Electronic cigarettes (EC) are commonly marketed as a safe alternative to traditional tobacco cigarettes (TC), but recent studies have observed adverse effects on vascular functions from EC vapours similar to those observed from TC. Currently, it is not clear if differing levels of nicotine and compounds from heating in e‐cigarettes influence vascular functions. Sixteen young, apparently healthy tobacco product naïve participants were studied. Each participant underwent three separate “vaping” trials with menthol‐flavoured quit smoking aid, electronically heated menthol‐flavoured EC with 0% or 5.4% nicotine. During each visit, measurements were performed at baseline, immediately post, 1, and 2 h post‐EC exposure. There were no significant changes in heart rate, systolic and diastolic blood pressure, endothelial function (via flow‐mediated dilation), and arterial stiffness (via cardio‐ankle vascular index) throughout the experiments. In conclusion, vaping electronic cigarettes did not produce acute and persistent effects on subclinical vascular functions over the course of a 2‐h trial in young, healthy, tobacco product naïve participants.

1 INTRODUCTION The use of electronic cigarettes (EC) has risen drastically over the last few years and is of particular concern because youths are especially drawn to their use.1 Traditional cigarettes are known to cause significant harm to the cardiovascular system. Just one traditional cigarette can increase arterial stiffness within minutes of smoking, and chronic use may lead to cardiovascular disease.2 Currently, little is known about the effects of EC in general. ECs are marketed to include favourable, mild vapours that pose little irritation and therefore provide a more enjoyable experience. However, a recent study has described adverse effects caused by ECs in regard to arterial stiffness for chronic cigarette smokers.3 More recently, significant increases in arterial stiffness were observed in an animal study when mice were continually exposed to EC vapour for 5 days per week over a span of 8 months.4 Electronic cigarettes are remarkably diverse in regards to ingredients, nicotine content, flavour, and combustion/heating mechanism. Chromatography studies have revealed a wide variety of concerning chemicals present in the vapour.5 In the case of tobacco cigarette (TC) smoke, some compounds from combustion/heating, such as formaldehyde and oxidant gases appear to be the main antagonists to vascular disease.6 Currently, it remains controversial if the ingredients in EC exert any adverse impacts on vascular function. Accordingly, the primary aim of the present study was to determine the influence of acute exposure to a variety of EC on key subclinical markers of vascular dysfunction. To maximize the effects of EC and to minimize the potential influence of downregulation associated with chronic exposure, we studied young healthy participants who were naïve to any tobacco products.

2 RESULTS Selected subject characteristics are presented in Table 1. Participants were young and healthy with clinically normal blood pressure values. All participants studied were naïve to regular use of any tobacco products in their lifetime. Many participants experience typical symptoms in exposure to nicotine (eg, coughing, dizziness, etc). Baseline levels of haemodynamic and vascular measures were not different among the three conditions (Table 2). Neither systolic nor diastolic blood pressure (BP) changed throughout the experimental protocol. There were no significant changes in cardio‐ankle vascular index (CAVI) or flow‐mediated dilation (FMD) with any of the ECs studied. Table 1. Selected subject characteristics Variable n or Mean ± SD Participants (n) 9 M and 7 F Age (year) 24 ± 3 Height (cm) 173 ± 8 Body weight (kg) 69.5 ± 10.3 BMI (kg/m2) 23.2 ± 2.8 Systolic BP (mmHg) 117 ± 8 Diastolic BP (mmHg) 68 ± 4 Heart rate (beats/min) 54 ± 8 Table 2. Changes in vascular and haemodynamic measures during the three experimental conditions Variable Group Baseline Immediately Post 1 h post 2 h post CAVI (U) Control 5.7 ± 0.6 5.9 ± 0.9 6.0 ± 0.8 6.0 ± 0.8 No nicotine 5.9 ± 0.6 6.0 ± 0.7 6.0 ± 0.5 6.1 ± 0.7 Nicotine 5.8 ± 0.7 6.2 ± 0.8 6.0 ± 0.9 5.9 ± 0.8 FMD (%) Control 5.6 ± 2.5 5.6 ± 2.4 5.6 ± 2.0 5.2 ± 3.2 No nicotine 5.7 ± 2.8 5.0 ± 2.0 5.0 ± 2.2 5.2 ± 2.5 Nicotine 5.6 ± 1.8 5.3 ± 1.7 6.1 ± 2.1 5.6 ± 2.6 Systolic BP (mmHg) Control 117 ± 6 119 ± 8 120 ± 7 120 ± 7 No nicotine 115 ± 8 118 ± 10 120 ± 8 119 ± 10 Nicotine 119 ± 10 124 ± 10 121 ± 10 121 ± 9 Diastolic BP (mmHg) Control 68 ± 3 68 ± 6 71 ± 6 69 ± 5 No nicotine 66 ± 4 68 ± 5 70 ± 5 68 ± 5 Nicotine 69 ± 4 73 ± 5 71 ± 6 70 ± 5

