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A comparative assessment of e-cigarette aerosols and cigarette smoke on in vitro endothelial cell migration
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Full Length Article
A comparative assessment of e-cigarette aerosols and cigarette smoke on in vitro endothelial cell migration ⁎
Mark Taylor , Tomasz Jaunky, Katherine Hewitt, Damien Breheny, Frazer Lowe, Ian M. Fearon, Marianna Gaca British American Tobacco (Investments) Ltd, Southampton, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Cigarette smoke E-cigarette Cardiovascular disease Endothelial migration Nicotine
Cigarette smoking is a risk factor for several diseases. There has been a steep increase in the use of e-cigarettes that may offer a safer alternative to cigarette smoking. In vitro models of smoking-related diseases may provide valuable insights into disease mechanisms associated with tobacco use and could be used to assess e-cigarettes. We previously reported the application of a ‘scratch wound’ assay, measuring endothelial cell migration rate following artificial wounding, in the presence or absence of cigarette smoke extracts. This study reports the comparative effects of two commercial e-cigarette products (Vype ePen and Vype eStick) and a scientific reference cigarette (3R4F) on endothelial migration in vitro. Puff-matched extracts were generated using the Health Canada Intense (HCI) regime for cigarettes and a modified HCI for e-cigarettes. Exposure to 3R4F extract (20 h) induced concentration-dependent inhibition of endothelial cell migration, with complete inhibition at concentrations > 20%. E-cigarette extracts did not inhibit migration, even at double the 3R4F extract nicotine concentration, allowing cells to migrate into the wounded area. Our data demonstrate that e-cigarettes do not induce the inhibition of endothelial cell migration in vitro when compared to 3R4F. The scratch wound assay enables the comparative assessment between tobacco and nicotine products in vitro.
1. Introduction According to the World Health Organisation (WHO), cardiovascular diseases (CVD) contributed to 17.5 million global deaths in 2012 (World Health Organization, 2015,). Cigarette smoking is a risk factor for CVD, which include atherosclerosis, ischaemic heart disease and acute coronary thrombosis (Ambrose and Barua, 2004; Black, 1995). However, the mechanistic link between cigarette smoking and CVD is yet to be fully elucidated. The incidence of endothelial dysfunction and structural damage to the endothelium in smokers is well documented and has been shown to involve the impairment of vascular repair mechanisms, such as the inhibition of endothelial cell migration (Newby et al., 1999; Bernhard et al., 2003). Several studies have reported the development and application of in vitro models of smoking-related diseases to help elucidate the mechanisms and key events, including cell migration inhibition that are associated with the development of atherosclerosis (Fearon et al., 2013; Snajdar et al., 2001). Some of these have been used to assess the potential impact of both cigarette smoking (Snajdar et al., 2001; McQuillan et al., 2015) and cigarette smoke
⁎
toxicant exposure reduction (Fearon et al., 2012) on disease processes. It is now generally acknowledged that exposure to some of the 6500 or more identified chemical constituents (Rodgman and Perfetti, 2013) in cigarette smoke, many of which are known toxicants, are responsible for the harmful effects of cigarette smoking. Reducing the exposure to these toxicants therefore is a key element of reducing both the individual and population-level health effects of cigarette smoking, and forms the basis of tobacco harm reduction. Over the last decade, the use of electronic nicotine delivery systems (ENDS) has risen exponentially. Electronic cigarettes (e-cigarettes) in particular, are seen as potentially safer alternatives to conventional cigarettes (Pepper and Brewer, 2014; Farsalinos et al., 2014; Hajek et al., 2014). E-cigarettes comprise a variety of devices from disposable ‘cig-a-like’ devices, rechargeable closed systems and modular open-tank systems, all with accompanying e-liquids. The principle of operating ecigarettes is similar across all devices. E-cigarette aerosol is produced by heating an e-liquid, usually comprised of propylene glycol and/or glycerol and commonly containing nicotine and/or flavorings. The heat is generated by an electrical filament atomizer, connected to a battery
Corresponding author. E-mail address: [email protected] (M. Taylor).
http://dx.doi.org/10.1016/j.toxlet.2017.06.001 Received 8 March 2017; Received in revised form 24 May 2017; Accepted 2 June 2017 0378-4274/ © 2017 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Taylor, M., Toxicology Letters (2017), http://dx.doi.org/10.1016/j.toxlet.2017.06.001
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within the device generating an aerosol, which is inhaled when a user puffs on the device. Since nicotine is transferred into the inhaled aerosol in the absence of both tobacco and combustion, e-cigarettes deliver an aerosol which is considered to be substantially less toxic than cigarette smoke (Azzopardi et al., 2016; Taylor and Carr, 2016) due to reductions in exposure to chemical toxicants (Margham et al., 2016; Goniewicz et al., 2014; McRobbie et al., 2015). Needless to say, there is growing body of evidence that suggest e-cigarettes pose a reduced relative risk to smokers (Nutt et al., 2014). Public Health England and the UK Royal College of Physicians (RCP) have both recently stated that ecigarettes were likely to be 95% less harmful than cigarettes and may provide a way for smokers to reduce their cigarette consumption. (Public Health England, 2017; Royal College of Physicians, 2017). However, further studies are still required to fully substantiate their harm reduction potential. We have previously reported on the development and application of a ‘scratch wound’ assay that measures the rate of human umbilical vein endothelial cell (HUVEC) migration following artificial wounding. This study demonstrated reduced rates of HUVEC migration in the presence of cigarette smoke extracts (Fearon et al., 2012). In this paper, we report a comparison of e-cigarette aqueous extracts (AqE) generated from commercial products (Vype ePen and Vype eStick) against AqE from a scientific reference cigarette (3R4F) on HUVEC migration.
Table 2 Test product AqE exposure concentration range.
2. Materials and methods
Nicotine was quantified in AqEs by Enthalpy Analytical Inc (Durban, NC, USA), using gas chromatography with mass spectrometry (GC–MS) on an Agilent GC 7890 Series system (Agilent, Santa Clara, CA, USA) to confirm the capture of test product and to ensure AqE batch-to-batch consistency, as reported previously (Taylor and Carr, 2016). These data were also used to calculate an AqE exposure concentration of equivalent nicotine dose between products (Table 3 and 4).
