In this investigation, we assessed the acute impacts of HTP-inhalation on EV-levels within a cohort of young, healthy individuals. This study demonstrates that brief exposure to nicotine-rich HTPs significantly increases EV levels, particularly those originating from endothelial and platelet cells. Conversely, EVs of leukocyte and neutrophil origin did not show significant changes following HTP exposure.
Endothelial and platelet-derived EVs are known to increase in response to endothelial activation or damage and are involved in processes such as platelet senescence and activation [21]. The presence of these EVs is a recognized marker of acute vascular distress, commonly observed in serious conditions like myocardial infarction and stroke, where elevated EV levels are correlated with poorer clinical outcomes [14–16]. Particularly noteworthy is the increase in P-selectin (CD62P) positive EVs, which indicates not only an increase in platelet shedding but also significant platelet activation [22].
The linkage between habitual cigarette consumption and an escalated cardiovascular disease risk is well-established, spotlighting endothelial dysfunction as a critical precursor to atherosclerotic pathology [23]. Our previous research confirmed that inhaling smoke from just one cigarette can elevate EV levels from endothelial, platelet, and leukocyte origins, accompanied by an increase in circulating endothelial progenitor cells (EPCs). This suggests acute vascular damage, inflammation, and thrombosis [18]. Moreover, in habitual smokers, elevated EVs of endothelial origin are consistently detected and have even been associated with the progression of chronic obstructive pulmonary disease (COPD) and emphysema [24]. Conversely, smoking cessation typically results in a reduction of endothelial EVs, provided there is no permanent pulmonary damage [25]. Acute inhalation of vapor from electronic cigarettes has also been shown to significantly increase endothelial- and platelet-derived EVs, underscoring that non-combustible nicotine delivery products, such as e-cigarettes, can adversely affect vascular health [19, 26]. Collectively, these findings indicate that HTPs elicit effects on EV shedding from endothelial and platelet cells that are comparable to those observed with traditional smoking and e-cigarette use.
Interestingly, our study noted that levels of EVs originating from leukocytes and neutrophils were unaffected. This is in contrast to previous findings where an increase in EVs from leukocytes was observed following cigarette inhalation [18, 27]. Inhalation of e-cigarettes, on the other hand, did not affect leukocyte-derived EVs [26]. The significant amounts of volatile organic compounds present in cigarette smoke could induce strong pro-inflammatory effects, which might explain the differential impact observed in traditional cigarette use compared to other nicotine delivery systems [28].
Historically, adverse vascular outcomes were attributed primarily to the combustible components of cigarettes [29]. However, evidence increasingly highlights a significant role for nicotine in promoting atherosclerotic conditions, challenging the perceived safety of combustion-free alternatives like e-cigarettes and HTPs [30, 31]. For instance, our previous study found that inhaling e-cigarette vapor without nicotine did not increase EV levels, unlike vapor containing nicotine [19]. Nicotine’s influence extends across a spectrum of biological responses. It not only exerts direct sympathomimetic effects but also triggers the release of endogenous catecholamines, which in turn, can activate platelets via adrenergic receptors [32]. These conditions potentially lead to increased production of reactive oxygen species (ROS), exacerbating endothelial inflammatory responses and subsequent EV release [33, 34]. This EV release might serve not merely as a marker of apoptosis but potentially as a protective mechanism, with EVs ferrying antioxidant enzymes to combat ROS [35]. Moreover, studies have shown that EVs isolated from users of e-cigarettes and traditional cigarettes exhibit in vitro vasoconstricting capabilities. This effect is mediated through the diminished activation of endothelial nitric oxide synthase (eNOS) and increased production of endothelin-1 (ET-1), both of which contribute to vascular dysfunction [36].
