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Research ArticleSignaling and Stress Response

A 6-month systems toxicology inhalation study in ApoE−/− mice demonstrates reduced cardiovascular effects of E-vapor aerosols compared with cigarette smoke

Published Online:03 Mar 2020https://doi.org/10.1152/ajpheart.00613.2019

Abstract

Smoking cigarettes is harmful to the cardiovascular system. Considerable attention has been paid to the reduced harm potential of alternative nicotine-containing inhalable products such as e-cigarettes. We investigated the effects of E-vapor aerosols or cigarette smoke (CS) on atherosclerosis progression, cardiovascular function, and molecular changes in the heart and aorta of female apolipoprotein E-deficient (ApoE−/−) mice. The mice were exposed to aerosols from three different E-vapor formulations: 1) carrier (propylene glycol and vegetable glycerol), 2) base (carrier and nicotine), or 3) test (base and flavor) or to CS from 3R4F reference cigarettes for up to 6 mo. Concentrations of CS and base or test aerosols were matched at 35 µg nicotine/L. Exposure to CS, compared with sham-exposed fresh air controls, accelerated atherosclerotic plaque formation, whereas no such effect was seen for any of the three E-vapor aerosols. Molecular changes indicated disease mechanisms related to oxidative stress and inflammation in general, plus changes in calcium regulation, and altered cytoskeletal organization and microtubule dynamics in the left ventricle. While ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following E-vapor aerosols exposure, the nicotine-containing base and test aerosols caused an increase in isovolumic relaxation time similar to CS. A nicotine-related increase in pulse wave velocity and arterial stiffness was also observed, but it was significantly lower for base and test aerosols than for CS. These results demonstrate that in comparison with CS, E-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular diseases.

NEW & NOTEWORTHY Analysis of key urinary oxidative stress markers and proinflammatory cytokines showed an absence of oxidative stress and inflammation in the animals exposed to E-vapor aerosols. Conversely, animals exposed to conventional cigarette smoke had high urinary levels of these markers. When compared with conventional cigarette smoke, E-vapor aerosols induced smaller atherosclerotic plaque surface area and volume. Systolic and diastolic cardiac function, as well as endothelial function, were further significantly less affected by electronic cigarette aerosols than conventional cigarette smoke. Molecular analysis demonstrated that E-vapor aerosols induce significantly smaller transcriptomic dysregulation in the heart and aorta compared with conventional cigarette smoke.

INTRODUCTION

Use of cigarettes is an established cause of clinical cardiovascular diseases (CVD) (79, 93, 114, 180, 183). Cigarette smoke (CS) is a complex chemical mixture containing more than 6,000 constituents (129). Excessive generation of harmful and potentially harmful constituents (HPHC) from combustion of tobacco and their inhalation have been identified as critical factors in the development of tobacco smoking-related diseases (14, 18, 63, 77, 84, 126, 136, 170, 178).

Quitting smoking reduces the risk for many of these diseases; however, smoking cessation programs, including nicotine replacement therapies (NRT), have been ineffective in many adult smokers attempting to quit (54, 62, 114). The concept of tobacco harm reduction relies on the use of potentially reduced-risk tobacco products, also termed modified risk tobacco products (MRTP), which deliver nicotine without the harmful constituents generated by tobacco combustion (65, 145, 152, 153). Among various MRTP candidates, electronic cigarettes (e-cigarettes) or E-vapor products have become popular as a potential alternative to cigarettes for smokers who are unable or unwilling to quit (10, 15, 111). E-cigarettes typically consist of battery-operated aerosolizing devices that generate an aerosol (often termed “E-vapor aerosol”) from a liquid mixture (contained in a cartridge or a tank) of vegetable glycerol (VG), propylene glycol (PG), flavors, and commonly nicotine (15, 111). Due to their relatively recent introduction to markets, conclusive long-term clinical studies on the health consequences of e-cigarette use are not yet available, which raises the need to characterize or screen the potentially harmful effects of E-vapor aerosol exposure in nonclinical and short-term clinical studies (15, 105, 111, 153).