3 DISCUSSION Electronic cigarettes are extremely diverse in terms of their nicotine contents, chemical and flavour compositions, and combustion temperatures. It is not feasible or even possible to evaluate all possible variants of ECs. In the present study, we tested two different popular ECs (with and without nicotine) in relation to the placebo control. We reasoned that high nicotine ECs would be used to evaluate the effects of nicotine in ECs, and ECs without nicotine could be used to assess the impact of the other chemical compounds. The present findings are in marked contrast to our working hypothesis as no significant changes in subclinical vascular functions were observed with the ECs with or without nicotine. A significant increase in arterial stiffness has been reported after acute exposure to vaping.3, 7 The present findings are in marked contrast to these previous small‐scale studies. One unique feature of the present study is that the participants studied were young, healthy, physically active participants who have not been exposed to any form of tobacco product in the past. A primary concern associated with the e‐cigarette use, particularly in media outlets, is centred around youths and young adults getting exposed to seemingly harmful stimulants. Yet it is not plausible to subject youths to electrical cigarettes from an ethical standpoint. As such, we chose to study this particular study population. Unlike the studies in arterial stiffness, much less information is available on the effect of acute exposure to ECs on endothelial function.8 Similar to arterial stiffness, we did not observe significant changes in FMD in response to any of the ECs examined in the present study. More specifically, there were no significant differences in FMD between ECs with and without nicotine. One strength of this study is the use of young participants, who were healthy, physically active, and naïve to both EC and tobacco smoking. Many of the previously mentioned studies utilized current or former smokers.3, 7 To facilitate the entry of these naïve participants, the use of a familiarization trial was implemented to ensure proper inhalation of the EC product. The use of smokers and smaller levels of nicotine in previous studies likely resulted in better inhalation of vapour product. Even though we observed slight changes in all variables, these changes were mild at best. It is very difficult to explain the discrepant findings from previous studies. However, it may be related to habituation of e‐cigarette use. Previous studies indicate that the susceptibility to oxidative stress was significantly greater in chronic EC users compared with non‐users.9, 10 There are also a number of important limitations of the present study that should be emphasized. First, this trial was constructed to assess acute effects of EC on peripheral/subclinical vascular function. Future studies should focus on chronic or longer‐term effects of e‐cigarette smoking on vascular function even though it is not clear if such study would be feasible or ethical to conduct in cigarette naïve participants. In this context, animal experiments that exposed mice to EC vapour for five days per week took as long as eight months to elicit significant increases in arterial stiffness since there was no significant change at the 4‐month mark.4 It is not known how long it would take to demonstrate arterial stiffening effects or vascular dysfunctions in humans. Second, due to the extreme difficulty in recruiting and convincing young healthy (ie, health‐conscious) participants to consume believe‐to‐be‐harmful ECs, the number of participants studied was relatively small. Third, blood markers of mechanistic variables (eg, oxidative stress, nitric oxide bioavailability) and blood concentrations of key toxins (eg, nicotine, cotinine) were not measured. In summary, in contrast to the prevailing notion that the EC are harmful, we demonstrated that an acute exposure to EC did not elicit significant changes in key subclinical markers of vascular dysfunction regardless of the EC ingredients.