In a previous analysis of our study participants, published previously, we observed that arterial stiffness increased following inhalation of HTPs [30]. Arterial stiffness serves as a surrogate marker for endothelial function and an independent indicator of vascular health. Increased arterial stiffness is robustly associated with the progression of vascular disease. These findings, coupled with the results from our current EV analysis, strengthen the assertion that HTP inhalation swiftly and significantly impacts vascular function, indicative of impaired endothelial function and pro-thrombotic activity [20, 31]. Given the accumulating evidence highlighting the acute adverse effects of nicotine inhalation, it becomes crucial to explore the long-term impacts of nicotine usage, particularly with the emergence of non-combustible nicotine delivery products.
In earlier research investigating the effects of nicotine on thrombus formation in healthy individuals, we observed that nicotine-containing e-cigarette inhalation led to increased thrombogenicity and impaired vascular function [37]. This growing body of evidence suggests that acute nicotine inhalation has detrimental effects on vascular health. However, the long-term vascular consequences of nicotine use, particularly from non-combustible nicotine delivery systems, remain less well-documented due to their novelty. In animal models, chronic nicotine administration has been shown to induce significant vascular changes and aortic remodeling [33, 38, 39]. In human populations, notably users of Swedish snus (oral moist snuff) - a product historically perceived as less harmful due to its lower content of tobacco nitrosamines - the prolonged use has been linked to increased cardiovascular disease mortality [40, 41]. Our previous findings corroborate this, showing that even apparently healthy chronic snus users exhibit significantly increased arterial stiffness compared to healthy, age-matched non-users [42]. These findings highlight nicotine's central role in vascular detriment.
Contrary to these findings, nicotine replacement therapy (NRT), often regarded as a safe cessation aid, has not been associated with increased mortality post-myocardial infarction, presenting a stark contrast to the risks related to snus use [40, 43, 44]. The administration route, which influences nicotine bioavailability and serum levels, appears critical in mediating these outcomes. Unlike NRT, which gradually elevates serum nicotine levels, products like HTPs or snus rapidly increase nicotine concentrations [45]. Moreover, nicotine may amplify the adverse effects of other aerosol constituents. For example, e-cigarette inhalation without nicotine led to a significant increase in activated platelet-derived EVs, an effect that was substantially intensified with the addition of nicotine [19].
It is also important to note that HTP aerosols contain other potentially hazardous components, such as nitrosamines, reactive oxygen species, volatile organic compounds, and carbon monoxide, albeit at lower levels compared to conventional cigarettes [3]. These primarily combustion-derived constituents are known to induce vascular stress and endothelial dysfunction [23, 46]. The interaction of these components with nicotine could exacerbate their negative impact on the vascular wall. Proposed mechanisms through which nicotine exacerbates these effects include increased adhesion of pro-inflammatory cells, heightened endothelial cell apoptosis, disruption of intercellular communication, and altered proliferation of vascular smooth muscle cells [31].
Limitations and Strengths
The study focuses on the acute effects of HTP inhalation, limiting its ability to predict long-term health outcomes or chronic vascular changes resulting from sustained HTP use. Without longitudinal data, it's challenging to ascertain to which degree the observed increase in EVs translates to an increased risk of cardiovascular disease over time. Furthermore, focusing on young, healthy individuals may not capture the full spectrum of potential effects in a more diverse population, including older adults or individuals with preexisting health conditions. The findings may not be generalizable to all HTP products or usage patterns, given the specific conditions under which the study was conducted (e.g., type of HTP used, duration and frequency of inhalation). The timing for EVs to revert to baseline values remains unclear. Previous analyses from cigarette smoking studies indicate that EV levels peak between 1 to 4 hours post-inhalation and return to baseline within 24 hours [18].
This study is among the first to investigate the acute effects of HTP inhalation on EV levels in young, healthy individuals, contributing novel insights into the cardiovascular implications of using such products. The acute exposure model and subsequent immediate measurement of EVs offer a controlled environment to directly assess the impact of HTP inhalation, minimizing confounding variables present in observational studies.