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Electronic nicotine delivery systems (ENDS) continue to rapidly evolve. Current products pose unique challenges and opportunities for researchers and regulators. This commentary aims to highlight research gaps, particularly in toxicity research, and provide guidance on priority research questions for the tobacco regulatory community. Disposable flavoured ENDS have become the most popular device class among youth and may contain higher nicotine levels than JUUL devices. They also exhibit enhanced harmful and potentially harmful constituents production, contain elevated levels of synthetic coolants and pose environmental concerns. Synthetic nicotine and flavour capsules are innovations that have recently enabled the circumvention of Food and Drug Administration oversight. Coil-less ENDS offer the promise of delivering fewer toxicants due to the absence of heating coils, but initial studies show that these products exhibit similar toxicological profiles compared with JUULs. Each of these topic areas requires further research to understand and mitigate their impact on human health, especially their risks to young users.

BackgroundThe emergence of a plethora of new tobacco products marketed as being less harmful than smoking, such as electronic cigarettes and heated tobacco products, and the increased popularity of recreational marijuana have raised concerns about the potential cardiovascular risk associated with their use.ObjectiveThe purpose of this study was to investigate whether the use of novel tobacco products or marijuana can cause the development of proarrhythmic substrate and eventually lead to arrhythmias.MethodsRats were exposed to smoke from tobacco, marijuana, or cannabinoid-depleted marijuana, to aerosol from electronic cigarettes or heated tobacco products, or to clean air once per day for 8 weeks, following by assays for blood pressure, cardiac function, ex vivo electrophysiology, and histochemistry.ResultsThe rats exposed to tobacco or marijuana products exhibited progressively increased systolic blood pressure, decreased cardiac systolic function with chamber dilation, and reduced overall heart rate variability, relative to the clean air negative control group. Atrial fibrillation and ventricular tachycardia testing by ex vivo optical mapping revealed a significantly higher susceptibility to each, with a shortened effective refractory period and prolonged calcium transient duration. Histological analysis indicated that in all exposure conditions except for air, exposure to smoke or aerosol from tobacco or marijuana products caused severe fibrosis with decreased microvessel density and higher level of sympathetic nerve innervation.ConclusionThese pathophysiological results indicate that tobacco and marijuana products can induce arrhythmogenic substrates involved in cardiac electrical, structural, and neural remodeling, facilitating the development of arrhythmias.

BackgroundExposure to tobacco or marijuana smoke, or e-cigarette aerosols, causes vascular endothelial dysfunction in humans and rats. We aimed to determine what constituent, or class of constituents, of smoke is responsible for endothelial functional impairment.MethodsWe investigated several smoke constituents that we hypothesized to mediate this effect by exposing rats and measuring arterial flow-mediated dilation (FMD) pre- and post-exposure. We measured FMD before and after inhalation of sidestream smoke from research cigarettes containing normal and reduced nicotine level with and without menthol, as well as 2 of the main aldehyde gases found in both smoke and e-cigarette aerosol (acrolein and acetaldehyde), and inert carbon nanoparticles.ResultsFMD was reduced by all 4 kinds of research cigarettes, with extent of reduction ranging from 20% to 46% depending on the cigarette type. While nicotine was not required for the impairment, higher nicotine levels in smoke were associated with a greater percent reduction of FMD (41.1±4.5% reduction versus 19.2±9.5%; P=0.047). Lower menthol levels were also associated with a greater percent reduction of FMD (18.5±9.8% versus 40.5±4.8%; P=0.048). Inhalation of acrolein or acetaldehyde gases at smoke-relevant concentrations impaired FMD by roughly 50% (P=0.001). However, inhalation of inert carbon nanoparticles at smoke-relevant concentrations with no gas phase also impaired FMD by a comparable amount (P