E-cigarette (closed modular or cartomizer style device)
3R4F Cigarette
AqE (%) 100 90 80 70 60 50 40
AqE (%) 30 25 20 15 12.5 10 5
produce a range of AqE concentrations for in vitro exposures (Table 2). All AqEs were generated on the day of each experiment and were used within 2 h post generation. The e-cigarette AqE concentration range for exposures was determined to include nicotine concentrations that were equivalent to that calculated for highest 3R4F cigarette AqE exposure concentration. In this way, e-cigarette AqE could be tested at equivalent and higher nicotine concentrations than 3R4F AqE (refer to results Table 4). 2.3. AqE nicotine quantification
2.1. Test products The 3R4F reference product (University of Kentucky) was used as a scientific reference cigarette in this study. This cigarette has a tar yield of 9.4 mg (International Organization for Standardization [ISO]). 3R4F cigarettes were conditioned for a minimum of 48 h at 22 ± 1 °C and 60 ± 3% relative humidity, in accordance with ISO 3402:1999 (International Organization for Standardization, 1999) before use. Two commercially available Vype e-cigarettes (Nicoventures, Blackburn, UK) were used in this study; a ‘cig-a-like’ cartomizer style product (Vype eStick) and a closed modular product (Vype ePen). For both devices, ‘Blended Tobacco’ flavoured variant was used containing 36 mg/mL nicotine (cartomizer style) and 18 mg/mL nicotine (closed modular). The cartomizer style product operated at 3.7 V and the closed modular device at 4.0 V. The e-cigarette product batteries were fully charged before use and a fresh e-liquid cartridge was used to obtain each batch of aqueous aerosol extracts (AqE).
2.4. Endothelial cell culture Primary human umbilical vein endothelial cells (HUVECs; Lifeline Cell Technology, California, USA) were maintained at 37 °C in a 5% CO2 humidified atmosphere. Cells were cultured in ‘complete’ VascuLife® cell culture media (Lifeline Cell Technology), supplemented with vascular endothelial growth factor (5 ng/mL), epidermal growth factor (5 ng/mL), basic fibroblast growth factor (5 ng/mL), insulin-like growth factor 1 (15 ng/mL), ascorbic acid (50 μg/mL), L-glutamine (10 mM), hydrocortisone hemisuccinate (1 μg/mL), heparin sulphate (0.75 units/mL) and foetal bovine serum (FBS) (2%). Cryopreserved HUVECs were recovered and cultured for 3 days before further subculturing. HUVECs were either seeded into new flasks, or into 24-well ImageLock™ Plates (Essen Instruments, Ann Arbor, MI, USA) for migration assays and grown to confluency before use. HUVECs were discarded after 4 passage cycles.
2.2. Preparation of aqueous aerosol extracts (AqE) Aqueous extracts (AqE) generated from 3R4F cigarette or e-cigarette whole aerosols were produced as previously described (Taylor and Carr, 2016). Briefly, products were machine-puffed on a Borgwaldt-KC RM20H smoking engine, following the HCI machine puffing regime or a modified HCI regime (Table 1). All AqEs were generated by bubbling 10 × 55 mL puffs through 20 mL of AqE capture media (VascuLife® media with added supplements and 0.1% foetal bovine serum (FBS)) contained in an impinger. This procedure provided a stock (100%) AqE that was diluted with appropriate volumes of VascuLife® media to
2.5. Endothelial cell scratch wound (migration) assay The scratch wound assay was utilized to detect and measure the inhibition of endothelial migration rates in vitro, as described previously (Fearon et al., 2012). 24-well ImageLock™ plates (Essen Instruments)
Table 1 Aerosol generation regimens. Product Cigarette e-cigarette
Puff Regimen 1
HCI CRM2
Puff Volume (mL)
Puff Frequency (secs)
Puff Duration (secs)
Puff Profile
Vent blocking
Coil pre-activation (secs)
55 55
30 30
2 3
Bell Square
100% N/A
N/A 0
1
= HCI T-115 (World Health Organization, 2015,). = CRM No 81 (Ambrose and Barua, 2004). N/A = metric not applicable 2
2
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3. Results
Table 3 Test product AqE nicotine levels. Nicotine was captured in the AqEs from 3R4F reference cigarette smoke and e-cigarette aerosols. Data shown are mean ( ± standard deviation (S.D.) of 6 individual batches of AqE for each test product.
3.1. AqE nicotine quantification
E-cigarette (closed modular device)
E-cigarette (cartomizer style device)
3R4F Cigarette
The amounts of nicotine in test product AqEs were quantified by GC–MS (Table 3). The 3R4F cigarette AqE contained an average of 6.31 ( ± 0.96) μg/mL nicotine and the e-cigarette AqEs contained an average of 3.91 ( ± 0.31) μg/mL and 4.76 ( ± 0.73) μg/mL for cartomizer style and modular devices, respectively. Assuming linear dilution of nicotine in the AqE when test concentrations were generated from the stock AqE, the maximum 3R4F AqE exposure concentration (30%) contained approximately 1.89 μg/mL nicotine. The closed modular device e-cigarette AqE contained 1.90 μg/mL at 40% dilution and the cartomizer style device AqE contained 1.95 μg/mL nicotine at 50% dilution (Table 4). This allowed a comparison of assay responses at equivalent nicotine doses across the test products.
AqE (%)
AqE (%)
AqE (%)
3.2. Time-dependent HUVEC cell migration
Test product AqE
3R4F reference cigarette
E-cigarette (cartomizer style device)
E-cigarette (closed modular device)
Nicotine (μg/ mL) S.D.
6.31
3.91
4.76
0.96
0.31
0.73
Table 4 Calculated nicotine levels in test product AqE exposure concentrations. Nicotine concentrations were calculated on the assumption of linear dilution of nicotine in the AqE.
100 90 80 70 60 50 40
Nicotine (μg/ mL) 4.76 4.28 3.81 3.33 2.85 2.38 1.90
100 90 80 70 60 50 40
Nicotine (μg/ mL) 3.91 3.52 3.13 2.74 2.35 1.95 1.56
30 25 20 15 12.5 10 5
Nicotine (μg/ mL) 1.89 0.47 0.38 0.28 0.24 0.19 0.09
The scratch wound assay was initially performed with AqE capture media as a negative control or cytochalasin D (2 μM) as a positive control to ascertain a baseline and maximal inhibition of migration response, respectively. The wound widths were measured hourly, which allowed calculations of rate of migration in vitro (μm/h). During the 20 h exposure, HUVECs incubated with AqE capture media migrated into the artificially induced wound, reducing the wound width in a time-dependent manner, leading to near complete closure of the wound after 20 h (Fig. 1). In the presence of cytochalasin D however, HUVEC migration was completely inhibited, resulting in no wound closure during the 20 h exposure period (Fig. 1). Representative images show the changes in wound widths at the indicated time points in response to the AqE capture media or cytochalasin D (Fig. 2).
