Won Hee Lee , PhD a , b , ∗∗∗ ( whlee{at}email.arizona.edu ) , Sang-Ging Ong , PhD a , c , d , ∗, Yang Zhou , MS a, Lei Tian , PhD a, Hye Ryeong Bae , BS a, Natalie Baker , BS a, Adam Whitlatch , BS e, Leila Mohammadi , MD, PhD f, Hongchao Guo , PhD a , g, Kari C. Nadeau , MD, PhD h, Matthew L. Springer , PhD e , f, Suzaynn F. Schick , PhD i, Aruni Bhatnagar , PhD j and Joseph C. Wu , MD, PhD a , g , ∗ ( joewu{at}stanford.edu ) , @StanfordCVI a Stanford Cardiovascular Institute , Stanford University School of Medicine , Stanford, California b Department of Basic Medical Sciences , University of Arizona College of Medicine , Phoenix, Arizona c Department of Pharmacology , University of Illinois College of Medicine , Chicago, Illinois d Division of Cardiology , Department of Medicine , University of Illinois College of Medicine , Chicago, Illinois e Department of Medicine , Division of Cardiology , University of California , San Francisco, California f Department of Medicine , Cardiovascular Research Institute , University of California , San Francisco, California g Department of Medicine, Division of Cardiology , Stanford University School of Medicine , Stanford, California h Sean N. Parker Center for Allergy and Asthma Research , Stanford University School of Medicine , Stanford, California i School of Medicine , Division of Occupational and Environmental Medicine , University of California , San Francisco, California j Division of Cardiovascular Medicine , University of Louisville School of Medicine , Louisville, Kentucky ↵ ∗ Address for correspondence:

Dr. Joseph C. Wu, 265 Campus Drive, G1120B, Stanford, California 94305-5344. ↵ ∗∗ Dr. Won Hee Lee, 425 North 5th Street, Room 426, Phoenix, Arizona 85004-2157.

Central Illustration

Abstract Background Electronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings. Objectives The purpose of this study was to investigate the effects of flavored e-cigarette liquids (e-liquids) and serum isolated from e-cigarette users on endothelial health and endothelial cell–dependent macrophage activation. Methods Human-induced pluripotent stem cell–derived endothelial cells (iPSC-ECs) and a high-throughput screening approach were used to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum from e-cigarette users. Results The cytotoxicity of the e-liquids varied considerably, with the cinnamon-flavored product being most potent and leading to significantly decreased cell viability, increased reactive oxygen species (ROS) levels, caspase 3/7 activity, and low-density lipoprotein uptake, activation of oxidative stress-related pathway, and impaired tube formation and migration, confirming endothelial dysfunction. Upon exposure of ECs to e-liquid, conditioned media induced macrophage polarization into a pro-inflammatory state, eliciting the production of interleukin-1β and -6, leading to increased ROS. After exposure of human iPSC-ECs to serum of e-cigarette users, increased ROS linked to endothelial dysfunction was observed, as indicated by impaired pro-angiogenic properties. There was also an observed increase in inflammatory cytokine expression in the serum of e-cigarette users. Conclusions Acute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which often precedes cardiovascular diseases.

Key Words

Cigarette smoking causes 1 of every 3 deaths that result from cardiovascular disease (CVD), leading to >480,000 premature deaths each year in the United States alone (1). While the detrimental effects of conventional cigarette smoking in CVD are well documented and smoking prevalence is declining, an explosive increase in the use of electronic cigarettes (e-cigarettes), especially among youth, is happening with scarce scientific evidence on their toxicity and health effects (2,3).

E-cigarettes are devices designed to deliver an aerosol containing nicotine by heating a liquid solution (often called e-liquid or e-juice) that typically uses propylene glycol (PG) and/or glycerol (glycerin) as a vehicle for nicotine and flavoring agents. Although e-cigarettes are relatively new to the market, they are an increasingly popular alternative to conventional tobacco cigarettes and encompass a wide range of product types and brands. As of 2014, there were >450 e-cigarette products with nearly 8,000 unique flavors available (4). Although most of the flavoring chemicals in e-liquids may meet the generally recognized safe standard for ingestion as food additives, they have not been adequately tested as inhalants. In fact, some popular flavorings are known to be toxic, including diacetyl (buttery flavor), acetyl propionyl (caramel or buttery flavor), and benzaldehyde (fruity taste). Recently, the Food and Drug Administration released a statement limiting the sales of flavored e-cigarettes except tobacco, menthol, and mint flavor in an effort to prevent a new generation of nicotine addicts (5). Despite the rapid increase in popularity, the potential for harmful cardiovascular effects following the use of inhaled e-cigarette flavoring chemicals has been largely unexplored.

