Impact of E-Cigarette Use on Circadian Proteins and Cardiovascular Risk Markers

The use of E-cigarettes or vaping has increased rapidly worldwide, particularly among adolescents and young adults [1]. The surge in utilizing E-cigarettes is due to its perceived safety compared to traditional tobacco-based cigarettes and hookah, as people inhale only the nicotine vapor without tobacco [2]. E-cigarette or vaping has been initially implemented to assist those who are considered tobacco addicted in smoking cessation [3]. Several studies reported controversial results regarding the long-run effects of E-cigarette or vaping on various body systems and regulatory pathways [2, 4, 5].

Circadian rhythm or system is an endogenous system regulated by the hypothalamus and the melatonin hormone to generate the 24-h biological clock, which synchronizes with the environmental daylight cycle and plays a pivotal role in controlling various cellular signaling pathways to ensure optimal physiological functions [6]. Several reports highlighted the importance of maintaining proper circadian rhythm, and any disruption or misalignment increases the risk of developing pathological changes and triggers serious illnesses [7, 8]. The circadian system includes various proteins that participate in controlling various biological processes and adjusting their function according to the body's needs [9]. These circadian proteins are crucial for modulating metabolism, survival, inflammation, apoptosis, proliferation, and autophagy, which are essential for cellular homeostasis [10]. The oscillation in the level of various circadian proteins depends on the level of two master regulators: Melatonin and Brain and Muscle Arnt-like (BMAL1) [10]. Hypothalamus signals the pineal gland to initiate the synthesis of Melatonin, which is integral for circadian system regulation [11]. Melatonin, in turn, increases the level of an important circadian protein, the BMAL1 [12]. BMAL1 forms a complex with another circadian protein, the CLOCK to stimulate the transcription of two groups of clock proteins; the Period proteins, which encompass three proteins, PER1, PER2, and PER3, and the Cryptochrome proteins, which comprise two proteins, CRY1 and CRY2 [13]. The formed CRY and PER proteins translocate to the nucleus and induce an inhibitory effect to suppress the activity of the BMAL1-CLOCK complex [14]. Based on that, BMAL1-CLOCK acts as an enhancer (positive pole) while CRY and PER are considered the suppressors (negative pole) of the circadian system feedback loop that is crucial for regulating the biological clock rhythmicity and functionality.

CRY proteins instigate a potent inhibitory effect on the activity of BMAL1-CLOCK complex compared to a mild one initiated by PER proteins [10]. The correlation between vaping and circadian dysregulation has not been thoroughly investigated. One study reported that E-cigarette vapor exposure can alter the level of Clock genes in the lung and negatively impact its metabolism and their defensive abilities against bacterial infections [15]. Scarcity in data about the systemic effects of vaping must be taken into consideration and carefully examined.

These circadian proteins are critically involved in cardiovascular regulation: BMAL1 and CLOCK modulate endothelial function, blood pressure, and heart rate variability, while PER and CRY protect the heart from oxidative stress, inflammation, and fibrosis. Disruption of these protein levels can lead to autonomic dysfunction, pro-inflammatory cytokine release, and increased cardiovascular risk [16, 17]. Given the central role of circadian proteins in cardiovascular homeostasis, this study aims to investigate the impact of E-cigarette use on circadian protein levels and inflammatory cytokines, providing insight into possible cardiovascular implications.

The disturbance in the circadian system markers is reported to be a risk factor for cardiovascular diseases (CVDs) by inducing chronic stress and modulating the functionality of the cardiovascular system. We compared the reduction in melatonin level in the vaping group with the level of melatonin detected in the cardiovascular non-vaping group. The results showed a significant decrease in melatonin level in the cardiovascular non-vaping group, and the reduction was comparable to the one detected in the vaping group (Fig. 1A), indicating that vaping may pose a risk to cardiovascular disorders.

We further investigated the level of the core clock protein BMAL1, which plays a vital role in regulating the level of downstream circadian markers, including PERs and CRYs, and considered the positive pole of the circadian system feedback loop [19]. The results indicated no significant difference in BMAL1 level between the control, vaping, and cardiovascular non-vaping groups (Fig. 1B).

