The principal finding of this study is that habitual E-cig use leads to vascular dysfunction, such as a significant increase in AS, reduced vascular relaxation to vasodilators, and enhanced responses to vascular constrictor agents. These findings are associated with the development of CVD (14) and qualitatively relate to other well-known CVD risk factors, including smoking traditional cigarettes.

Vascular and cardiac responses. The key observation from this study is that even low levels (see below) of exposure to E-cig vapor increased AS and impaired ex vivo vascular responses. The clinical relevance of our findings can be demonstrated by relating the degree of the arterial dysfunction we observed in the present study to other well-known CVD risk factors (Fig. 4). 1) Previous reports indicate that smoking increases central AS from 0.6 to 1.1 m/s (6, 40, 57, 66). In context, a 1-m/s increase in central PWV corresponds to an age-, sex-, and risk factor-adjusted risk increase of 15% in cardiovascular and all-cause mortality (67). Thus the 1.14- and 1.28-m/s increase in AS (i.e., ΔPWV shown in Fig. 1B) we observed in the present study would reflect an ∼17–19% increased risk of all-cause mortality with chronic use of E-cigs and conventional cigarettes in humans. 2) When comparing our results with other studies assessing aortic reactivity in rodents with either overt CVD (e.g., hypertension and atherosclerosis) or known CVD risk factors (e.g., stress, hyperlipidemia, and diabetes), we found that E-cig exposure for 8 mo created a risk for CVD similar to several other well-known risk factors, including smoking (Fig. 4). Fig. 4.Magnitude of aortic vascular dysfunction from E-cig vapor compared with other rodent studies (using the same or similar ex vivo methodology) evaluating aortic responses to various knowm cardiovascular disease (CVD) risk factors and/or overt CVD (e.g., atherosclerosis and hypertension). All data represent maximal methacholine dose reported (e.g., 10−5 M) compared with control conditions within each respective study, where controls were set to equal 100 and change in the treatment/disease condition is calculated. UCMS, unpredictable chronic mild stress; IP, intraperotineal; SHR, spontaneously hypertensive rats; HSD, high-salt diet. **Data from the present study. Download figureDownload PowerPoint

Because a measurable deficit in AS was only seen in our mice after 4.5 mo of exposure, it might be tempting to think that the same relative duration in humans (i.e., ~15 yr) will be the time frame needed to achieve vascular dysfunction in humans. This notion should be viewed with caution, since it is likely that our mice experienced much lower levels of E-cig vapor than are likely to occur in humans. For chronic smoke chamber studies, daily total particulate matter (TPM) in a range of 100–250 mg/m3 has typically been used to induce chronic obstructive pulmonary disease (COPD) in small animals (20, 39, 69). Our average daily chamber TPM from the E-cigs was much lower [i.e., 59 ± 14 (SD) mg/m3]. This means that our exposure was less than half of the average concentration range typically used to elicit COPD symptoms with cigarette smoke in animal studies (20, 39, 69). Yet the fact that vascular dysfunction was observed, despite the lower exposure level, suggests that the threshold to induce vascular injury may be “very low.” This finding is consistent with evidence showing a marked increase in cardiovascular risk even at “low levels” of cigarette exposure (7, 37). A recent study in humans also reports an acute effect of E-cig exposure (to just 9 puffs) of impaired FMD, a noninvasive measure of endothelium-mediated vascular function linked to NO bioavailability that is frequently used to assess vascular function in humans (10). Given that endothelial dysfunction, even if temporary, can be seen early in atherogenesis (14), we believe that these data collectively suggest that the threshold for damage from E-cigs is likely low with respect to the vascular system, similar to that observed with traditional cigarettes (7, 37). A potential explanation for increased risk from low-level exposures may be that E-cigs produce elevated levels of particulate matter (PM) in the ultrafine (<100 nm) and PM 2.5 (<2.5 μm) range (47, 48). While some studies indicate similar distribution of the particle size from E-cigs and tobacco cigarettes in the submicrometer range (~125–160 nm) (29, 70, 76), there is also evidence that E-cigs deliver more ultrafine (<1-µm) particles (23, 42, 43). Ultrafine and submicrometer particles are more easily brought into and out of the lung and penetrate more deeply than larger (micrometer) particles (42). Nanoparticles also easily traverse the alveolar-capillary interface and gain direct access to vascular