To date, the toxicological studies conducted on e-cigarettes have been mostly limited to cytotoxicity studies using established cell lines. Early on, limited studies of e-cigarette vapor showed that e-cigarettes delivered much lower levels of carcinogens in aerosols when compared with conventional cigarette smoke (6,7). At this time, the cardiovascular risk of e-cigarettes is not clear; however, some e-liquid flavorings have been shown to be cytotoxic in cellular models including pulmonary fibroblasts, human embryonic stem cells, and neural stem cells (6,8).

The vascular endothelium plays an important role in vascular function through elaboration of paracrine factors that regulate vascular tone, cell adhesion, fibrinolysis, and blood flow (9). Smoking causes endothelial dysfunction, which is a risk factor for CVD (10). It has been hypothesized that the use of e-cigarettes is associated with endothelial cell damage leading to acute endothelial dysfunction (11), but further in-depth studies are warranted. Despite the extensive use of induced pluripotent stem cells (iPSCs) in other areas of research, no previous studies have leveraged the powerful platform of human-induced pluripotent stem cell–derived endothelial cells (iPSC-ECs) to assess the potential health risks of e-cigarettes on endothelial integrity. Here, we used iPSC-ECs to evaluate the effects of flavored e-liquids on endothelial health and to understand the cross-talk between endothelial cells and macrophages. We further validated our results using serum collected from e-cigarette users and conventional cigarette smokers to assess the potential effects of e-cigarette use on cardiovascular function.

Methods Detailed methods and supporting data are available in the Online Appendix. Differentiation of iPSC-ECs The iPSCs (over passage 20) from 3 healthy individuals were split in a 1:12 ratio using EDTA, as described previously (12), and were grown for 3 to 4 days until they reached ∼75% confluence. For the endothelial cell differentiation and characterization protocol, please refer to the Online Appendix. E-cigarette liquids (e-liquids) Six different e-liquids were purchased online: Freedom Smoke USA (Tucson, Arizona), Johnson Creek (Johnson Creek, Wisconsin), and E Liquid Market (Birmingham, Alabama) in 0, 6, and 18 mg/ml of nicotine concentrations and were stored at 4°C in the dark (Online Table 1). The bottles were chosen to represent the 3 most common vehicle types (50% PG/50% vegetable glycerin [VG], 80% PG/20% VG, and 100% VG) and a range of popular flavors. Patients’ serum sample collection The subject population consisted of 5 healthy nonsmokers (nonsmoker), 5 active cigarette smokers (cigarette), and 2 dual users of e-cigarettes and cigarettes as well as 2 sole users of e-cigarettes (e-cigarette). We included serum from both sole users of e-cigarettes and dual users of e-cigarettes and combustible cigarettes in our study because both patterns of use are common. We combined the 2 into a single group (e-cigarette) as most recruited participants were traditionally long-term cigarette smokers who started the use of e-cigarettes as an alternative to quitting the use of cigarettes. This is also due to the recruited population having a mean average age of 29 years, whereas most solely e-cigarette users with no prior smoking history tend to be younger. Depending on the population and the wording of the questions, 25% to 70% of e-cigarette users describe themselves as dual users (13,14). The participants classified as e-cigarette users reported using their e-cigarettes an average of 27.5 ± 5.0 days/month, with 9.8 ± 3.3 e-cigarette sessions (>2 puffs) per day. Dual users smoked <2 packs of cigarettes/month. Cigarette smokers reported 29.8 ± 0.4 days of smoking in the past 30 days and 10.1 ± 3.4 cigarettes/day. All subjects were healthy individuals free of other major cardiovascular risk factors. Demographic and clinical characteristics of subjects are summarized in Online Table 2. Informed consent was obtained from all subjects, and the conduct of the study was approved by the University of California, San Francisco Institutional Review Board. Participants in the study were asked not to use combustible cigarettes for 7 days before all study visits and to abstain from the use of cigarettes, e-cigarettes, food, and caffeinated drinks for 12 h prior to the study day. To ensure abstinence from tobacco products, carbon monoxide (CO) and nicotine levels were tested at the start of each visit. For the e-cigarette study, the subjects used an e-cigarette containing RY4-flavored e-liquid (Changning Dekang Ltd, Shenzhen, China) with 16 mg/ml nicotine and instructed to use it for a total of 10 min (1 puff every 30 s, each puff lasting 2 s). For the cigarette condition, subjects smoked a Marlboro cigarette for 10 min or until the cigarette went out, by taking 2 puffs/min, each puff lasting 2 s. Statistical analysis Statistical analysis and graphs of data were performed with SigmaPlot version 13.0 (Systat Software, San Jose, California). Either a paired or unpaired 2-tailed Student’s t-test for normal distributed variables and Mann-Whitney U test for non-normal distributed variables were used for 2-group comparisons. Differences of >2 groups were performed using analysis of variance (ANOVA) with Bonferroni post hoc analysis. When >2 independent variables were present, a 2-way ANOVA with Bonferroni correction was used. To test for serial changes, a 1-way repeated-measures ANOVA was used. Normal distribution was tested with the Shapiro-Wilks test. All data were summarized as mean ± SD or mean ± SEM, and Bonferroni correction was used for multiple comparisons as indicated. A p value <0.05 was considered statistically significant.

