response impaired by both VG and PG.</td><td></td><td>57</td></tr></tbody></table>
<table><tr><td></td><td></td><td>vegetable glycerin (VG) or pure propylene glycol (PG) for 60-100 min.</td><td></td><td></td><td></td></tr><tr><td>Shabestari et al.</td><td>2023</td><td>Wistar rats exposed to e-cigarette for 40 min, 3 times a day for 4 mo.</td><td>Serum parameters, prostate pathology, and gene expression (ex vivo analyses).</td><td>Hyperemia, induced inflammatory cell infiltration and hypertrophy of smooth muscle of the vascular wall.</td><td>58</td></tr><tr><td>Pitzer et al.</td><td>2023</td><td>C57BL/6 mice exposed to 10 puffs of e-cigarette aerosol (single exposure).</td><td>Endothelial function in the arterioles of the gluteus maximus muscle (vessels diameters, arteriole diameters and reactivity).</td><td>Peripheral vasoconstriction.</td><td>59</td></tr><tr><td>Sifat et al.</td><td>2022</td><td>Male C57BL/6 mice exposed to e-cigarette aerosol for 14 d followed by a transient middle cerebral artery occlusion (tMCAO) with reperfusion to mimic ischemic stroke.</td><td>Plasma levels of IL-6 and thrombomodulin, BBB tight junction proteins, key inflammatory and oxidative stress markers expression (ex vivo analyses).</td><td>Upregulation of IL-6 and decreased plasma thrombomodulin levels. Post-ischemic brain injury following tMCAO significantly worsened by e-cig aerosol pre-exposure. Downregulation of tight junction proteins and antioxidant Nrf2 expression. Upregulation of ICAM-1.</td><td>60</td></tr><tr><td>Mills et al.</td><td>2022</td><td>Male Sprague-Dawley rats exposed to e-cigarette (20 or 60 puffs).</td><td>Middle cerebral artery (MCA) reactivity.</td><td>Effect on endothelial-dependent dilation within 24h of exposure, impairment persistent for up to 3 days.</td><td>61</td></tr><tr><td>El-Mahdy et al.</td><td>2022</td><td>C57BL/6 male mice were exposed to e-cigarette aerosol generated from e-cig for 16 and 60 wk.</td><td>Vascular reactivity, blood pressure, cardiac function, in situ detection of superoxide and nitric oxide in the aorta.</td><td>Time-dependent elevation in blood pressure and systemic vascular resistance, impairment of acetylcholine-induced aortic relaxation. Decreased intravascular nitric oxide levels and increased superoxide generation.</td><td>62</td></tr><tr><td>Rao et al.</td><td>2022</td><td>Male and female Sprague-Dawley rats exposed to aerosols from different e-cigarettes (single exposure of 5 min).</td><td>Arterial flow-mediated dilation (FMD).</td><td>Significantly impaired FMD.</td><td>63</td></tr><tr><td>Zhang et al.</td><td>2022</td><td>C57BL/6 male mice exposed to Juul electronic nicotine aerosol (3 s puff</td><td>Expression of inflammatory cytokines (Western blot) in brain tissue (ex vivo analyses).</td><td>Immunomodulator IL-5 significantly upregulated.</td><td>64</td></tr></table>