were seeded with HUVECs in complete VascuLife® media and allowed to grow to confluency over a period of 24–48 h prior to performing the assay. Monolayers were confirmed for confluency by well-scanning using the IncuCyte imaging apparatus (Essen Instruments). On the morning of the scratch wound assay the complete VascuLife® media was replaced with AqE capture media (VascuLife® media containing 0.1% FBS) to minimise proliferation. Following a 6 h incubation, scratch wounds were created on the monolayers using the WoundMaker apparatus (Essen Instruments). This was achieved by attaching sterile 10 μL pipette tips to the WoundMaker followed by moving the ImageLock™ plate backwards and forwards three times to create linear scratch wounds. Immediately following wounding, media were replaced with either test product AqE, the positive control cytochalasin D (2 μM) or AqE capture media, which was the negative control for the assay. Cytochalasin D is a cell-permeable inhibitor of actin polymerization, which is a critical requirement for endothelial cell migration. HUVEC monolayers in duplicate wells were exposed to test product AqE or the assay controls. The migration rates were compared to the baseline migration rates calculated in the wells exposed to the AqE capture media only. This experiment was repeated a minimum of four times with different HUVEC passage numbers (passage 1–4). Experimental plates were placed inside the IncuCyte live-cell imaging system in a 5% CO2 humidified atmosphere at 37 °C, where individual images of each scratch wound were taken at hourly intervals over a 20 h period.
3.3. Cigarette smoke AqE inhibited HUVEC migration into an artificially induced wound The effect of cigarette smoke AqE on HUVEC migration was assessed across concentrations ranging from 0 to 30% 3R4F AqE, which represented exposure to 0.09–1.98 μg/mL nicotine. A concentration-dependent inhibition of HUVEC migration was observed across the AqE exposure range, and confirmed previous findings which demonstrated HUVEC migration inhibition following exposure to cigarette smoke extracts (Fearon et al., 2012). Exposure to 3R4F AqE at concentrations between 5% and 20% significantly inhibited migration rates in a concentration-dependent manner, and a complete inhibition of endothelial migration was observed when the cells were exposed to 3R4F AqE
2.6. Statistical analysis and rate calculation methods Numerical data were obtained by measuring the scratch wound widths (μm) at each hourly time point, allowing the reductions in wound width (μm/h) to be calculated for each test product AqE exposure. A repeated measures mixed model statistical analysis of the data was performed with SAS software (version 9.3). Dunnett’s test was employed to compare the AqE-treated cellular responses to the untreated cellular response to identify the AqE concentration required to induce a significant change in migration rate. The data were expressed as a percentage of the initial wound width or as a percentage of the baseline migration rate.
Fig. 1. Untreated HUVECs migrated into the artificially induced wound, reducing wound width over 20 h. In the presence of cytochalasin D (2 μM) HUVEC migration was completely inhibited. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells from a minimum of 4 independent experiments.
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Fig. 2. Representative images from the scratch wound assay at 0, 5, 15 and 20 h timepoints in the presence of the AqE capture media or cytochalasin D (2 μM). Untreated HUVECs migrated into the wound over 20 h, which was not observed with cytochalasin D treatment.
Fig. 3. Reference 3R4F cigarette AqE inhibited HUVEC migration in a concentrationdependent manner. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells and from a minimum of 4 independent experiments.
Fig. 4. E-cigarette AqE from a closed modular device did not inhibit HUVEC migration. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells and from a minimum of 4 independent experiments.
concentrations greater than 20%. The normalized wound widths are shown in Fig. 3.
tested. This AqE concentration range contained 1.56–3.91 μg/mL nicotine or 1.90–4.76 μg/mL nicotine for the cartomizer or modular devices, respectively (Table 4). As illustrated in Figs. 4 and 5, e-cigarette AqE did not cause any significant reductions in HUVEC migration rate, irrespective of the device type from which the AqE were obtained.
3.4. E-cigarette AqE treated- HUVECs migrated into an artificially induced wound We next assessed the effect of AqE generated from two e-cigarettes on HUVEC migration. AqE concentrations between 40% and 100% were 4
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Ambrose and Barua, 2004; Black, 1995; Bakhru and Erlinger, 2005), although the exact mechanisms are yet to be fully defined. A number of in vitro models have been employed to study cardiovascular disease mechanisms and to examine the role of smoking in disease initiation and progression (Fearon et al., 2013, 2012; Snajdar et al., 2001; McQuillan et al., 2015; Soghomonians et al., 2004). Such models could be used to help understand and assess novel tobacco and nicotine delivery products such as e-cigarettes. Historically cigarette smoke total particulate matter (TPM) has been used as an exposure matrix for in vitro models of smoking-related disease processes. However, this exposure matrix does not include the vapour phase components and therefore this approach has been criticised for not providing a biologically-relevant system of exposing vascular cells to smoke toxicants, since only those chemical species that can readily pass into the circulation would gain access to vascular cells (Fearon et al., 2013; Adamson and Thorne, 2013). For this reason, AqE, the water-soluble fraction of smoke or e-cigarette aerosols, was employed as an exposure matrix for the submerged HUVEC cultures in this study, to more closely represent in vivo exposure. The endothelial scratch wound (migration) assay has been described as a simple and well-developed method to measure cell migration in vitro (Liang et al., 2007). This technique has been used extensively to assess tobacco products and has been demonstrated to be responsive, in a concentration-dependent manner to extracts of cigarettes smoke (Snajdar et al., 2001; Fearon et al., 2012; Soghomonians et al., 2004). In this study, we used the scratch wound assay to examine effects on HUVEC migration rates of exposure to AqE generated from cigarette smoke and e-cigarette aerosols. The results from the scratch wound assay demonstrated that cigarette smoke AqE had an inhibitory effect on endothelial migration in vitro. The concentration-dependent inhibition in HUVEC migration observed in this study upon treatment with 3R4F AqE suggests that chemical species present within the 3R4F AqE were responsible for this inhibition, and that this effect could be proportionally reduced via dilution of the AqE. No significant inhibition of HUVEC migration was measured following exposure to e-cigarette AqEs (cartomizer style or closed modular device), under consistent experimental conditions that included puff-matched AqE. Notably, the exposure concentration range for the e-cigarette AqE included an equivalent nicotine dose to the 3R4F AqE and the undiluted (100%) stock AqE, which contained at least double nicotine of the 30% 3R4F AqE. Whilst some variation in the endothelial migration rates could be observed following exposure to ecigarette AqE, crucially these fluctuations did not follow an AqE concentration-dependent pattern and were within the inherent variability of the assay. Therefore, it can be hypothesised that chemical species present in the 3R4F smoke AqE that were responsible for the inhibition of endothelial migration were either absent in the e-cigarette AqEs or present in insufficient concentrations to elicit any significant assay response. The scratch assay has been previously utilized to assess the effects of individual cigarette smoke components, such as cadmium and nicotine (Snajdar et al., 2001). This study reported that exposure of HUVECs to a cigarette smoke extract induced concentration-dependent decreases in endothelial migration rates, whilst in the same study, pure nicotine (0.4–20 μg/mL) had no effect. This supports our observation that nicotine did not appear to be responsible for the inhibition of endothelial migration that was induced by 3R4F AqE exposure. This conclusion was based on the observation that when HUVECs were exposed to AqEs containing double the nicotine concentration of the 3R4F AqE, no inhibition of endothelial migration was observed. Moreover, recent studies have demonstrated that nicotine may actually induce migration and proliferation of vascular cells by binding to specific nicotinic acetylcholine receptors (Park et al., 2008; Liu and Nicotine, 2014). A potential mechanism for the adverse effects of smoking on the cardiovascular system has been described, which involves oxidative stress (Messner and Bernhard, 2014). Tobacco smoke contains tar and
Fig. 5. E-cigarette AqE from a cartomizer style device did not inhibit HUVEC migration. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells and from a minimum of 4 independent experiments.