Discussion Understanding the health effects of e-liquids, especially flavorings, is important for establishing the short- and long-term safety of e-cigarettes. In this study, we compared the biological effects of flavored e-liquids (both with and without nicotine) on iPSC-ECs and found that some flavorings had toxic effects on endothelial cell viability and function (Central Illustration). The cinnamon-flavored product (Marcado) was the most toxic sample tested, producing strong cytotoxicity in iPSC-ECs that led to decreased cell survival, impaired angiogenic responses, and increased ROS levels and caspase 3/7 activity. Our findings are concordant with the results of recent studies showing that cinnamon-flavored e-liquids and aerosols are highly volatile, cytotoxic, and genotoxic to human embryonic cells and adult lung cells (8,18). Notably, our results showed that the effects of e-liquid flavorings on endothelial phenotypes and function including cytotoxicity, ROS level, caspase 3/7 activity, pro-angiogenic properties, and migration, were stronger than those of nicotine concentration. Similar biological effects of e-liquid flavoring on iPSC-ECs were also observed in a gene set enrichment and GO enrichment analyses. We identified changes in the expression of genes involved in multiple biological functions, including response to toxic substances, membrane-bounded vesicle, growth factor binding, pentose biosynthetic process, and oxidation reduction process 24 h post-exposure to e-liquid flavor, Marcado, with or without nicotine. Some of these enriched functional categories from our study were also highlighted in previously reported studies performed on a primary 3-dimensional airway cell system and mouse lungs exposed to e-cigarette aerosol or cigarette smoking, respectively (19,20). Central Illustration Human-Induced Pluripotent Stem Cell-Derived Endothelial Cells for Evaluating E-Cigarette Risk Mechanistic overview by which e-cigarette use might cause acute endothelial dysfunction. Exposure of endothelial cells to e-cigarette flavorings or serum of e-cigarette users leads to endothelial dysfunction associated with increased apoptosis, reactive oxygen species, and inflammation. ICAM = intracellular adhesion molecule; IL = interleukin; MCP = monocyte chemoattractant protein; MCSF = macrophage colony-stimulating factor; ROS = reactive oxygen species. Nicotine, 1 of the major active constituents in most smoking products, is primarily metabolized by the liver into cotinine (21). Although it has been reported that nicotine absorption rate and plasma nicotine levels are lower from e-cigarette use compared with conventional cigarette smoking, it could still reach a delayed but comparable level compared to a tobacco cigarette (21). As intensive puffing (i.e., more puffs and greater puff volume) may influence nicotine delivery, we applied a similar study design for e-cigarette users and smokers (one 2-s puff every 30 s for 10 min). We found that the effect of e-cigarette use on mean plasma nicotine levels was similar to tobacco cigarette smoking. Peak plasma nicotine concentration for both conventional cigarette and e-cigarette users was observed immediately after smoking, which is consistent with previous reports (21,22), indicating that e-cigarettes provide nicotine via rapid pulmonary absorption. Although studies with conventional cigarettes have shown that acute and long-term exposure to smoking increases the risk of CVD via enhanced oxidative stress (9,23), inflammation (9), endothelial dysfunction (9,10), and elevated blood pressure (24), studies on the effects of e-cigarette nicotine and liquid flavors have been somewhat limited. In the present study, we observed that pro-inflammatory markers such as IL-6, ICAM-1, macrophage colony-stimulating factor, and monocyte chemoattractant protein-1 were elevated in the serum of users of e-cigarettes and conventional cigarettes 3 h after use compared with serum collected prior to smoke, highlighting the danger of even short-term exposure to e-cigarette aerosol in vivo. These inflammatory markers are known to play a critical role in the pathogenesis of vascular disease. We also demonstrated that e-cigarette and cigarette users’ serum expressed elevated basal ROS when compared with nonsmokers, which may be linked to activated inflammatory mediators. The already elevated basal ROS levels in e-cigarette users’ serum increased rapidly after smoking. Accumulating evidence suggests that ROS plays an important role in physiological and pathological angiogenesis (9,25). In this study, we found that conditioned media from iPSC-ECs with exposure to e-liquid treatment promoted polarization of both M1 and M2 macrophages, with a higher tendency of M1 polarization. These M1 polarized macrophages may be, in part, responsible for strong pro-inflammatory profiles, which are linked to increased ROS generation. This is consistent with previous studies demonstrating that macrophages produce high levels of ROS, which also acts in a feedback-loop regulating macrophage phenotype (25,26). However, the exact role of ROS in macrophages activation still requires further investigation. Importantly, our data further validated that the presence of ROS in e-cigarette users’ serum after vaping flavored e-liquids was associated with endothelial cell dysfunction, as shown by altered tube formation in iPSC-ECs. Consistent with this finding, Carnevale et al. (23) observed negative effects on markers of oxidative stress (i.e., soluble NOX2-derived peptide and 8-iso-prostaglandin F2α) and flow-mediated dilation after a single use of e-cigarette, raising concerns regarding vascular safety of e-cigarettes. The results from these studies lend further support to the harmful effects of e-cigarette use and cigarette smoking in increasing oxidative stress and endothelial dysfunction. Finally, we performed a characterization of leukocyte responses in smokers, as it has been reported that acute and chronic cigarette smoking is followed by a transient elevation of WBC count, considered to be a well-established cardiovascular risk factor in epidemiological studies (27). In our study, there were no statistically significant differences in either total or subpopulation leukocyte counts between nonsmokers and smokers of either e-cigarettes or conventional cigarettes, which is consistent with a previous study (28). However, we did find a significantly lower PLT count in smokers compared with nonsmokers, which could be due to the decreased thrombopoietic activity induced by nicotine commonly seen in chronic smokers (29). Regardless of the underlying mechanisms, these changes are important indications of how e-cigarette use could compromise host immunity. Study limitations Despite its many strengths, our study has several limitations. First, as we were interested in assessing the direct effects of these liquids on vascular health, the results from our in vitro evaluation of e-liquid flavoring using iPSC-ECs were restricted to e-liquids and not their aerosols, as no heating or combustion was involved. We did, however, add serum from e-cigarette users into culture media of iPSC-ECs to mimic cellular exposure to e-cigarette aerosol that occurs in vivo, including exposure to toxicants that have undergone metabolic activation or deactivation. The presence or absence of aerosols may explain the difference observed in nicotine effects: nicotine present in e-liquids seemed to only have a slight effect on ROS production and no effect on tube formation, whereas serum collected from both e-cigarette and cigarette smokers after smoking, which contains increased levels of nicotine, significantly increased ROS production and impaired tube formation. These effects may be due to the lack of combustion used in the in vitro settings, which could make e-cigarettes potentially more toxic compared with serum collected from both e-cigarette and cigarette smokers that involves combustion. Another factor that may contribute to the differential responses is the increased molecular complexity of serum collected from patients due to systemic effects of other cell types beyond endothelial cells that may be affected by e-cigarettes or cigarettes. Further studies exploring the chemical toxicants in the e-liquids, e-cigarette aerosol, and serum in relation to the endothelial cytotoxicity and function are needed to better understand the potential risk from toxic compounds generated by e-cigarette products. Second, a fixed puffing protocol used in our study at an experimental setting may not reflect actual user puff practice. However, this fixed protocol was necessary to allow us to directly compare the results between different subjects across separate studies in a standardized manner. It should be noted that our results on 6 selected flavors may not be universally applicable to all e-cigarettes, given the wide variety of e-liquids in the market that can vary in composition by brand. Third, the relatively small volume and sample size of sera available precluded the possibility of performing additional assays, and additional sample collection may be needed to extrapolate the findings of our study to the general population of e-cigarette users.