<table><tr><td>Burrage et al.</td><td>2021</td><td>Pregnant female Sprague Dawley rats exposed to e-cigarette aerosol (1 h/day, 5 days/wk, starting on gestational day 2 until pups were weaned).</td><td>Middle cerebral artery (MCA) reactivity.</td><td>Reduction in endothelial-dependent dilation response to acetylcholine in 1, 3, and 7-mo-old offspring.</td><td></td><td>65</td></tr><tr><td>El-Mahdy et al.</td><td>2021</td><td>Male C57BL/6 male mice exposed 2 h/day, 5 days/wk to e-cigarette vaping up to 60 wk.</td><td>Blood pressure, vascular reactivity, cardiac function and morphology.</td><td>After 16 wk, impaired endothelium-dependent and -independent vasodilation in aortic segments, onset of systemic hypertension, elevations in vascular resistance. After 32 wk, cardiac hypertrophy. All of these abnormalities increased out to 60 wk, with increased heart weight, aortic thickness and superoxide production.</td><td></td><td>66</td></tr><tr><td>Jin et al.</td><td>2021</td><td>Male and female C57BL/6J mice exposed by inhalation to PG-VG-derived aerosol for 4 d.</td><td>Biomarkers of exposure and cardiopulmonary injury.</td><td>Significantly altered endothelium-dependent relaxation, decreased white blood cells, increased red blood cells and hemoglobin.</td><td></td><td>67</td></tr><tr><td>Szostak et al.</td><td>2020</td><td>Female apolipoprotein E-deficient (ApoE-/-) mice exposed to e-cigarette aerosols for up to 6 mo.</td><td>Atherosclerosis progression, cardiovascular function, molecular changes in the heart and aorta.</td><td>No accelerated atherosclerotic plaque formation. Ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged.</td><td></td><td>68</td></tr><tr><td>Kuntic et al.</td><td>2020</td><td>Male C57BL/6 mice exposed to e-cigarette aerosol over 1, 3, or 5 days for 2 h/day (6 times 20 min).</td><td>Endothelial function, oxidative stress.</td><td>Significant impairment of the endothelial function, increased aortic ROS formation.</td><td></td><td>69</td></tr><tr><td>Rao et al.</td><td>2020</td><td>Male and female Sprague-Dawley rats exposed to aerosols produced by an e-cigarette using unflavored freebase nicotine e-liquid and to aerosols produced by a JUUL pod (10 cycles of 2 s inhalation over 5 min).</td><td>Endothelial function assessed as arterial flow-mediated dilation (FMD).</td><td>Impaired FMD.</td><td></td><td>70</td></tr></table>
<table><tr><td>Heldt et al.</td><td>2020</td><td>Male C57BL/6 mice exposed to e-cigarette (tobacco-flavored e-liquid without or with nicotine (18 mg/mL)), 2 h daily, 5 days a week for 8 weeks.</td><td>Brain histology, gene expression in cerebral microvessel, expression of tight junction proteins, BBB permeability (ex vivo analyses).</td><td>Expression of occludin at vessel tight junctions significantly decreased in the cortex, BBB permeability increased following nicotine-free e-cigarette exposure.</td><td></td><td>71</td></tr><tr><td>Shi et al.</td><td>2019</td><td>Adult male and female C57BL/6 mice exposed to e-cigarette vaping for 2-weeks, 3 h/day.</td><td>Cardiac function.</td><td>No significant effect on cardiac contractility. Significantly increased angiogenesis in heart tissue. Increased endothelial cell markers CD31 and CD34.</td><td></td><td>72</td></tr><tr><td>Olfert et al.</td><td>2018</td><td>Female mice (C57BL/6) exposed daily to e-cigarette aerosol 4 h/day, 5 days/wk for 8 mo.</td><td>Cardiac function and aortic arterial stiffness (AS).</td><td>Accelerated AS, significantly impaired aortic endothelial function.</td><td></td><td>73</td></tr></table>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