Circadian system markers are reported to control the level of inflammation and oxidative stress in the human body [22]. The disturbance in circadian system markers observed in the vaping group prompted us to examine the level of inflammation and oxidative stress in vaping samples by measuring the levels of interferon gamma (IFN-γ), a cytokine involved in the inflammatory response, and malondialdehyde (MDA), a marker of lipid peroxidation and oxidative stress.

To explore potential relationships among the measured biomarkers, pairwise Spearman correlation coefficients were calculated between the six circadian proteins (Melatonin, BMAL1, PER1, PER2, CRY1, CRY2) and the inflammatory/oxidative stress markers (IFN-γ, MDA). Significant positive correlations were observed between Melatonin and PER1 (r = 0.42, p < 0.001) and CRY1 (r = 0.38, p = 0.01), while IFN-γ showed an inverse correlation with CRY2 (r = –0.35, p = 0.02). These results indicate that disruptions in circadian protein levels are associated with elevated inflammatory and oxidative stress markers, highlighting the interconnected effects of vaping on circadian regulation and cardiovascular risk.

Vaping has emerged as a safe and appealing alternative to traditional smoking cigarettes. It is widely used by younger adults and teenagers [23]. Unlike traditional cigarettes, which burn tobacco to produce smoke, vaping involves heating a liquid that contains nicotine mixed with flavoring spices and other chemicals and then the aerosol is inhaled [24]. The rapid rise in vaping has raised significant public health concerns, as its risks remain incompletely studied and poorly understood. Emerging evidence suggests that vaping may have extensive negative health effects on various body systems [25, 26]. The circadian system is an endogenous system that regulates various physiological functions in the human body mediated by upstream and downstream circadian-related proteins in response to local environmental cues, mainly the daylight [27]. The main role of this system is to control the 24-h biological clock [27]. The regulation of the internal molecular clock is found to profoundly impact many cellular functions, and it is crucial to ensure cellular homeostasis [10]. As vaping has increased rapidly, particularly among the young population, we opted to investigate the impact of vaping on various circadian system parameters and correlate the results with the possibility of increasing the risk of heart problems among vaping users.

Melatonin, often called the "sleep hormone," is produced by the pineal gland and helps signal the body to prepare for sleep, regulating sleep onset and quality [28]. It is considered the upstream inciter for the level of other circadian–related proteins, including BMAL1. Melatonin also has roles independent of initiating the circadian rhythm [28]. It has antioxidant properties and immune regulatory functions for helping the body to defend against cellular damage and infections [29]. Additionally, melatonin influences mood, reproductive health, and metabolism [30]. Disruptions in melatonin are reported to have downstream negative effects on cardiovascular functions [31]. Melatonin acts as a scavenger for reactive oxygen species (ROS), which is beneficial to curb the myocardial damage associated with ischemia–reperfusion injury [32]. Further, melatonin can improve the vagal tone of the heart and control heart rate and arterial pressure [33]. BMAL1 is a key component of the molecular clock system that regulates circadian rhythms in the human body [19]. By acting as a transcription factor, BMAL1 forms a complex with another protein called CLOCK to drive the level of various clock-controlled proteins, thus it is considered the positive pole of the circadian feedback loop [34]. Beyond its role in circadian rhythm regulation, BMAL1 also regulates various cellular processes, including cell proliferation, DNA repair, and regeneration [35]. BMAL1 is crucial for marinating the contractile functions of the heart by regulating the myocardial metabolic pathways [36]. It has been reported that deleting BMAL1 can instigate many remodeling changes in the heart and promote pressure overload [36]. Our study indicated that vaping reduced the level of melatonin and did not change the level of BMAL1. The reduction in melatonin seen in vaping users was similar to that shown in cardiovascular non-vaping patients indicating that vaping may increase the risk of heart disease. These findings raise concerns that vaping could increase the risk of heart problems by decreasing melatonin and omitting its heart-protective role.