Conclusions Our data demonstrated that selected e-liquid flavorings have detrimental effects on endothelial cell viability and function, changes that are accompanied by increased ROS and caspase 3/7 activity. We also showed that the use of e-cigarettes alone is capable of increasing plasma nicotine concentrations comparable to levels achieved via conventional cigarettes, indicating that e-cigarettes provide effective and measurable nicotine delivery. In addition, our results show increased ROS generation and inflammatory cytokines present in serum was observed in concert with acute e-cigarette use–induced endothelial dysfunction, as indicated by impaired tube formation of iPSC-ECs. As e-cigarette use becomes more widespread, additional studies of their health effects become more urgent as this understanding could inform both public health policy and regulation. Nonetheless, our current findings are an important first step in filling this gap by providing mechanistic insights on how e-cigarettes cause endothelial injury and dysfunction, which are an important risk factors for the development of CVD. Perspectives COMPETENCY IN MEDICAL KNOWLEDGE: Human iPSC-ECs exposed to the compounds in e-cigarette liquid or serum from e-cigarette users develop endothelial dysfunction associated with decreased viability, accumulation of ROS, and impaired proangiogenic properties. TRANSLATIONAL OUTLOOK: Because endothelial dysfunction often precedes clinical manifestations of disease, clinical studies are needed to examine the long-term effects of e-cigarettes on cardiovascular outcomes.

