Date on Master's Thesis/Doctoral Dissertation

8-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Degree Program

Pharmacology and Toxicology, PhD

Committee Chair

Conklin, Daniel

Committee Co-Chair (if applicable)

Siskind, Leah

Committee Member

Siskind, Leah

Committee Member

Srivastave, Sanjay

Committee Member

O'Toole, Timothy

Committee Member

D'Souza, Stanley

Author's Keywords

platelet activation; thrombosis; cardiovascular disease; tobacco; aldehydes; cigarettes

Abstract

Cardiovascular disease (CVD) remains the world’s leading cause of morbidity and mortality. Conventional smoking of tobacco cigarettes is the number one risk factor for onset of CVD. Active cigarette users as well as those individuals who are exposed to second-hand cigarette smoke are at increased risk of CVD. Studies have shown that conventional smoking significantly increases a user’s risk of thrombosis, atherosclerosis, peripheral arterial disease, and coronary artery disease. Any of these diseases may trigger myocardial infarctions, stroke, or hypertension that may lead to cardiovascular morbidity and mortality. New and emerging electronic nicotine delivery systems (ENDS), such as electronic cigarettes (e-cigs), are advertised as a less toxic alternative to smoking conventional, combustible tobacco cigarettes. To address potential toxicities associated with e-cig exposure, there are ongoing studies assessing CVD risk of e-cig use. This research includes studies of the chemical constituents present in the e-cig aerosols and their role in cardiovascular toxicity of exposure to e-cig-derived aerosols.

Epidemiological and experimental results suggest that harmful or potentially harmful constituents (HPHCs), such as aldehydes and flavorants, potentially mediate tobacco smoke- and e-cig aerosol-induced cardiovascular toxicity. Some HPHCs are shared among tobacco product types, including formaldehyde, acetaldehyde, acrolein, and menthol to name a few. However, their contribution to platelet activation, a marker of thrombosis, is largely unknown. The transient receptor potential ankyrin 1 (TRPA1) channel is a sensory receptor that mediates some of tobacco smoke-induced cardiopulmonary toxicity, however, whether TRPA1 mediates platelet activation of tobacco smoke is unknown. Irritating aldehydes and flavorants such as acrolein, formaldehyde, and cinnamaldehyde are agonists of TRPA1. The goal of this dissertation research is to investigate the effects of ecigs and their HPHCs on platelet activation and to test whether the TRPA1 channel mediates or contributes to tobacco-derived aerosols- or HPHC-induced platelet activation. Thus, the central hypothesis is that aldehydes (and flavorants) in tobaccoderived aerosols enhance platelet activation via stimulation of the TRPA1 channel. Methods Healthy human volunteers as well as male and female mice were used for this research project. For the ex vivo studies, platelet-rich plasma (PRP) from healthy human volunteers was isolated and used in a platelet aggregometry assay. For this study, the effects of 15 different parent flavorants on adenosine diphosphate (ADP)-induced biphasic platelet aggregation were tested. For the in vivo studies, mice were exposed in whole-body inhalation chambers to mainstream cigarette smoke (MCS), e-cig aerosols, propylene glycol (PG), vegetable glycerin (VG), gaseous aldehydes, or HEPA-filtered air

for 4 days, 2 weeks, or 12 weeks. After euthanization, blood was drawn from the right ventricle, and platelet-leukocyte aggregates (PLAs) were measured by flow cytometry. In addition, plasma platelet factor 4 (PF-4) levels were measured by ELISA. Other endpoints in these studies include complete blood counts (CBCs), toxicology screening (Tox Screen), and vascular reactivity. Statistical analyses For the ex vivo studies involving the platelet aggregometry assay, the effects of each flavorant compound on ADP-induced human biphasic platelet aggregation were measured using a One-Way ANOVA or One-Way ANOVA with repeated measures where appropriate (SigmaPlot ver. 12). For the in vivo studies using flow cytometry and PF-4 ELISA, the effects of wholebody exposure to individual HPHCs were compared with air exposures or across genotypes using the Mann-Whitney U test (SigmaPlot ver. 12) or the Two-Way ANOVA with Tukey post hoc test (GraphPad Prism ver. 9) where appropriate. Statistical significance was accepted where p

ADP (10 μM). Aggregation curves were analyzed for flavorant-induced effects on total (%) aggregation, Phase 1 and Phase 2 components, and compared with their ADP-only control via One Way ANOVA with repeated measures. Our results show that eugenol significantly inhibited total aggregation in part by sole inhibition of the Phase 2 response. No other flavorant tested had significant effects. For the in vivo studies, C57BL/6 male and female mice were acutely exposed to MCS, e-cig aerosols, PG:VG (30:70%, v:v) formaldehyde (FA; 2 or 5 ppm), acetaldehyde (AA; 5 ppm), acrolein (AC; 1 ppm), crotonaldehyde (CR; 1 or 3 ppm), or HEPA-filtered air (control) for 4 days, 2 weeks, or 12 weeks. Blood was isolated and PLAs were quantified as double positive events (CD41+/CD45+) via flow cytometry (Chapter III). These data reveal no statistically significant changes in % PLAs between exposures to e-cig aerosols or the saturated aldehydes (FA and AA) compared with filtered air control groups by the Mann-Whitney U test. There were, however, statistically significant changes in % PLAs in mice exposed to MCS, PG:VG, and the unsaturated aldehydes (AC and CR) when each was compared with its air control group via Mann- Whitney U test. In addition, results from PF-4 ELISA reveal that PG:VG exposure in female mice significantly elevated plasma PF-4 levels. Lastly, results show significant changes in % PLAs and plasma PF-4 levels between WT and TRPA1-null animals exposed to AC and CR (Chapter IV). Summary & Conclusions Results of the ex vivo studies suggest that parent flavorant compounds commonly found in e-cig liquids neither activate nor inhibit ADP-induced human platelet

aggregation. As eugenol is a banned flavor, its inhibition of platelet activation proved a useful positive control. The results of in vivo studies support that short-term exposure of mice to unsaturated aldehydes but not the 2 most abundant saturated aldehydes lead to platelet activation, and thus, these aldehydes (AC and CR) may contribute to the overall MCS-induced thrombosis. Furthermore, the TRPA1 receptor appears to be an important mediator of unsaturated aldehyde-induced platelet activation in vivo. Future studies should explore other agonists of platelet activation (as done with ADP) as well as explore the contribution of nicotine to better understand how HPHCs adversely impact thrombosis and overall cardiovascular health. More experiments are needed in order to understand the role of TRPA1 in the mechanism of tobacco smokeinduced thrombosis and CVD. For example, in isolated PRP, testing direct effects of HPHCs on platelet aggregation in vitro in the presence of a TRPA1 antagonist such as A967079 or HC030031. In conclusion, my research demonstrates that HPHCs (likely unsaturated aldehydes) are present in tobacco aerosols and at levels that activate platelets via stimulation of TRPA1. Thus, these HPHCs are toxic to the cardiovascular system and exposure to these aldehydes whether from e-cigs or combustible cigarettes increases CVD risk. These data regarding levels of unsaturated aldehydes that trigger platelet activation can help inform the FDA to better regulate tobacco products that emit HPHCs to lower levels that do not trigger pro-thrombotic changes.

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