Many current studies vary in the methodologies used to collect and use e-Cig aerosol15. In this study, we have condensed the e-Cig aerosol in order to form treatments that can replicate e-Cig vaping. Many studies exhibit the effect of e-Cig aerosol in individual cultures of a single cell type in which they can examine, for example, the respiratory tract or the endothelial effect35. In this study, we used both A549 epithelial lung cells and HCAECs to emulate the process of contacting the epithelial layer of the lung first before the metabolites reach the endothelial cells in the blood vessel. Importantly, our study shows that endothelial cells and markers of cardiac health are affected by e-Cig aerosol both in vitro and in vivo.
In this study, we demonstrated a significant reduction in cell viability of HCAECs following direct exposure to EAC generated from the e-liquid base constituents, PG and VG, alone. Our results agree with Anderson et al. (2016)7 and Putzhammer et al. (2016)36, who similarly showed significantly reduced cell viability in human umbilical vein endothelial cells (HUVEC) exposed to tobacco flavour and a variety of e-liquids. Of interest in our study, however, is that we showed significant cytotoxic effects in HCAECs exposed to conditioned media from lung cells exposed to EAC generated from the base/tobacco e-liquid (with or without nicotine), indicating the EAC likely induced pro-inflammatory conditions in lung epithelial cells that subsequently induced a detrimental effect on the HCAECs.
Oxidative damage as a result of an imbalance in antioxidants and ROS levels has been shown to play an important role in atherogenesis and endothelial dysfunction during cigarette smoking37–39. In this study, we showed that HCAEC exposed directly to EAC at high concentrations with or without nicotine resulted in increased ROS levels in endothelial cells compared to the controls. Our results corroborate with previous studies, which showed e-Cig vapour extracts increased levels of ROS expression in varying types of endothelial cells, and that the pre-treatment of antioxidants on cells abrogated this effect40–43. Nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) plays a crucial role in maintaining vascular physiology. In an oxidative stress state, eNOS uncoupling occurs, which results in ROS rather than NO being produced, cascading into the production of peroxynitrite (ONOO−) that has oxidative and cytotoxic effects, exacerbating endothelial dysfunction44. It is, therefore, likely that EAC induced eNOS uncoupling in HCAECs subsequently resulted in increased ROS; however, this remains to be confirmed. Decreased cell viability was similarly observed with HCAECs exposed to conditioned media from lung cells, demonstrating significantly increased ROS levels. Whether lung cells exposed to EAC result in an increase in the secretion of pro-inflammatory cytokines and therefore induce further adverse effects on the HCAECs requires further investigation.
The oxidative stress response is linked to the inflammatory pathway, both of which lead to a disruption in the endothelial equilibrium and subsequently endothelial dysfunction, pivotal in the early stages of atherosclerosis. The first step in endothelial dysfunction is the expression of molecules that aid in the adhesion of monocytes to the endothelium and subsequent migration into the subendothelial space45. Whilst VCAM-1 mRNA expression was significantly increased after exposure to 1:50 dilution of EAC (18mg) treatment, ICAM-1 mRNA levels showed a significant increase at 1:50 (0mg) and 1:25, 1:50 (18mg) dilutions. Nicotine has been demonstrated to have anti-inflammatory properties suggesting that other factors, such as flavouring, are responsible for the increased inflammatory response46
Although no significant difference was shown for differences in ICAM-1 protein levels in the in vitro samples following exposure to EAC with or without nicotine, our results contrast with those observed in a study by Makwana et al. (2021)47, who showed a significant increase in ICAM-1 expression in aortic endothelial cells (HAEC) in a cardiovascular microfluidic model, following treatment with traditional cigarette conditioned media, but not e-Cig conditioned media. This discrepancy in ICAM-1 expression may be a result of a difference in collection and treatment method of e-Cig aerosol. However, Makwana et al. (2021)47 also determined a significant dose-dependent increase in THP-1 monocyte adhesion to HAECs within 10 minutes of the adhesion period that diminished over time. Contrastingly, Muthumalage et al. (2017)48 found significant dose-dependent increases in the pro-inflammatory cytokine IL-8 following in vitro treatment of monocytic cells with flavoured e-liquid. IL-8 and ICAM-1 are respectively chemoattractant and adhesion molecules that are involved in monocyte adhesion47, however, it is noted that expression of these molecules can be determinant on the specific cell type. It is noted that ROS generation reportedly increases ICAM-1 transcription in endothelial cells, but not epithelial cells49. This is possibly the reason for the difference between the findings of Makwana et al. (2021)47 and Muthumalage et al. (2017)48. This presents a possible ICAM-1 specific role in adhesion regulation after exposure to e-Cig condensate in endothelial cells. However, further investigation is required such as a monocyte adhesion assay that was performed by Makwana et al. (2021)47 to determine the effect of EAC on THP-1 adhesion to HCAECs. Nevertheless, the assessment of murine hearts obtained from an in vivo model where mice were exposed to e-Cig aerosol with or without nicotine for 12 weeks showed an increase in cardiac ICAM-1 in mice exposed to e-Cig aerosol containing nicotine, suggesting that in vivo ICAM-1 could be initiating these early atherosclerosis changes.
In relation to angiogenesis, although no changes were observed at the mRNA level, FKBPL at the protein level was significantly increased following exposure to nicotine e-Cig aerosol. Similarly, CD3122,50, was also increased following exposure to e-Cig aerosol with nicotine, perhaps as part of the compensatory mechanism. The determinant factor for these results appears to involve the presence of nicotine, which has been shown to have pro-angiogenic properties14. Nicotine exhibits dose-dependent impacts on endothelial cell homeostasis and exhibits angiogenic effects that may be responsible for the pathogenesis of diseases like atherosclerosis16,51,5. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels abundant in endothelial cells and mediate functions such as proliferation, migration and angiogenesis in vivo51. The effects of nicotine binding to these receptors include endothelium vasodilation, reduced NO availability and eNOS uncoupling, and directly acting on the elements involved in plaque formation52,53. Increases in FKBPL as a key anti-angiogenic regulator54,55, and CD31 in the presence of nicotine are indicative of restrictive angiogenesis and perhaps a compensatory increase in the number of endothelial cells23, suggesting that E-cigs with nicotine are damaging to the cardiac vasculature causing early endothelial cell damage. This was also demonstrated in vitro. Ultimately, nicotine plays a critical role in cell migration and vascular permeability, all of which can stimulate the development of atherosclerotic CVD53.
While unable to determine the exact effects of e-Cig aerosol through this project, we were able to determine the effects of e-liquid constituents, flavouring, and nicotine on cardiovascular health. Unlike our in vivo results, our in vitro findings suggest that e-Cig aerosols affect endothelial homeostasis independent of nicotine. It has been shown that endothelial cell sensitivity of particulates, independent of nicotine, can elicit pro-inflammatory responses that disrupt endothelial cell homeostasis and progress CVD pathogenesis17.
To the best of our knowledge this is the first study to demonstrate the disruption of endothelial homeostasis following exposure to conditioned media from lung epithelial cells exposed to EAC. This result is significant as it demonstrates that e-Cig use can potentially lead to activation of endothelial cells, even after the EAC undergoes first-pass metabolism by lung epithelial cells. We are also reporting, for the first time, changes in a key anti-angiogenic mechanism mediated through FKBPL in murine hearts following exposure to E-cig condensate with nicotine, suggesting that this combination can lead to cardiac damage and diastolic dysfunction, which we have previously shown in human studies where FKBPL was increased in the presence of diastolic dysfunction 22.