PER1 and PER2 are downstream circadian-related proteins that are tightly regulated by BMAL1 [10]. The PERs are central to the "core" machinery of the circadian system, regulating the timing of various biological processes in alignment with the 24-h day-night cycle [37]. PERs are integral for ensuring healthy cardiovascular functions and preventing excessive cardiac damage during injury [38]. The cardiac–related functions of PERs are mediated by regulating cardiac fatty acid metabolism and preventing intense inflammatory response [39]. Our results showed that vaping significantly reduced the level of PER1 and PER2 while the level of both proteins was not changed in cardiovascular non-vaping patients indicating that vaping produces profound disruption in the circadian markers. Another significant set of circadian parameters examined includes the CRY1 and CRY2 proteins. CRYs act as the negative pole of the circadian feedback loop to repress the level of BMAL1 and other clock-related proteins to ensure proper synchronization of circadian rhythms [40]. CRYs also control various biological functions, such as metabolism and gene level. CRYs are essential for regulating cardiac metabolism, triglycerides, oxidative stress levels, and blood pressure [41]. The levels of CRY1 and CRY2 in vaping participants and cardiovascular non-vaping patients were remarkably reduced. The reduction in CRYs disrupted the negative pole of the circadian feedback loop and explained the unchanged and high level of BMAL1 reported in vaping users [19]. As circadian–related proteins control the level of systemic inflammation and oxidative stress and prevent their harmful elevation, we studied the level of inflammation and oxidative stress in vaping users. Our study revealed that the disruption in the circadian markers was correlated with marked upregulation in lipid peroxidation and INF-γ, which can negatively induce adverse effects in various cells, including cardiac cells. The observation that the levels of both IFN-γ and MDA in vaping users were notably comparable to those seen in cardiovascular non-vaping patients suggests that vaping may be associated with substantial inflammatory and oxidative stress responses in the heart, potentially increasing the risk of cardiovascular events over time. Although vaping is often perceived as a less harmful alternative to smoking, these findings indicate that it may still have significant adverse effects on the body system, especially the cardiovascular system. Vaping appears to disrupt the circadian system and induce imbalances in inflammatory and oxidative stress pathways [42]. Recent evidence supports our findings that vaping induces inflammatory and oxidative pathways similar to cardiovascular pathology [25, 26, 43]. These mechanisms involve immune activation and mitochondrial dysfunction, reinforcing our hypothesis of a potential association between vaping and increased cardiovascular risk. These clinically important results highlighted the need to plan future research to validate these results on larger population samples and correlate the results with other diseases. Further, the findings of our study implicate the necessity to implement new strategies to encourage young people to quit vaping by highlighting their unfavorable, long-term outcomes, particularly in disrupting the circadian system and inducing systemic inflammatory and oxidative stress responses that can worsen heart function.

Although our study provides important insights into the impact of E-cigarette use on circadian and inflammatory markers; several limitations should be acknowledged. First, this is a cross-sectional study which may limit the ability to infer causality between vaping and the observed molecular alterations. Longitudinal studies are necessary to validate whether these changes lead to long-term physiological dysfunctions that increase the risk of cardiovascular diseases. Second, while we controlled several confounding variables, there are several unmeasured factors, such as diet, physical activity, body mass index, and environmental exposures, that may influence the results. Third, as the vaping and the medical history are self-reported, there is a high chance of recall or reporting bias. Fourth, the circadian markers were measured at a single time point, which may not fully capture their diurnal variations. Fifth, correlation analyses were not performed due to limited data on BMI and other lifestyle variables. Time-series sampling and a larger population may be used in future research to validate and extend these findings. Finally, the incomplete covariate data (including BMI, physical activity, and dietary information) precluded formal adjustment for potential confounders in our analyses. Consequently, we cannot fully rule out the influence of these factors on the observed group differences.

Our study revealed a novel correlation between vaping and the possibility of developing heart diseases mediated by disturbing the endogenous circadian system regulators. As the level of circadian-related markers decreased, regulatory control on inflammatory and oxidative stress was lost. These findings underscore the unexpected safety concerns related to vaping, as it can cause significant harm to the circadian system and impact its protective role on various body systems, including the cardiovascular system.