BackgroundThe harmful vascular effects of smoking are well established, but the effects of chronic use of electronic cigarettes (e-cigarettes) on endothelial function are less understood. We hypothesized that e-cigarette use causes changes in blood milieu that impair endothelial function.MethodsEndothelial function was measured in chronic e-cigarette users, chronic cigarette smokers, and nonusers. We measured effects of participants' sera, or e-cigarette aerosol condensate, on NO and H2O2 release and cell permeability in cultured endothelial cells (ECs).ResultsE-cigarette users and smokers had lower flow-mediated dilation (FMD) than nonusers. Sera from e-cigarette users and smokers reduced VEGF (vascular endothelial growth factor)-induced NO secretion by ECs relative to nonuser sera, without significant reduction in endothelial NO synthase mRNA or protein levels. E-cigarette user sera caused increased endothelial release of H2O2, and more permeability than nonuser sera. E-cigarette users and smokers exhibited changes in circulating biomarkers of inflammation, thrombosis, and cell adhesion relative to nonusers, but with distinct profiles. E-cigarette user sera had higher concentrations of the receptor for advanced glycation end products (RAGE) ligands S100A8 and HMGB1 (high mobility group box 1) than smoker and nonuser sera, and receptor for advanced glycation end product inhibition reduced permeability induced by e-cigarette user sera but did not affect NO production.ConclusionsChronic vaping and smoking both impair FMD and cause changes in the blood that inhibit endothelial NO release. Vaping, but not smoking, causes changes in the blood that increase microvascular endothelial permeability and may have a vaping-specific effect on intracellular oxidative state. Our results suggest a role for RAGE in e-cigarette-induced changes in endothelial function.

IntroductionElectronic nicotine delivery systems (ENDS; ie, vaping devices) such as e-cigarettes, heated tobacco products, and newer coil-less ultrasonic vaping devices are promoted as less harmful alternatives to combustible cigarettes. However, their cardiovascular effects are understudied. We investigated whether exposure to aerosol from a wide range of ENDS devices, including a new ultrasonic vaping device, impairs endothelial function.Aims and methodsWe measured arterial flow-mediated dilation (FMD) in rats (n = 8/group) exposed to single session of 10 cycles of pulsatile 5-second exposure over 5 minutes to aerosol from e-liquids with and without nicotine generated from a USONICIG ultrasonic vaping device, previous generation e-cigarettes, 5% nicotine JUUL pods (Virginia Tobacco, Mango, Menthol), and an IQOS heated tobacco product; with Marlboro Red cigarette smoke and clean air as controls. We evaluated nicotine absorption and serum nitric oxide levels after exposure, and effects of different nicotine acidifiers on platelet aggregation.ResultsAerosol/smoke from all conditions except air significantly impaired FMD. Serum nicotine varied widely from highest in the IQOS group to lowest in USONICIG and previous generation e-cig groups. Nitric oxide levels were not affected by exposure. Exposure to JUUL and similarly acidified nicotine salt e-liquids did not affect platelet aggregation rate. Despite lack of heating coil, the USONICIG under airflow conditions heated e-liquid to ~77°C.ConclusionsA wide range of ENDS, including multiple types of e-cigarettes with and without nicotine, a heated tobacco product, and an ultrasonic vaping device devoid of heating coil, all impair FMD after a single vaping session comparably to combusted cigarettes.ImplicationsThe need to understand the cardiovascular effects of various ENDS is of timely importance, as we have seen a dramatic increase in the use of these products in recent years, along with the growing assumption among its users that these devices are relatively benign. Our conclusion that a single exposure to aerosol from a wide range of ENDS impairs endothelial function comparably to cigarettes indicates that vaping can cause similar acute vascular functional impairment to smoking and is not a harmless activity.

Introduction: Electronic nicotine delivery systems (ENDS; i.e., vaping devices) such as e-cigarettes, heated tobacco products, and newer coil-less ultrasonic vaping devices are promoted as less harmful alternatives to combustible cigarettes. However, the cardiovascular health effects of these devices are understudied. We investigated whether exposure to aerosol from a wide range of ENDS devices with and without nicotine, including a new ultrasonic vaping device, impairs endothelial function. Hypothesis: ENDS aerosols irrespective of nicotine, flavors, and heating coil impair endothelial function comparably to smoke from cigarettes. Methods: We exposed 11 groups (n=8), of anaesthetized rats to aerosol from propylene glycol (PG), vegetable glycerin (VG), PG+VG without nicotine, a USONICIG Zip ultrasonic vaping device, previous generation e-cigarettes (tank style with freebase nicotine), 5% nicotine JUUL pods of three flavors (Virginia tobacco, Mango, and Menthol), and an IQOS heated tobacco product; with Marlboro Red cigarette smoke and clean air as controls, to a single session of 10 cycles of pulsatile 5s exposure over 5 minutes. Endothelial function was quantified as arterial flow-mediated dilation (FMD) using micro-ultrasound. Results: Aerosol/smoke from all conditions except air significantly impaired FMD (Figure: each colored line denotes one rat, black horizontal bars denote means). The extent of impairment ranged from 40%-67% although there were no significant differences between groups. Nicotine absorption into the blood varied widely from these undiluted aerosols (those containing nicotine), with IQOS being the highest, and USONICIG and previous generation e-cig being the lowest. Conclusions: A single session of exposure to aerosol from a wide range of ENDS, including multiple types of e-cigarettes, a heated tobacco product, and an ultrasonic vaping device, all impair endothelial vascular function comparably to combusted cigarettes. Key words: JUUL, IQOS, ultrasonic vaping device, e-liquids, flow-mediated dilation