3.5. 3R4F AqE treated HUVECs demonstrated inhibition of migration compared to e-cigarette AqE To compare the inhibition of endothelial migration by the different test product AqEs, the mean HUVEC migration rates (μm/h) over 20 h were calculated for each AqE exposure concentration and normalized to the baseline migration rate, where cells were exposed to AqE capture media (Fig. 6). Exposure to 3R4F AqE significantly inhibited migration rates in a concentration-dependent manner; ranging from the uninhibited baseline migration rate of 21.06 ( ± 5.52) μm/h, to an almost complete inhibition of endothelial migration with a rate of 1.06 ( ± 2.77) μm/h. Exposure to either the closed modular or cartomizer style e-cigarette AqE did not induce any significant reductions in HUVEC migration rates. Significant differences between responses to 3R4F AqE and e-cigarette AqEs were observed (p < 0.05). 4. Discussion Over the last decade there has been an increase in the numbers of consumers switching to novel tobacco and nicotine products, including e-cigarettes. Public Health England have recently stated that e-cigarettes could provide a 95% reduced risk compared to cigarettes and the UK Royal College of Physicians have supported the use of e-cigarettes as replacement products for smokers (Public Health England, 2017; Royal College of Physicians, 2017). There currently exists insufficient longterm epidemiological data to support these health claims. The deleterious effects of tobacco smoking to the cardiovascular system are well recognised and widely reported (World Health Organization, 2015;
Fig. 6. Comparison of endothelial cell migration rates following exposure to AqE from 3R4F cigarettes or AqE from e-cigarettes over a 20 h exposure period. 3R4F AqE induced a concentration-dependant inhibition of HUVEC migration, compared to no inhibition of migration following exposure to e-cigarette AqE. Data are means ± SEM from duplicate wells and from a minimum of 4 independent experiments.
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various gas phase species, including carbonyls, which provide a source of oxidants and free-radicals (Faux et al., 2009; Pryor, 1997). These highly reactive and damaging chemicals have been shown to induce endothelial dysfunction through oxidative damage to protein and lipids and to elicit both apoptosis and necrosis (Messner et al., 2012), which results from oxidative damage to mitochondria or DNA (Kannan and Jain, 2016). The increasingly widespread use of e-cigarette as an alternative to cigarette smoking has the potential to reduce CVD by reducing a consumer’s exposure to pro-oxidant species. Previously reported studies have described the reduced number of individual chemicals within e-cigarette aerosols as compared to tobacco smoke and when present, have been quantified to exist at very low concentrations (Taylor and Carr, 2016; Margham et al., 2016; Goniewicz et al., 2014; Callahan-Lyon, 2014; Cheng, 2014). Carbonyl species have been detected and quantified in e-cigarette aerosols (Taylor and Carr, 2016; Bekki et al., 2014; Kosmider et al., 2014; Jensen et al., 2015; Farsalinoes et al., 2015) and found to exist in far lower concentration than those found in cigarette smoke. Our data has demonstrated that AqEs of two commercially available e-cigarettes did not induce the inhibition of endothelial cell migration in vitro as compared to 3R4F AqE. Further investigations might incorporate the testing of a wide range of e-cigarette devices and e-liquids in order to better reflect the diversity of products in the e-cigarette market. The scratch wound assay enables a comparative assessment between conventional cigarettes and e-cigarettes in vitro and demonstrates the usefulness and versatility of this assay for the assessment of e-cigarettes. Further in vitro studies incorporating aerosol exposures, dosimetry techniques, use of 3D tissue models, multi-endpoint analysis and broader global omics approaches can provide researchers with invaluable data to support the assessment of novel tobacco and nicotine products. Author contributions T.J, K.H and M.T performed the main experiments; M.T and F.L designed the experiments and analysed the data; M.T, M.G and I.F wrote and edited the manuscript; D.B and M.G revised the manuscript. Funding All experimental work was funded by Nicoventures Ltd., UK, a wholly-owned subsidiary of British American Tobacco Investments Ltd. All study products were provided by Nicoventures. Disclosure statement The authors are employees of British American Tobacco Investments Ltd. The authors alone are responsible for the content and writing of this article. References Adamson, J., Thorne, D., 2013. A review of in vitro cigarette smoke exposure systems. Exp. Toxicol. Pathol. 65. Ambrose, J.A., Barua, R.S., 2004. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J. Am. Coll. Cardiol. 43 (May(10)), 1731–1737. Azzopardi, D., Patel, K., Jaunky, T., et al., 2016. Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke. Toxicol. Mech. Methods 26 (July (6)), 477–491. Bakhru, A., Erlinger, T.P., 2005. Smoking Cessation and Cardiovascular Disease Risk Factors: Results from the Third National Health and Nutrition Examination Survey. PLoS Med. 2 (June(6)), e160. Bekki, K., Uchiyama, S., Ohta, K., Inaba, Y., Nakagome, H., Kunugita, N., 2014. Carbonyl compounds generated from electronic cigarettes. Int. J. Environ. Res. Public Health 11 (October(11)), 11192–111200. Bernhard, D., Pfister, G., Huck, C.W., et al., 2003. Disruption of vascular endothelial homeostasis by tobacco smoke: impact on atherosclerosis. FASEB J. 17 (December (15)), 2302–2304. Black, H.R., 1995. Smoking and cardiovascular disease. In: Laragh, J.H., Brenner, B.M. (Eds.), Hypertension: Pathophysiology, Diagnosis and Management, 2nd edition.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Full Length Article
A comparative assessment of e-cigarette aerosols and cigarette smoke on in vitro endothelial cell migration ⁎
Mark Taylor , Tomasz Jaunky, Katherine Hewitt, Damien Breheny, Frazer Lowe, Ian M. Fearon, Marianna Gaca British American Tobacco (Investments) Ltd, Southampton, UK
A R T I C L E I N F O
A B S T R A C T
Keywords: Cigarette smoke E-cigarette Cardiovascular disease Endothelial migration Nicotine
Cigarette smoking is a risk factor for several diseases. There has been a steep increase in the use of e-cigarettes that may offer a safer alternative to cigarette smoking. In vitro models of smoking-related diseases may provide valuable insights into disease mechanisms associated with tobacco use and could be used to assess e-cigarettes. We previously reported the application of a ‘scratch wound’ assay, measuring endothelial cell migration rate following artificial wounding, in the presence or absence of cigarette smoke extracts. This study reports the comparative effects of two commercial e-cigarette products (Vype ePen and Vype eStick) and a scientific reference cigarette (3R4F) on endothelial migration in vitro. Puff-matched extracts were generated using the Health Canada Intense (HCI) regime for cigarettes and a modified HCI for e-cigarettes. Exposure to 3R4F extract (20 h) induced concentration-dependent inhibition of endothelial cell migration, with complete inhibition at concentrations > 20%. E-cigarette extracts did not inhibit migration, even at double the 3R4F extract nicotine concentration, allowing cells to migrate into the wounded area. Our data demonstrate that e-cigarettes do not induce the inhibition of endothelial cell migration in vitro when compared to 3R4F. The scratch wound assay enables the comparative assessment between tobacco and nicotine products in vitro.