Appendix Online Data [S0735109719346960_mmc1.pdf]

Footnotes This work was supported by the American Heart Association (AHA) Scientist Development Grant 16SDG27560003 (to Dr. Lee), a Pilot Award from the Stanford Diabetes Research Center from a grant sponsored by the National Institutes of Health (NIH) P30DK116074 (to Dr. Lee), NIH R00 HL130416 (to Dr. Ong), NIH R01 HL141371 (to Dr. Wu), University of California Tobacco Related Disease Research Program 27IR-0012 (to Dr. Wu), AHA 17MERIT33610009 (to Dr. Wu), NIH P50-CA-180890-01 and the U.S. Food and Drug Administration Center for Tobacco Products (to Dr. Schick), University of California Tobacco Related Disease Research Program 24RT-0039 (to Dr. Schick), NIH R01 HL120062 and the U.S. Food and Drug Administration Center for Tobacco Products (to Dr. Springer), and NIH U54 HL120163 (to Dr. Bhatnagar). Dr. Springer’s wife is on advisory boards for Bayer, ADC Therapeutics, and Seattle Genetics. Dr. Wu is a co-founder of Khloris Biosciences but has no competing interests, as the work presented here is completely independent. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Listen to this manuscript's audio summary by Editor-in-Chief Dr. Valentin Fuster on JACC.org.

Abbreviations and Acronyms CVD cardiovascular disease e-cigarette electronic cigarette e-liquids electronic cigarette liquids iPSC-ECs induced pluripotent stem cell–derived endothelial cells PG propylene glycol PLT platelet ROS reactive oxygen species VG vegetable glycerin

Received June 20, 2018.

June 20, 2018. Revision received February 28, 2019.

February 28, 2019. Accepted March 3, 2019.