ObjectivesJUUL and earlier generation electronic cigarettes (e-cigs) are promoted as being less hazardous than cigarettes. While JUUL Labs, in particular, claims that switching from smoking to vaping has beneficial impacts, the health effects of such products are not well understood. We investigated whether exposure to JUUL and previous generation e-cig aerosol impairs endothelial function comparably to cigarette smoke.MethodsWe exposed rats to aerosol from Virginia Tobacco flavor JUUL, an e-cig tank system using unflavored freebase nicotine e-liquid, Marlboro Red combustible tobacco cigarettes, or clean air for 10 cycles of 2 second inhalation over 5 minutes. Endothelial function (FMD) was measured pre- and post-exposure. Blood was collected 20 mins post-exposure for serum nicotine analysis.ResultsAerosol/smoke from JUUL, previous generation e-cigs, and cigarettes all impaired FMD. The extent of impairment ranged from 34%-58%, although the differences between groups were insignificant. Nicotine was highest in serum from the JUUL group; for the other e-cig and cigarette groups, nicotine levels were lower and comparable to each other.ConclusionsAerosol from JUUL and previous generation e-cigs impairs endothelial function in rats, comparable to impairment by cigarette smoke.

Introduction: Acute exposure to tobacco or marijuana secondhand smoke (SHS) causes endothelial dysfunction. The identity of specific SHS constituents that cause vascular toxicity is unclear. Heavy metals are present in SHS and at elevated levels in the blood of smokers, and may mediate acute endothelial dysfunction through reactive oxygen species formation and decreasing NO bioavailability. We assessed the effects of exposure to cadmium, lead, mercury, and arsenic at levels present in the blood of human smokers on flow-mediated dilation (FMD) as a measure of endothelial function in rats. We also evaluated the effects of heavy metal exposure on intimal structure, protein localization pattern, and inflammatory gene expression in the aortic endothelium. Hypothesis: Sub-cytotoxic levels of heavy metals impair FMD, alter intimal structure, and induce pro-inflammatory signaling in endothelium. Methods: We injected rats (n=8/group) with heavy metal cocktail or vehicle intravenously and quantitated pre- and post-exposure FMDs measures defined as percent vasodilation of femoral artery after transient ischemia. We performed en face aorta immunostaining and assessed endothelial cell axis alignment, cell length ratio, and localization pattern of PECAM-1, VE-cadherin, and vimentin. We also quantified gene expression of key endothelial proteins in aorta homogenates. Results: FMD was not impaired in the heavy metal group (8.8±3.6(SD)% vs. 12.9±8.0%, p=.31 or controls (7.5±2.7% vs. 8.8±5.8%, p=.63). No significant difference in cell length ratio and endothelial x and y axes of alignment were detected between groups (p>.8) and localization of PECAM-1, VE-cadherin, and vimentin in the aorta endothelium remained unaltered following heavy metal injection. However, expression of PECAM-1 and VE-cadherin was significantly lower in the heavy metal-treated rats, while VCAM-1 gene expression was significantly higher (p

BackgroundElectronic cigarettes (e-cigarettes) have experienced a tremendous increase in use. Unlike cigarette smoking, the effects of e-cigarettes and their constituents on mediating vascular health remain understudied. However, given their increasing popularity, it is imperative to evaluate the health risks of e-cigarettes, including the effects of their ingredients, especially nicotine and flavorings.ObjectivesThe purpose of this study was to investigate the effects of flavored e-cigarette liquids (e-liquids) and serum isolated from e-cigarette users on endothelial health and endothelial cell-dependent macrophage activation.MethodsHuman-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a high-throughput screening approach were used to assess endothelial integrity following exposure to 6 different e-liquids with varying nicotine concentrations and to serum from e-cigarette users.ResultsThe cytotoxicity of the e-liquids varied considerably, with the cinnamon-flavored product being most potent and leading to significantly decreased cell viability, increased reactive oxygen species (ROS) levels, caspase 3/7 activity, and low-density lipoprotein uptake, activation of oxidative stress-related pathway, and impaired tube formation and migration, confirming endothelial dysfunction. Upon exposure of ECs to e-liquid, conditioned media induced macrophage polarization into a pro-inflammatory state, eliciting the production of interleukin-1β and -6, leading to increased ROS. After exposure of human iPSC-ECs to serum of e-cigarette users, increased ROS linked to endothelial dysfunction was observed, as indicated by impaired pro-angiogenic properties. There was also an observed increase in inflammatory cytokine expression in the serum of e-cigarette users.ConclusionsAcute exposure to flavored e-liquids or e-cigarette use exacerbates endothelial dysfunction, which often precedes cardiovascular diseases.