1. Introduction According to the World Health Organisation (WHO), cardiovascular diseases (CVD) contributed to 17.5 million global deaths in 2012 (World Health Organization, 2015,). Cigarette smoking is a risk factor for CVD, which include atherosclerosis, ischaemic heart disease and acute coronary thrombosis (Ambrose and Barua, 2004; Black, 1995). However, the mechanistic link between cigarette smoking and CVD is yet to be fully elucidated. The incidence of endothelial dysfunction and structural damage to the endothelium in smokers is well documented and has been shown to involve the impairment of vascular repair mechanisms, such as the inhibition of endothelial cell migration (Newby et al., 1999; Bernhard et al., 2003). Several studies have reported the development and application of in vitro models of smoking-related diseases to help elucidate the mechanisms and key events, including cell migration inhibition that are associated with the development of atherosclerosis (Fearon et al., 2013; Snajdar et al., 2001). Some of these have been used to assess the potential impact of both cigarette smoking (Snajdar et al., 2001; McQuillan et al., 2015) and cigarette smoke
⁎
toxicant exposure reduction (Fearon et al., 2012) on disease processes. It is now generally acknowledged that exposure to some of the 6500 or more identified chemical constituents (Rodgman and Perfetti, 2013) in cigarette smoke, many of which are known toxicants, are responsible for the harmful effects of cigarette smoking. Reducing the exposure to these toxicants therefore is a key element of reducing both the individual and population-level health effects of cigarette smoking, and forms the basis of tobacco harm reduction. Over the last decade, the use of electronic nicotine delivery systems (ENDS) has risen exponentially. Electronic cigarettes (e-cigarettes) in particular, are seen as potentially safer alternatives to conventional cigarettes (Pepper and Brewer, 2014; Farsalinos et al., 2014; Hajek et al., 2014). E-cigarettes comprise a variety of devices from disposable ‘cig-a-like’ devices, rechargeable closed systems and modular open-tank systems, all with accompanying e-liquids. The principle of operating ecigarettes is similar across all devices. E-cigarette aerosol is produced by heating an e-liquid, usually comprised of propylene glycol and/or glycerol and commonly containing nicotine and/or flavorings. The heat is generated by an electrical filament atomizer, connected to a battery
Corresponding author. E-mail address: [email protected] (M. Taylor).
http://dx.doi.org/10.1016/j.toxlet.2017.06.001 Received 8 March 2017; Received in revised form 24 May 2017; Accepted 2 June 2017 0378-4274/ © 2017 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
Please cite this article as: Taylor, M., Toxicology Letters (2017), http://dx.doi.org/10.1016/j.toxlet.2017.06.001
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within the device generating an aerosol, which is inhaled when a user puffs on the device. Since nicotine is transferred into the inhaled aerosol in the absence of both tobacco and combustion, e-cigarettes deliver an aerosol which is considered to be substantially less toxic than cigarette smoke (Azzopardi et al., 2016; Taylor and Carr, 2016) due to reductions in exposure to chemical toxicants (Margham et al., 2016; Goniewicz et al., 2014; McRobbie et al., 2015). Needless to say, there is growing body of evidence that suggest e-cigarettes pose a reduced relative risk to smokers (Nutt et al., 2014). Public Health England and the UK Royal College of Physicians (RCP) have both recently stated that ecigarettes were likely to be 95% less harmful than cigarettes and may provide a way for smokers to reduce their cigarette consumption. (Public Health England, 2017; Royal College of Physicians, 2017). However, further studies are still required to fully substantiate their harm reduction potential. We have previously reported on the development and application of a ‘scratch wound’ assay that measures the rate of human umbilical vein endothelial cell (HUVEC) migration following artificial wounding. This study demonstrated reduced rates of HUVEC migration in the presence of cigarette smoke extracts (Fearon et al., 2012). In this paper, we report a comparison of e-cigarette aqueous extracts (AqE) generated from commercial products (Vype ePen and Vype eStick) against AqE from a scientific reference cigarette (3R4F) on HUVEC migration.
Table 2 Test product AqE exposure concentration range.
2. Materials and methods
Nicotine was quantified in AqEs by Enthalpy Analytical Inc (Durban, NC, USA), using gas chromatography with mass spectrometry (GC–MS) on an Agilent GC 7890 Series system (Agilent, Santa Clara, CA, USA) to confirm the capture of test product and to ensure AqE batch-to-batch consistency, as reported previously (Taylor and Carr, 2016). These data were also used to calculate an AqE exposure concentration of equivalent nicotine dose between products (Table 3 and 4).