4 MATERIALS AND METHODS Sixteen young healthy adults were studied. Participants with overt cardiovascular and pulmonary diseases (hypertension, diabetes, asthma, etc) and those who had regularly used tobacco and nicotine products within the last 6 months and who were taking cardiovascular acting drugs were excluded. None of the participants was weaned from any form of tobacco products prior to the study participation. Additionally, female participants were tested only during the follicular phase of their menstrual cycles to minimize the potential confounding effects of hormonal milieu. Experimental protocols were approved by the local institutional review board, and informed consents were obtained. The present study (NCT03209661) was registered with the ClinicalTrials.gov. Participants reported to the laboratory having fasted for a minimum of 8 h and having abstained from vigorous activity, caffeine, and alcohol for a minimum of 12 h. After a supine resting period, baseline measurements were taken. Upon completion of baseline assessments, participants were then escorted to a previously selected outdoor area (per institutional restriction) and asked to vape an electronic cigarette. The three EC used were: (a) control: menthol‐flavoured cigarette‐like pipe (Harmless Cigarette Quit Smoking Aid); (b) Nicotine EC: a combination of a battery (Cirrus 3, White Cloud Cigarette) and cartridge (Menthol Flavour Clear Draw Max 5.4% nicotine); and (c) No nicotine EC: a combination of a battery (Cirrus 3) and cartridge (Menthol Flavour Clear Draw Max 0% nicotine). EC containing 5.4% nicotine by volume (50 mg of nicotine in 1000 mg of e‐liquid) was selected as it is considered a higher strength vapour that would be likely to produce a robust response. The vaping protocol was a total of 6 minutes in length, consisting of 4‐second inhalations every 20 s culminating into 18 puffs in total. These sessions were closely monitored and supervised by one of the investigators to ensure that the required numbers of inhalations and puffs were taken by the participants. The order in which ECs were tested was randomized. Participants were then asked to return to the laboratory for an immediately‐post‐EC assessment. Assessments were repeated at 1 hour and two hours after EC exposure to determine residual effect on the vasculature. EC conditions were separated by a minimum of 48 h. All measurements were conducted by a single‐blinded researcher to eliminate inter‐individual variabilities. In an attempt to further reduce investigator bias, all the measurements and analyses were conducted using semi‐automated devices. The CAVI is a measure of arterial stiffness with a computerized, semi‐automatic, non‐invasive monitoring device VaSera (Fukuda Denshi).11, 12 CAVI is a measure of arterial stiffness and was calculated from pulse wave distance divided by transit time. Blood pressure cuffs placed on ankles and arms were inflated to a pressure of 30–50 mmHg in order to record pulse waves but not affect haemodynamics.11 Pulse wave velocity was measured by summing closure of the aortic valve and brachial notch appearance upon the wave form, plus the time between the rise of the brachial and ankle pulse wave.11 Heart rate and blood pressure were also measured using this device. Flow‐mediated dilation (FMD) is a non‐invasive measure of vascular endothelium‐dependent vasodilation which utilizes ultrasound recordings of the brachial artery's responsiveness to ischaemic stress. All measurements were taken using an automated diagnostic ultrasound system (UNEX EF‐38G, UNEX Corporation).13 The system is equipped with an electric linear‐array transducer operating at 10 MHz. While subjects rested in supine position, a blood pressure cuff was placed on the forearm with the proximal edge of the cuff above the subject's antecubital fossa and second blood pressure cuff was placed on the contralateral arm for standard blood pressure measurements.13 Cross‐sectional images of the artery were acquired utilizing the automated probe, which self‐adjusted to provide a clear longitudinal image of the artery and began baseline measurements.13 After the acquisition of baseline measurement, the cuff inflated to 50 mmHg above resting systolic blood pressure for 5 min effectively occluding the arm below the antecubital fossa. Upon cuff deflation, ultrasound‐derived measurements of the brachial artery diameters and blood flow velocity were taken for 2 min. FMD was calculated by the following equation: (maximum diameter – baseline diameter)/baseline diameter × 100.13 Results were analyzed utilizing a repeated measures ANOVA (SPSS version 24; IBM, Armonk, NY, USA). All P‐values were reported utilizing Greenhouse‐Geisser correction with any significant effects analyzed utilizing pairwise comparisons.

ACKNOWLEDGMENTS The present study (NCT03209661) was registered with the ClinicalTrials.gov.

CONFLICT OF INTEREST The authors have no conflict of interest to disclose.