ABSTRACT Objectives JUUL and earlier generation electronic cigarettes (e-cigs) are promoted as being less hazardous than cigarettes. While JUUL Labs, in particular, claims that switching from smoking to vaping has beneficial impacts, the health effects of such products are not well understood. We investigated whether exposure to JUUL and previous generation e-cig aerosol impairs endothelial function comparably to cigarette smoke. Methods We exposed rats to aerosol from Virginia Tobacco flavor JUUL, an e-cig tank system using unflavored freebase nicotine e-liquid, Marlboro Red combustible tobacco cigarettes, or clean air for 10 cycles of 2 second inhalation over 5 minutes. Endothelial function (FMD) was measured pre- and post-exposure. Blood was collected 20 mins post-exposure for serum nicotine analysis. Results Aerosol/smoke from JUUL, previous generation e-cigs, and cigarettes all impaired FMD. The extent of impairment ranged from 34%-58%, although the differences between groups were insignificant. Nicotine was highest in serum from the JUUL group; for the other e-cig and cigarette groups, nicotine levels were lower and comparable to each other. Conclusions Aerosol from JUUL and previous generation e-cigs impairs endothelial function in rats, comparable to impairment by cigarette smoke.

BackgroundHeated tobacco products (also called 'heat-not-burn' products) heat tobacco at temperatures below that of combustion, causing nicotine and other compounds to aerosolise. One such product, IQOS from Philip Morris International, is being marketed internationally with claims of harm reduction. We sought to determine whether exposure to IQOS aerosol impairs arterial flow-mediated dilation (FMD), a measure of vascular endothelial function that is impaired by tobacco smoke.MethodsWe exposed anaesthetised rats (n=8/group) via nose cone to IQOS aerosol from single HeatSticks, mainstream smoke from single Marlboro Red cigarettes or clean air for a series of consecutive 30 s cycles over 1.5-5 min. Each cycle consisted of 15 or 5 s of exposure followed by removal from the nose cone. We measured pre-exposure and postexposure FMD, and postexposure serum nicotine and cotinine.ResultsFMD was impaired comparably by ten 15 s exposures and ten 5 s exposures to IQOS aerosol and to cigarette smoke, but not by clean air. Serum nicotine levels were similar to plasma levels after humans have smoked one cigarette, confirming that exposure conditions had real-world relevance. Postexposure nicotine levels were ~4.5-fold higher in rats exposed to IQOS than to cigarettes, despite nicotine being measured in the IQOS aerosol at ~63% the amount measured in smoke. When IQOS exposure was briefer, leading to comparable serum nicotine levels to the cigarette group, FMD was still comparably impaired.ConclusionsAcute exposures to IQOS aerosol impairs FMD in rats. IQOS use does not necessarily avoid the adverse cardiovascular effects of smoking cigarettes.

Evidence is accumulating that exposure to cigarette smoke (CS) increases the risk of developing acute respiratory distress syndrome (ARDS). Streptococcus pneumoniae is the most common cause of bacterial pneumonia, which in turn is the leading cause of ARDS. Chronic smokers have increased rates of pneumococcal colonization and develop more severe pneumococcal pneumonia than nonsmokers; yet mechanistic connections between CS exposure, bacterial pneumonia, and ARDS pathogenesis remain relatively unexplored. We exposed mice to 3 wk of moderate whole body CS or air, followed by intranasal inoculation with an invasive serotype of S. pneumoniae. CS exposure alone caused no detectable lung injury or bronchoalveolar lavage (BAL) inflammation. During pneumococcal infection, CS-exposed mice had greater survival than air-exposed mice, in association with reduced systemic spread of bacteria from the lungs. However, when mice were treated with antibiotics after infection to improve clinical relevance, the survival benefit was lost, and CS-exposed mice had more pulmonary edema, increased numbers of BAL monocytes, and elevated monocyte and lymphocyte chemokines. CS-exposed antibiotic-treated mice also had higher serum surfactant protein D and angiopoietin-2, consistent with more severe lung epithelial and endothelial injury. The results indicate that acute CS exposure enhances the recruitment of immune cells to the lung during bacterial pneumonia, an effect that may provide microbiological benefit but simultaneously exposes the mice to more severe inflammatory lung injury. The inclusion of antibiotic treatment in preclinical studies of acute lung injury in bacterial pneumonia may enhance clinical relevance, particularly for future studies of current or emerging tobacco products.