E-cigarette (closed modular or cartomizer style device)
3R4F Cigarette
AqE (%) 100 90 80 70 60 50 40
AqE (%) 30 25 20 15 12.5 10 5
produce a range of AqE concentrations for in vitro exposures (Table 2). All AqEs were generated on the day of each experiment and were used within 2 h post generation. The e-cigarette AqE concentration range for exposures was determined to include nicotine concentrations that were equivalent to that calculated for highest 3R4F cigarette AqE exposure concentration. In this way, e-cigarette AqE could be tested at equivalent and higher nicotine concentrations than 3R4F AqE (refer to results Table 4). 2.3. AqE nicotine quantification
2.1. Test products The 3R4F reference product (University of Kentucky) was used as a scientific reference cigarette in this study. This cigarette has a tar yield of 9.4 mg (International Organization for Standardization [ISO]). 3R4F cigarettes were conditioned for a minimum of 48 h at 22 ± 1 °C and 60 ± 3% relative humidity, in accordance with ISO 3402:1999 (International Organization for Standardization, 1999) before use. Two commercially available Vype e-cigarettes (Nicoventures, Blackburn, UK) were used in this study; a ‘cig-a-like’ cartomizer style product (Vype eStick) and a closed modular product (Vype ePen). For both devices, ‘Blended Tobacco’ flavoured variant was used containing 36 mg/mL nicotine (cartomizer style) and 18 mg/mL nicotine (closed modular). The cartomizer style product operated at 3.7 V and the closed modular device at 4.0 V. The e-cigarette product batteries were fully charged before use and a fresh e-liquid cartridge was used to obtain each batch of aqueous aerosol extracts (AqE).
2.4. Endothelial cell culture Primary human umbilical vein endothelial cells (HUVECs; Lifeline Cell Technology, California, USA) were maintained at 37 °C in a 5% CO2 humidified atmosphere. Cells were cultured in ‘complete’ VascuLife® cell culture media (Lifeline Cell Technology), supplemented with vascular endothelial growth factor (5 ng/mL), epidermal growth factor (5 ng/mL), basic fibroblast growth factor (5 ng/mL), insulin-like growth factor 1 (15 ng/mL), ascorbic acid (50 μg/mL), L-glutamine (10 mM), hydrocortisone hemisuccinate (1 μg/mL), heparin sulphate (0.75 units/mL) and foetal bovine serum (FBS) (2%). Cryopreserved HUVECs were recovered and cultured for 3 days before further subculturing. HUVECs were either seeded into new flasks, or into 24-well ImageLock™ Plates (Essen Instruments, Ann Arbor, MI, USA) for migration assays and grown to confluency before use. HUVECs were discarded after 4 passage cycles.
2.2. Preparation of aqueous aerosol extracts (AqE) Aqueous extracts (AqE) generated from 3R4F cigarette or e-cigarette whole aerosols were produced as previously described (Taylor and Carr, 2016). Briefly, products were machine-puffed on a Borgwaldt-KC RM20H smoking engine, following the HCI machine puffing regime or a modified HCI regime (Table 1). All AqEs were generated by bubbling 10 × 55 mL puffs through 20 mL of AqE capture media (VascuLife® media with added supplements and 0.1% foetal bovine serum (FBS)) contained in an impinger. This procedure provided a stock (100%) AqE that was diluted with appropriate volumes of VascuLife® media to
2.5. Endothelial cell scratch wound (migration) assay The scratch wound assay was utilized to detect and measure the inhibition of endothelial migration rates in vitro, as described previously (Fearon et al., 2012). 24-well ImageLock™ plates (Essen Instruments)
Table 1 Aerosol generation regimens. Product Cigarette e-cigarette
Puff Regimen 1
HCI CRM2
Puff Volume (mL)
Puff Frequency (secs)
Puff Duration (secs)
Puff Profile
Vent blocking
Coil pre-activation (secs)
55 55
30 30
2 3
Bell Square
100% N/A
N/A 0
1
= HCI T-115 (World Health Organization, 2015,). = CRM No 81 (Ambrose and Barua, 2004). N/A = metric not applicable 2
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3. Results
Table 3 Test product AqE nicotine levels. Nicotine was captured in the AqEs from 3R4F reference cigarette smoke and e-cigarette aerosols. Data shown are mean ( ± standard deviation (S.D.) of 6 individual batches of AqE for each test product.
3.1. AqE nicotine quantification
E-cigarette (closed modular device)
E-cigarette (cartomizer style device)
3R4F Cigarette
The amounts of nicotine in test product AqEs were quantified by GC–MS (Table 3). The 3R4F cigarette AqE contained an average of 6.31 ( ± 0.96) μg/mL nicotine and the e-cigarette AqEs contained an average of 3.91 ( ± 0.31) μg/mL and 4.76 ( ± 0.73) μg/mL for cartomizer style and modular devices, respectively. Assuming linear dilution of nicotine in the AqE when test concentrations were generated from the stock AqE, the maximum 3R4F AqE exposure concentration (30%) contained approximately 1.89 μg/mL nicotine. The closed modular device e-cigarette AqE contained 1.90 μg/mL at 40% dilution and the cartomizer style device AqE contained 1.95 μg/mL nicotine at 50% dilution (Table 4). This allowed a comparison of assay responses at equivalent nicotine doses across the test products.
AqE (%)
AqE (%)
AqE (%)
3.2. Time-dependent HUVEC cell migration
Test product AqE
3R4F reference cigarette
E-cigarette (cartomizer style device)
E-cigarette (closed modular device)
Nicotine (μg/ mL) S.D.
6.31
3.91
4.76
0.96
0.31
0.73
Table 4 Calculated nicotine levels in test product AqE exposure concentrations. Nicotine concentrations were calculated on the assumption of linear dilution of nicotine in the AqE.
100 90 80 70 60 50 40
Nicotine (μg/ mL) 4.76 4.28 3.81 3.33 2.85 2.38 1.90
100 90 80 70 60 50 40
Nicotine (μg/ mL) 3.91 3.52 3.13 2.74 2.35 1.95 1.56
30 25 20 15 12.5 10 5
Nicotine (μg/ mL) 1.89 0.47 0.38 0.28 0.24 0.19 0.09
The scratch wound assay was initially performed with AqE capture media as a negative control or cytochalasin D (2 μM) as a positive control to ascertain a baseline and maximal inhibition of migration response, respectively. The wound widths were measured hourly, which allowed calculations of rate of migration in vitro (μm/h). During the 20 h exposure, HUVECs incubated with AqE capture media migrated into the artificially induced wound, reducing the wound width in a time-dependent manner, leading to near complete closure of the wound after 20 h (Fig. 1). In the presence of cytochalasin D however, HUVEC migration was completely inhibited, resulting in no wound closure during the 20 h exposure period (Fig. 1). Representative images show the changes in wound widths at the indicated time points in response to the AqE capture media or cytochalasin D (Fig. 2).