* Underlying mechanism(s) of toxicity:

<figure><img src="image_23.png" /></figure>

Change in gene/
protein expression

<figure><img src="image_24.png" /></figure>

Oxidative stress

<figure><img src="image_25.png" /></figure>

Vascular effect (dysfunction,
reactivity, dilation,
constriction, etc.)

<figure><img src="image_26.png" /></figure>

No significant toxic effect
observed

<figure><img src="image_27.png" /></figure>

Barrier disruptive effect

<figure><img src="image_28.png" /></figure>

Pro-inflammatory effect

<figure><img src="image_29.png" /></figure>

Blood pressure
<table><caption>**Table 3** – Summary of the studies assessing the effects of e-cigarette on endothelial function both *in vitro* (light blue) and *in vivo* (dark blue), ROS: reactive oxygen species.</caption><thead><tr><th>Authors</th><th>Year</th><th>Biological models and exposure</th><th>Biological endpoints</th><th>Main results / conclusion</th><th>Underlying mechanism(s) of toxicity*</th><th>Ref</th></tr></thead><tbody><tr><td rowspan="2">Dai et al.</td><td rowspan="2">2025</td><td>Human pulmonary microvascular endothelial cells (HPMVECs) exposed to e-cigarette extract diluted 10, 20 and 30% for 24h.</td><td>Mitochondrial function, endothelial cell viability.</td><td>Increase in mitochondrial reactive oxygen species (ROS), mitochondrial membrane depolarization.</td><td></td><td rowspan="2">74</td></tr><tr><td>Male C57BL/6J mice exposed to e-cigarette aerosol (4 times daily, 5 days/week for 16 weeks).</td><td>Lung histological analysis.</td><td>Vascular remodeling.</td><td></td></tr><tr><td rowspan="2">Meister et al.</td><td rowspan="2">2023</td><td>Human microvascular endothelial cells (HMVECs) exposed to 0.5% (v/v) e-cigarette condensate for 24h.</td><td>ROS production, NO measurement.</td><td>Reduction in NO production.</td><td></td><td rowspan="2">75</td></tr><tr><td>C57BL/6 male mice exposed to e-cigarette aerosol 1 h/day, 5 times/week for 12 weeks.</td><td>Histological analysis, protein and mRNA expression analysis in tissue.</td><td>E-cigarette exposure induced vascular oxidative stress.</td><td></td></tr><tr><td rowspan="2">Roxlau et al.</td><td rowspan="2">2023</td><td>Human and mouse primary pulmonary arterial smooth muscle cells (PASMC) exposed to nicotine-containing e-cigarette aerosol extract or nicotine-free extract (15 puffs).</td><td>Metabolic activity, cell proliferation, gene expression patterns.</td><td>E-cigarette aerosol extract but not nicotine-free e-cigarette aerosol extract increased the mRNA expression of inducible nitric oxide synthase (Nos2) and cyclin A1 (Ccna1) specifically in mPASMC.</td><td></td><td rowspan="2">76</td></tr><tr><td>C57BL/6J mice exposed to nicotine-containing e-cigarette aerosol extract or nicotine-free e-cigarette aerosol extract for 6 h/day, 5 days/week for 8 months.</td><td>Pulmonary vascular function and structure, heart function, inflammatory mediators in bronchoalveolar lavage fluid, hemodynamic measurements.</td><td>Pulmonary inflammatory response, structural and functional pulmonary changes without effects on the lung vasculature.</td><td></td></tr></tbody></table>
<table><tr><td>Chhor et al.</td><td>2023</td><td>Human coronary artery endothelial cells (HCAEC) exposed to e-cigarette aerosol condensate for 24h.<br>Balb/c female mice exposed to e-cigarette aerosol generated from tobacco flavored e-liquid with or without nicotine, twice daily for 12 weeks.</td><td>Cell viability, ROS production, expression of cell adhesion molecules.<br>Immunohistochemistry of the heart tissue, mRNA expression.</td><td>Decrease in cell viability, increased ROS.<br>Upregulation of anti-angiogenic marker FKBPL and endothelial cell adhesion molecule ICAM-1 expression.</td><td></td><td>77</td></tr><tr><td>Wölkart et al.</td><td>2023</td><td>Porcine aortic endothelial cells (PAECs) exposed to diluted vapor from e-cigarette using an unflavored e-liquid containing nicotine (20 mg/ml) in 50/50 (vol/vol.) glycerol/propylene glycol for 24h.<br>Rings of thoracic aorta isolated from Sprague-Dawley rats and rings isolated from porcine coronary artery exposed to e-cigarette aerosol for 24h.</td><td>NO synthase (NOS) activity, accumulation of intracellular cGMP, endothelial cell viability.<br>Ex vivo analysis of vessel function (maximal contractility, endothelial-dependent relaxation).</td><td>No effect on cell viability, nor on the endothelial NO/cGMP signaling.<br>No significant effect on vessel function.</td><td></td><td>78</td></tr><tr><td>Liu et al.</td><td>2022</td><td>Bovine aortic endothelial cells (BAECs) exposed for 24h to e-cigarette aerosol extract from e-cigarette with various contents of nicotine (0, 1.2%, 2.4%), and various flavors.<br>Male C57BL/6 streptozotocin-induced diabetic mice had a 6-mm skin wound made by a biopsy punch. The wound area was treated with VEGF in the presence or absence of e-cigarette aerosol extract every other day till day 12.</td><td>Determination of superoxide production, endothelial cell apoptosis, endothelial cell monolayer wound closure assay.<br>Wound healing assay.</td><td>Flavored and nicotine containing e-cigarette aerosol extract increased endothelial superoxide production, decreased endothelial NO bioavailability, induced endothelial cell apoptosis and impaired endothelial cell wound healing.<br>Flavored and nicotine containing e-cigarette aerosol extract induced impaired diabetic wound healing in vivo.</td><td></td><td>79</td></tr></table>
<table><tr><td rowspan="2">Schweitzer et al.</td><td rowspan="2">2015</td><td>Primary rat lung endothelial cells (RLEC), primary mouse lung endothelial cells (MLEC) and primary human microvascular cells-lung derived (HMVEC) exposed to e-cigarette aerosol (1–20 mM nicotine).</td><td>Trans-endothelial electrical resistance, cell proliferation/metabolic activity, cytotoxicity.</td><td>Dose-related loss of endothelial barrier.</td><td></td></tr><tr><td>Female C57Bl/6 mice exposed to condensed e-cigarette aerosol to for 30 min, or 24 h.</td><td>Oxidative damage quantified in plasma or bronchoalveolar lavage fluid, ROS level.</td><td>Inflammation and oxidative stress observed.</td><td></td></tr></table>