BackgroundDespite public awareness that tobacco secondhand smoke (SHS) is harmful, many people still assume that marijuana SHS is benign. Debates about whether smoke-free laws should include marijuana are becoming increasingly widespread as marijuana is legalized and the cannabis industry grows. Lack of evidence for marijuana SHS causing acute cardiovascular harm is frequently mistaken for evidence that it is harmless, despite chemical and physical similarity between marijuana and tobacco smoke. We investigated whether brief exposure to marijuana SHS causes acute vascular endothelial dysfunction.Methods and resultsWe measured endothelial function as femoral artery flow-mediated dilation (FMD) in rats before and after exposure to marijuana SHS at levels similar to real-world tobacco SHS conditions. One minute of exposure to marijuana SHS impaired FMD to a comparable extent as impairment from equal concentrations of tobacco SHS, but recovery was considerably slower for marijuana. Exposure to marijuana SHS directly caused cannabinoid-independent vasodilation that subsided within 25 minutes, whereas FMD remained impaired for at least 90 minutes. Impairment occurred even when marijuana lacked cannabinoids and rolling paper was omitted. Endothelium-independent vasodilation by nitroglycerin administration was not impaired. FMD was not impaired by exposure to chamber air.ConclusionsOne minute of exposure to marijuana SHS substantially impairs endothelial function in rats for at least 90 minutes, considerably longer than comparable impairment by tobacco SHS. Impairment of FMD does not require cannabinoids, nicotine, or rolling paper smoke. Our findings in rats suggest that SHS can exert similar adverse cardiovascular effects regardless of whether it is from tobacco or marijuana.

ObjectivesLittle cigars and cigarillos are gaining in popularity as cigarette use wanes, mainly due to relaxed regulatory standards that make them cheaper, easier to buy individually, and available in a variety of flavors not allowed in cigarettes. To address whether they should be regulated as strictly as cigarettes, we investigated whether little cigar secondhand smoke (SHS) decreases vascular endothelial function like that of cigarettes.MethodsWe exposed rats to SHS from little cigars, cigarettes, or chamber air, for 10 minutes and measured the resulting acute impairment of arterial flow-mediated dilation (FMD).ResultsSHS from both little cigars and cigarettes impaired FMD. Impairment was greater after exposure to little cigar SHS than by cigarette SHS relative to pre-exposure values, although the post-exposure FMD values were not significantly different from each other.ConclusionsExposure to little cigar SHS leads to impairment of FMD that is at least equal to that resulting from similar levels of cigarette SHS. Our findings support the need to prevent even brief exposure to little cigar SHS, and support tobacco control policies that regulate little cigars as strictly as cigarettes.

IntroductionWe sought to determine the effects of brief exposures to low concentrations of tobacco secondhand smoke (SHS) on arterial flow-mediated dilation (FMD, a nitric oxide-dependent measure of vascular endothelial function), in a controlled animal model never before exposed to smoke. In humans, SHS exposure for 30 min impairs FMD. It is important to gain a better understanding of the acute effects of exposure to SHS at low concentrations and for brief periods of time.MethodsWe measured changes in FMD in rats exposed to a range of real-world levels of SHS for durations of 30 min, 10 min, 1 min, and 4 breaths (roughly 15 s).ResultsWe observed a dose-response relationship between SHS particle concentration over 30 min and post-exposure impairment of FMD, which was linear through the range typically encountered in smoky restaurants and then saturated at higher concentrations. One min of exposure to SHS at moderate concentrations was sufficient to impair FMD.ConclusionsBrief SHS exposure at real-world levels reversibly impairs FMD. Even 1 min of SHS exposure can cause reduction of endothelial function.