were seeded with HUVECs in complete VascuLife® media and allowed to grow to confluency over a period of 24–48 h prior to performing the assay. Monolayers were confirmed for confluency by well-scanning using the IncuCyte imaging apparatus (Essen Instruments). On the morning of the scratch wound assay the complete VascuLife® media was replaced with AqE capture media (VascuLife® media containing 0.1% FBS) to minimise proliferation. Following a 6 h incubation, scratch wounds were created on the monolayers using the WoundMaker apparatus (Essen Instruments). This was achieved by attaching sterile 10 μL pipette tips to the WoundMaker followed by moving the ImageLock™ plate backwards and forwards three times to create linear scratch wounds. Immediately following wounding, media were replaced with either test product AqE, the positive control cytochalasin D (2 μM) or AqE capture media, which was the negative control for the assay. Cytochalasin D is a cell-permeable inhibitor of actin polymerization, which is a critical requirement for endothelial cell migration. HUVEC monolayers in duplicate wells were exposed to test product AqE or the assay controls. The migration rates were compared to the baseline migration rates calculated in the wells exposed to the AqE capture media only. This experiment was repeated a minimum of four times with different HUVEC passage numbers (passage 1–4). Experimental plates were placed inside the IncuCyte live-cell imaging system in a 5% CO2 humidified atmosphere at 37 °C, where individual images of each scratch wound were taken at hourly intervals over a 20 h period.
3.3. Cigarette smoke AqE inhibited HUVEC migration into an artificially induced wound The effect of cigarette smoke AqE on HUVEC migration was assessed across concentrations ranging from 0 to 30% 3R4F AqE, which represented exposure to 0.09–1.98 μg/mL nicotine. A concentration-dependent inhibition of HUVEC migration was observed across the AqE exposure range, and confirmed previous findings which demonstrated HUVEC migration inhibition following exposure to cigarette smoke extracts (Fearon et al., 2012). Exposure to 3R4F AqE at concentrations between 5% and 20% significantly inhibited migration rates in a concentration-dependent manner, and a complete inhibition of endothelial migration was observed when the cells were exposed to 3R4F AqE
2.6. Statistical analysis and rate calculation methods Numerical data were obtained by measuring the scratch wound widths (μm) at each hourly time point, allowing the reductions in wound width (μm/h) to be calculated for each test product AqE exposure. A repeated measures mixed model statistical analysis of the data was performed with SAS software (version 9.3). Dunnett’s test was employed to compare the AqE-treated cellular responses to the untreated cellular response to identify the AqE concentration required to induce a significant change in migration rate. The data were expressed as a percentage of the initial wound width or as a percentage of the baseline migration rate.
Fig. 1. Untreated HUVECs migrated into the artificially induced wound, reducing wound width over 20 h. In the presence of cytochalasin D (2 μM) HUVEC migration was completely inhibited. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells from a minimum of 4 independent experiments.
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Fig. 2. Representative images from the scratch wound assay at 0, 5, 15 and 20 h timepoints in the presence of the AqE capture media or cytochalasin D (2 μM). Untreated HUVECs migrated into the wound over 20 h, which was not observed with cytochalasin D treatment.
Fig. 3. Reference 3R4F cigarette AqE inhibited HUVEC migration in a concentrationdependent manner. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells and from a minimum of 4 independent experiments.
Fig. 4. E-cigarette AqE from a closed modular device did not inhibit HUVEC migration. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells and from a minimum of 4 independent experiments.
concentrations greater than 20%. The normalized wound widths are shown in Fig. 3.
tested. This AqE concentration range contained 1.56–3.91 μg/mL nicotine or 1.90–4.76 μg/mL nicotine for the cartomizer or modular devices, respectively (Table 4). As illustrated in Figs. 4 and 5, e-cigarette AqE did not cause any significant reductions in HUVEC migration rate, irrespective of the device type from which the AqE were obtained.
3.4. E-cigarette AqE treated- HUVECs migrated into an artificially induced wound We next assessed the effect of AqE generated from two e-cigarettes on HUVEC migration. AqE concentrations between 40% and 100% were 4
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Ambrose and Barua, 2004; Black, 1995; Bakhru and Erlinger, 2005), although the exact mechanisms are yet to be fully defined. A number of in vitro models have been employed to study cardiovascular disease mechanisms and to examine the role of smoking in disease initiation and progression (Fearon et al., 2013, 2012; Snajdar et al., 2001; McQuillan et al., 2015; Soghomonians et al., 2004). Such models could be used to help understand and assess novel tobacco and nicotine delivery products such as e-cigarettes. Historically cigarette smoke total particulate matter (TPM) has been used as an exposure matrix for in vitro models of smoking-related disease processes. However, this exposure matrix does not include the vapour phase components and therefore this approach has been criticised for not providing a biologically-relevant system of exposing vascular cells to smoke toxicants, since only those chemical species that can readily pass into the circulation would gain access to vascular cells (Fearon et al., 2013; Adamson and Thorne, 2013). For this reason, AqE, the water-soluble fraction of smoke or e-cigarette aerosols, was employed as an exposure matrix for the submerged HUVEC cultures in this study, to more closely represent in vivo exposure. The endothelial scratch wound (migration) assay has been described as a simple and well-developed method to measure cell migration in vitro (Liang et al., 2007). This technique has been used extensively to assess tobacco products and has been demonstrated to be responsive, in a concentration-dependent manner to extracts of cigarettes smoke (Snajdar et al., 2001; Fearon et al., 2012; Soghomonians et al., 2004). In this study, we used the scratch wound assay to examine effects on HUVEC migration rates of exposure to AqE generated from cigarette smoke and e-cigarette aerosols. The results from the scratch wound assay demonstrated that cigarette smoke AqE had an inhibitory effect on endothelial migration in vitro. The concentration-dependent inhibition in HUVEC migration observed in this study upon treatment with 3R4F AqE suggests that chemical species present within the 3R4F AqE were responsible for this inhibition, and that this effect could be proportionally reduced via dilution of the AqE. No significant inhibition of HUVEC migration was measured following exposure to e-cigarette AqEs (cartomizer style or closed modular device), under consistent experimental conditions that included puff-matched AqE. Notably, the exposure concentration range for the e-cigarette AqE included an equivalent nicotine dose to the 3R4F AqE and the undiluted (100%) stock AqE, which contained at least double nicotine of the 30% 3R4F AqE. Whilst some variation in the endothelial migration rates could be observed following exposure to ecigarette AqE, crucially these fluctuations did not follow an AqE concentration-dependent pattern and were within the inherent variability of the assay. Therefore, it can be hypothesised that chemical species present in the 3R4F smoke AqE that were responsible for the inhibition of endothelial migration were either absent in the e-cigarette AqEs or present in insufficient concentrations to elicit any significant assay response. The scratch assay has been previously utilized to assess the effects of individual cigarette smoke components, such as cadmium and nicotine (Snajdar et al., 2001). This study reported that exposure of HUVECs to a cigarette smoke extract induced concentration-dependent decreases in endothelial migration rates, whilst in the same study, pure nicotine (0.4–20 μg/mL) had no effect. This supports our observation that nicotine did not appear to be responsible for the inhibition of endothelial migration that was induced by 3R4F AqE exposure. This conclusion was based on the observation that when HUVECs were exposed to AqEs containing double the nicotine concentration of the 3R4F AqE, no inhibition of endothelial migration was observed. Moreover, recent studies have demonstrated that nicotine may actually induce migration and proliferation of vascular cells by binding to specific nicotinic acetylcholine receptors (Park et al., 2008; Liu and Nicotine, 2014). A potential mechanism for the adverse effects of smoking on the cardiovascular system has been described, which involves oxidative stress (Messner and Bernhard, 2014). Tobacco smoke contains tar and
Fig. 5. E-cigarette AqE from a cartomizer style device did not inhibit HUVEC migration. Data shown are mean wound widths as a% of the initial wound width (μm) from duplicate wells and from a minimum of 4 independent experiments.