endothelial cells and the bloodstream, which could explain the robust and rapid systemic vascular effects that have recently been observed in response to acute E-cig use (10). One aspect that cannot be addressed by our study is determination of the component(s) in E-cig vapor responsible for mediating these vascular effects. For example, nicotine is known to induce significant effects on the cardiovascular system. In humans and rodents, nicotine increases blood pressure and has been linked to arterial remodeling (75). The arterial response to phenylephrine-induced contraction is greater in nicotine-treated than control rats, and nicotine-treated animals showed impaired endothelium-dependent relaxation to acetylcholine compared with control rats (75), demonstrating that nicotine alone is capable of inducing vascular dysfunction. However, the role of nicotine in CVD risk is controversial, since CVD risk is low (or lower) in individuals who use nicotine medications or smokeless tobacco products compared with active smokers (5). However, very few long-term exposure studies have been conducted with inhaled or aerosolized nicotine, and this route involves less contact with other cells and nicotine metabolism before contact with the vascular endothelium. So, while nicotine replacement therapies do not appear to increase CVD risk, the long-term effects of inhaling nicotine (in the absence of combustion of tobacco) are still poorly understood. Since nicotine is capable of acutely increasing vascular wall stiffness (due to its effects on the central nervous system), temporal increases in AS that do not reflect vessel remodeling can be observed immediately following acute exposures. However, the changes in AS and ex vivo aortic ring tension observed in our study are not likely related to the acute exposure effects, since all our assessments were made ≥24 h after smoke/vapor exposure. Moreover, the halfway time-point assessment for AS (i.e., PWV; Fig. 1) does not show significant change compared with baseline; therefore, we do not believe that our data are the result of lingering acute effects. Also, the level of urine cotinine (a stable by-product of nicotine used as a biomarker for nicotine due to its short half-life) in our E-cig-exposed mice was almost half that in 3R4F-exposed mice, yet the degree of vascular dysfunction was very similar between the groups. This could suggest that some component of the E-cig liquid (other than nicotine) may have a greater influence on vascular impairment. Further studies are required to elucidate these effects. Our chronic exposure resulted in small, but statistically significant, decreases in FS% and EF% in 3R4F-exposed mice, with a similar (although not significant) trend for E-cig-exposed mice (P < 0.10; Table 1). Based on these data, this level of E-cig exposure did not result in overt cardiac dysfunction. However, LV mass was greater in the E-cig- than air- or 3R4F-exposed mice. Although this finding can signify cardiac remodeling, its significance is unclear, as reduced cardiac performance was observed only in 3R4F-exposed mice (in which LV mass was not different from controls). It may be tempting to speculate that E-cigs have little impact on cardiac function, but we cannot rule out the possibility that the subtle changes we observed could progress to pathological outcomes with more intense and/or longer exposure. Further research is needed to elucidate such effects, as well as other responses (i.e., development of hypertension and histological assessment of aortic remodeling) that was not determined from our present study.

Respiratory responses. We observed changes in lung histology (Table 3) and respiratory system compliance (Fig. 4) in 3R4F-exposed, but not E-cig- or air-exposed, mice. The changes in 3R4F-exposed mice (i.e., higher compliance, more pigmented macrophages, and higher emphysema score) are consistent with the well-known effects of cigarette smoke. While our pulmonary findings are consistent with findings from at least one other study that used the C57BL/6 murine model (54), studies using other mouse strains (e.g., A/J or BALB/c) demonstrate that E-cig vapor does induce histological changes and impairment in airway and lung mechanical properties similar to a COPD phenotype (24, 36). Thus it is possible that the lack of pulmonary effects from E-cigs in our study may be due to 1) the selected inbred mouse strain we used and/or 2) the relatively mild TPM exposure in our paradigm (see above). Given growing evidence from cellular and in vivo animal studies, as well as acute studies in humans, showing the toxicity of E-cig vapor in airway cells and respiratory function [see review (16)], we would caution against the interpretation (based on our data) that E-cigs are safe for the lung.