* Underlying mechanism(s) of toxicity:

<figure><img src="image_30.png" /></figure>

Change in gene/
protein expression

<figure><img src="image_31.png" /></figure>

Cell migration

<figure><img src="image_32.png" /></figure>

Mitochondrial dysfunction

<figure><img src="image_33.png" /></figure>

Nitric Oxide production

<figure><img src="image_34.png" /></figure>

Cell death/cytotoxicity

<figure><img src="image_35.png" /></figure>

Pro-inflammatory effect

<figure><img src="image_36.png" /></figure>

Vascular effect (dysfunction, reactivity, dilation, constriction, etc.)

<figure><img src="image_37.png" /></figure>

No significant toxic effect
observed

<figure><img src="image_38.png" /></figure>

Barrier disruptive effect

<figure><img src="image_39.png" /></figure>

Oxidative stress
Then, we performed a quantitative analysis of the results of all these preclinical studies, as reported in Figure 2.

<figure><img src="image_40.png" /><figcaption>**Figure 2** – Quantitative analysis of the results of the preclinical studies.</figcaption></figure>

Results are clearly heterogenous between *in vitro* and *in vivo* studies.

Over the 23 *in vitro* studies, 7 (30%) showed no effect of e-cigarette aerosols on endothelial cells 33,36,38,40,42,46,50, 6 (26%) showed variable effects 34,39,41,44,45,49 and 10 (44%) showed a significant adverse effect on the endothelial function 28–32,35,37,43,47,48.

The latter report a broad spectrum of negative effects on endothelial cells: reduced cell viability, induction of oxidative stress (intracellular ROS production), decreased metabolic activity, reduced cell proliferation, down-regulation of tight junction protein expression, reduced barrier integrity, DNA damage, etc.

*In vivo* studies are more consistent, with 22 out of 23 studies (96%) reporting negative effects of e-cigarettes on endothelial function 51–67,69–73, one study (4%) found no change caused by e-cigarettes 68.

The main effects observed *in vivo* are an impairment of flow-mediated dilation (FMD, a conventional measure of vascular function) and inflammatory response.

Finally, in the group of papers reporting both *in vitro* and *in vivo* studies, 1 out of 7 reported no effects (14.2%) 78, 1 reported variable effects (14.2%) 76 and 5 reported adverse effects (71.4%) 74,75,77,79,80.

**3. Discussion**
Despite varying results from preclinical studies, acute adverse effects of e-cigarettes on cardiovascular health have been evidenced. Figure 3 schematically summarizes the main biological effects of e-cigarette aerosols on the cardiovascular system. The main underlying mechanisms are oxidative stress and inflammation, causing endothelial dysfunction and associated adverse cardiovascular outcomes ¹,⁷,⁸¹⁻⁸⁴. Endothelial cells indeed play a key role in the regulation of vascular tone, inflammation, vascular growth, platelet aggregation, and blood coagulation. They produce vasodilatory substances such as nitric oxide and prostacyclin, which have anti-atherosclerotic and anti-aggregatory effects, and ensure a healthy vascular system. Impaired endothelial function is a hallmark of all cardiovascular diseases ²³,⁸¹. It has also been shown that the toxicity of e-cigarette aerosols on endothelial cells is influenced by the composition of the e-liquids (i.e. the presence and type of flavors) ²²,³⁹,⁴⁵,⁴⁹,⁷⁹.

<figure><img src="image_41.png" /><figcaption>**Figure 3** – Graphical summary of the biological effects of e-cigarette aerosols and their impact on the cardiovascular system. Created with BioRender.com.</figcaption></figure>
When analyzing the preclinical studies, their limitations should be kept in mind. While acute effects are frequently reported, fewer studies address the long-term effects of e-cigarettes on cardiovascular risk and the results are controversial ⁸¹. While abnormal flow-mediated dilation or aortic stiffness are strong markers for future cardiovascular events, an acute stimulus mediated by e-cigarette use that causes transient abnormalities might not be a relevant marker, especially if e-cigarettes are only used intermittently ².