3.5. 3R4F AqE treated HUVECs demonstrated inhibition of migration compared to e-cigarette AqE To compare the inhibition of endothelial migration by the different test product AqEs, the mean HUVEC migration rates (μm/h) over 20 h were calculated for each AqE exposure concentration and normalized to the baseline migration rate, where cells were exposed to AqE capture media (Fig. 6). Exposure to 3R4F AqE significantly inhibited migration rates in a concentration-dependent manner; ranging from the uninhibited baseline migration rate of 21.06 ( ± 5.52) μm/h, to an almost complete inhibition of endothelial migration with a rate of 1.06 ( ± 2.77) μm/h. Exposure to either the closed modular or cartomizer style e-cigarette AqE did not induce any significant reductions in HUVEC migration rates. Significant differences between responses to 3R4F AqE and e-cigarette AqEs were observed (p < 0.05). 4. Discussion Over the last decade there has been an increase in the numbers of consumers switching to novel tobacco and nicotine products, including e-cigarettes. Public Health England have recently stated that e-cigarettes could provide a 95% reduced risk compared to cigarettes and the UK Royal College of Physicians have supported the use of e-cigarettes as replacement products for smokers (Public Health England, 2017; Royal College of Physicians, 2017). There currently exists insufficient longterm epidemiological data to support these health claims. The deleterious effects of tobacco smoking to the cardiovascular system are well recognised and widely reported (World Health Organization, 2015;
Fig. 6. Comparison of endothelial cell migration rates following exposure to AqE from 3R4F cigarettes or AqE from e-cigarettes over a 20 h exposure period. 3R4F AqE induced a concentration-dependant inhibition of HUVEC migration, compared to no inhibition of migration following exposure to e-cigarette AqE. Data are means ± SEM from duplicate wells and from a minimum of 4 independent experiments.
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various gas phase species, including carbonyls, which provide a source of oxidants and free-radicals (Faux et al., 2009; Pryor, 1997). These highly reactive and damaging chemicals have been shown to induce endothelial dysfunction through oxidative damage to protein and lipids and to elicit both apoptosis and necrosis (Messner et al., 2012), which results from oxidative damage to mitochondria or DNA (Kannan and Jain, 2016). The increasingly widespread use of e-cigarette as an alternative to cigarette smoking has the potential to reduce CVD by reducing a consumer’s exposure to pro-oxidant species. Previously reported studies have described the reduced number of individual chemicals within e-cigarette aerosols as compared to tobacco smoke and when present, have been quantified to exist at very low concentrations (Taylor and Carr, 2016; Margham et al., 2016; Goniewicz et al., 2014; Callahan-Lyon, 2014; Cheng, 2014). Carbonyl species have been detected and quantified in e-cigarette aerosols (Taylor and Carr, 2016; Bekki et al., 2014; Kosmider et al., 2014; Jensen et al., 2015; Farsalinoes et al., 2015) and found to exist in far lower concentration than those found in cigarette smoke. Our data has demonstrated that AqEs of two commercially available e-cigarettes did not induce the inhibition of endothelial cell migration in vitro as compared to 3R4F AqE. Further investigations might incorporate the testing of a wide range of e-cigarette devices and e-liquids in order to better reflect the diversity of products in the e-cigarette market. The scratch wound assay enables a comparative assessment between conventional cigarettes and e-cigarettes in vitro and demonstrates the usefulness and versatility of this assay for the assessment of e-cigarettes. Further in vitro studies incorporating aerosol exposures, dosimetry techniques, use of 3D tissue models, multi-endpoint analysis and broader global omics approaches can provide researchers with invaluable data to support the assessment of novel tobacco and nicotine products. Author contributions T.J, K.H and M.T performed the main experiments; M.T and F.L designed the experiments and analysed the data; M.T, M.G and I.F wrote and edited the manuscript; D.B and M.G revised the manuscript. Funding All experimental work was funded by Nicoventures Ltd., UK, a wholly-owned subsidiary of British American Tobacco Investments Ltd. All study products were provided by Nicoventures. Disclosure statement The authors are employees of British American Tobacco Investments Ltd. The authors alone are responsible for the content and writing of this article. References Adamson, J., Thorne, D., 2013. A review of in vitro cigarette smoke exposure systems. Exp. Toxicol. Pathol. 65. Ambrose, J.A., Barua, R.S., 2004. The pathophysiology of cigarette smoking and cardiovascular disease: an update. J. Am. Coll. Cardiol. 43 (May(10)), 1731–1737. Azzopardi, D., Patel, K., Jaunky, T., et al., 2016. Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke. Toxicol. Mech. Methods 26 (July (6)), 477–491. Bakhru, A., Erlinger, T.P., 2005. Smoking Cessation and Cardiovascular Disease Risk Factors: Results from the Third National Health and Nutrition Examination Survey. PLoS Med. 2 (June(6)), e160. Bekki, K., Uchiyama, S., Ohta, K., Inaba, Y., Nakagome, H., Kunugita, N., 2014. Carbonyl compounds generated from electronic cigarettes. Int. J. Environ. Res. Public Health 11 (October(11)), 11192–111200. Bernhard, D., Pfister, G., Huck, C.W., et al., 2003. Disruption of vascular endothelial homeostasis by tobacco smoke: impact on atherosclerosis. FASEB J. 17 (December (15)), 2302–2304. Black, H.R., 1995. Smoking and cardiovascular disease. In: Laragh, J.H., Brenner, B.M. (Eds.), Hypertension: Pathophysiology, Diagnosis and Management, 2nd edition.
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