Clinical relevance to humans and study limitations. Some might argue that intermittent E-cig use for a total of 4 h/day seems too high or unrealistic for the average E-cig user. However, when examining data anonymously volunteered from >180,000 Evolv DNA-series E-cig devices via the ECigStats data collection program (www.ecigstats.org, accessed April 6, 2017) that records user usage characteristics, we found that the average number of puffs per day reported across all devices is 172 ± 131 (SD). This is actually higher than the 152–156 puffs/day used in this study (38–39 puffs per hour × 4 h). However, the ECigStats data also report that devices/users have an average puff duration time of 2.31 ± 2.11 (SD) s. The nearly identical mean and SD of the puff duration (2.31 and 2.11, respectively) indicates a wide variability in individual usage characteristics, with nearly one-third of the average users adopting long (up to 4.4-s) puff durations. Our E-cig usage characteristics (i.e., 5-s, 55-ml puff with the device set at 4.9 V, 14.1 W) are actually similar to those of several recent scientifically controlled studies examining E-cig puff topography, showing that the average “experienced” E-cig user adopts longer (e.g., 4–8 s) puff durations (61, 63). Moreover, an “average-experienced” E-cig user (with a 4-s puff duration) is reported to generate 29.4–152.7 mg of TPM, depending on puff velocity and voltage (i.e., 3.3 vs. 5.2 V, respectively), but an “extremely experienced” E-cig user (with an 8-s puff duration) can generate 68.8–333.2 mg of TPM (63). So, when comparing the total daily TPM achieved by experienced E-cig users with our chamber exposure, we find that the mice (with a daily TPM average of 59 mg) received the lower end of TPM compared with most experienced E-cig users. Thus, despite the similarities in E-cig topology between our animal exposure and human usage, it is possible that our exposure paradigm underestimates the effect that will be experienced by a human E-cig user. Our use of a chamber exposure paradigm may also have a dampening effect on our outcomes, because rodents are obligate nasal breathers, and the nose can effectively filter many airborne particles compared with direct inhalation via the mouth (as humans would experience). Together with the caveat that our animals experienced a lower level of TPM exposure than the average E-cig user, this could also potentially explain why we saw minimal pulmonary abnormalities in our study. Nevertheless, the mice developed significant vascular dysfunction, suggesting that the degree of vascular dysfunction may not be substantially different between E-cigs and conventional cigarettes. The fact that we observed minimal cardiac and pulmonary changes could mean that these organs have a greater threshold and/or resilience to functional impairments and that endothelial and vascular dysfunction is simply the first step and harbinger in the etiology of CVD (15, 25, 49). An additional clinical consideration is that we studied only female mice. The chamber exposure method we used necessitated the use of female mice to reduce fighting and injury when mice (otherwise housed in separate cages) were temporarily grouped communally for several hours each day during the exposures. We do not know if male mice would have exhibited the same level of vascular dysfunction. Consistent with human studies (53), rodent studies have also found that females are less sensitive than males to the pharmacological effects of nicotine (27, 58), in part because of protective effects of female hormones (8). Thus one could speculate that female mice may have a dampened response and that E-cig exposure in males may result in greater CVD risk and worse outcomes (3, 8). Future studies must include both sexes in these evaluations. A broad concern is the relevance of E-cig-related cardiovascular responses in mice compared with humans, especially given the potential difference we have noted with respect to mouse strains. Indeed, it is too early to know if vaping effects in rodents will faithfully recapitulate those ultimately seen in humans. Given the wide variations and options possible with E-cig vapor exposures (i.e., varying quality of devices, different device settings, and varying concentration of constituents in the E-cig liquid and/or formulation of the base solution), it is likely that varying and, possibly, divergent outcomes may be observed, depending on the exposure paradigm. However, broadly speaking, we would emphasize that decades of evidence from cigarette smoking demonstrate good fidelity with pulmonary and cardiovascular outcomes observed between rodents and humans (22, 71, 72). We would also suggest, even given the limited evidence that currently exists, from the preponderance of data obtained from interventional studies (including this study), that it seems counterintuitive to believe or expect that long-term use of E-cigs will likely prove to be safe in terms of overall human health.