Very importantly, comparisons between studies are hampered by the lack of standardized methods for assessing the effects of e-cigarette on the cardiovascular system, making it difficult to draw firm conclusions. Indeed, the studies are hardly replicable in terms of both exposure and chemical analysis protocols. This is partly due to the wide variety of parameters involved ⁸⁵. For example, there are many types and brands of e-cigarettes with different settings (low or high battery voltage, composition of e-liquid, possible presence of nicotine, presence and type of flavor, etc.) and individual use patterns (i.e. amount and duration of aerosol inhaled). A central issue is the role of nicotine, as it has been demonstrated to have a negative impact on the cardiovascular system ⁴,⁸. It is important to determine whether the adverse effects observed in *in vitro* and *in vivo* studies using e-cigarette aerosol are due to nicotine or other components present in the aerosol. This requires rigorous study protocols with appropriate control conditions to address this specific effect and to isolate the potential impact of nicotine from other e-liquid ingredients or neoformed compounds in the aerosol. Therefore, the e-cigarette cannot be considered as a single standardized device and the effects observed under one experimental condition (one type of e-cigarette with a specific e-liquid composition) cannot be generalized to all combinations of device and e-liquid formulation ²,⁴,²³,⁸¹,⁸⁶. The high variability and diversity of parameters to consider in studies of e-cigarettes make them much more challenging compared to studies of tobacco cigarettes, which involve very little product variability.

In addition, concentrations of aerosol extracts and condensates vary widely across different studies, ranging from extremely diluted to highly concentrated. If the concentrations are orders of magnitude
higher than those encountered by intimal endothelial cells in the bloodstream, meaningful comparisons are difficult, and only very limited conclusions can be drawn.

Similarly, different biological endpoints are considered depending on the study, as shown in Tables 1 to 3, although some of them are frequently investigated (cell viability, oxidative stress, pro-inflammatory response, cell migration as an indicator of vascular repair that may be impaired due to endothelial dysfunction, flow-mediated dilation, arterial stiffness, etc.).

Finally, one limitation should be mentioned that applies specifically to *in vitro* models: they are too simple, do not represent the complexity of the entire organism and do not reproduce toxicokinetics⁸⁷.

Therefore, they may not be predictive of what actually happens *in vivo*, which explains the observed discrepancies between *in vitro* and *in vivo* studies (Figure 2). For instance, exposure to the toxicants contained in e-cigarette aerosol may differ substantially *in vitro* and *in vivo* due to the contribution of clearance and metabolization *in vivo*⁴. *In vivo* models also allow the investigation of the defense mechanisms that could counter-balance a biological response (e.g. anti-oxidant defense, tissue repair mechanisms, etc.). In addition, long-term effects can hardly be assessed *in vitro*, whereas they can be observed *in vivo*. Despite these drawbacks, *in vitro* models remain useful, because they are easier, faster and cheaper than animal testing. They are suitable for studying mechanisms and allow extensive screening of effects; therefore, they can be used in a first attempt before *in vivo* testing. The development of advanced *in vitro* models (3D models, co-cultures, organoids, organ on a chip, etc.) will hopefully make it possible to narrow the gap between *in vitro* and *in vivo* testing⁸⁷.

Therefore, *in vivo* studies and clinical trials seem to be the most reliable source of information when it comes to assessing the effects of e-cigarettes on human health. On the other hand, such studies also have their limitations. Ethical reasons and international legislation tend to limit the use of *in vivo* models, strongly recommending compliance with the “3R” rule: Reduce, Refine, and Replace animal experiments.⁸⁸ In addition, such models are expensive and require facilities and personnel to house, feed and care for the animals. Experiments with animals can be lengthy and complex. And due to the
aforementioned enormous amount and variety of factors involved in vaping, the systematic
assessment of their toxicity profile *in vivo* is simply not feasible and not sustainable ⁴.

In addition, caution should be exercised when drawing conclusions about the effects of e-cigarettes
on human health based on *in vivo* studies, as extrapolations may be challenging due to the anatomical,
physiological and biochemical differences between species (especially mice or rats, which are
commonly used, and humans) ⁸⁷.

4. Conclusion

Although there is evidence that e-cigarette use may be safer than traditional tobacco smoking, e-cigarettes may still have adverse cardiovascular effects through various mechanisms such as oxidative stress, inflammation, DNA damage, arterial stiffness, altered hemodynamics and platelet activity ⁴,⁸³.

In particular, our analysis has shown that using *in vivo* models, which are more relevant for predicting
what could happen in humans, most studies highlight that e-cigarettes can impair endothelial function,
and should therefore not be considered harmless ²³. Complementary preclinical studies are needed to
better elucidate this sanitary question, including assessment in other cell models to assess the full
extent of cardiovascular damage, and possibly using standardized protocols to allow comparisons
between different studies. Such standardized studies could also help determine the role of nicotine or
other compounds in e-cigarette aerosol, such as flavors or thermal degradation products, through
systematic comparisons of groups to identify the driver(s) of toxicity.

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