Analytical studies have demonstrated that E-vapor aerosols contain significantly lower levels of HPHCs than CS, and carcinogen metabolite levels have been shown to be substantially lower in the urine of e-cigarette users than that of smokers (48, 58, 101, 149). The major constituents of most E-vapor aerosols, PG and VG, have been classified as “generally recognized as safe” when used as food additives. Their toxicity, when inhaled as an aerosol, has been investigated in a few studies, and to date, no adverse effects on the lungs, kidney, or reproductive system have been observed in animal and human studies (83, 121, 127, 153, 167, 168). Nevertheless, Lerner and colleagues reported an increase in oxidative stress and inflammation [related to interleukin (IL)-8 and IL-6 secretion) in both lung cells and mouse lungs in response to exposure to flavored e-cigarette aerosols (88).

Some cardiovascular effects of nicotine, administered via NRTs or MRTPs, cannot be excluded, particularly in vulnerable individuals already suffering from CVDs (1, 9, 111). However, mechanistic in vitro studies have revealed no signs of oxidative stress and only weak cytotoxicity in cultured cardiac myocytes and endothelial cells exposed to extracts from E-vapor aerosols, relative to the effects of CS extracts (36, 148, 150). In the present study, we used the apolipoprotein E-deficient (ApoE−/−) mouse model as a model of CVD to quantify atherosclerosis, identify molecular and structural changes in the aorta and heart ventricle, and assess functional alterations in the cardiovascular system following exposure to E-vapor aerosols or CS for 6 mo.

Data Availability

MATERIALS AND METHODS

General Study Design

This work is part of a comprehensive inhalation study that also included assessment of systemic and respiratory effects in the same mouse model. The experimental animals were approximately 12–14 wk old at the start of exposure. The mice were randomized into five exposure groups: sham (exposed to fresh air), 3R4F reference cigarette (3R4F), PG/VG (carrier), PG/VG/nicotine (base), and PG/VG/nicotine/flavoring (test). Exposed reserve mice (n = 35) were used to replace moribund animals or premature deaths at any point during the study (Table 1). The aerosol for the base group contained PG, VG, and 4% nicotine, whereas that for the test group contained PG, VG, 4% nicotine, and flavor mix. The animals were exposed to 3R4F CS or E-vapor aerosols for 3 h/day, 5 days/wk, in whole body exposure chambers. The base and test group exposures were configured to deliver a nicotine concentration of 35 µg/L [equivalent to the nicotine level in 560 µg/L total particulate matter (TPM) from 3R4F CS]. The acclimatization exposure regimens for each 3R4F CS and E-vapor aerosols group, implemented for 7 days, are highlighted in Supplementary Fig. S1, A and B. The exposure time and fresh air-break regimens were consistent across exposure groups are shown in Fig. 1. The maximum exposure duration was 6 mo, and interim dissection points were scheduled after 3 mo (Fig. 1). The terminal anesthesia at 3 and 6 mo of exposure was realized with pentobarbital sodium (100 mg/kg, Jurox, Rutherford, NSW, Australia) via intraperitoneal injection. Cardiovascular tissues were collected from various groups for transcriptomics, histopathological, and immunohistochemical (Table 1).

Table 1. Allocation of mice to groups and study end points

BALF AnalysisHistopathological AnalysisOMICS AnalysisMicro-CTExposedTotal Number of Mice
Sham
    3 mo1012101648
    6 mo1012101648
    Reserve77
3R4F
    3 mo1012101648
    6 mo1012101648
    Reserve77
Carrier (PG/VG)
    3 mo1012101648
    6 mo1012101648
    Reserve77
Base (PG/VG/N)
    3 mo1012101648
    6 mo1012101648
    Reserve77
Test (PG/VG/N/F)
    3 mo1012101648
    6 mo1012101648
    Reserve77
Total10012010016035515

BALF, bronchoalveolar lavage fluid; PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring.

Fig. 1.

Fig. 1.Study design, groups, and exposure. A: timeline of the study showing the different groups. 3R4F is the standard reference cigarette; carrier (PG/VG group), base (PG/VG/N), and test (PG/VG/N/F) represent E-vapor aerosols from the test formulation. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; M, months; CS, cigarette smoke. B: whole body exposure protocol. Red dashed square, exposure time; blue square, exposure break.

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Test Atmosphere Generation

3R4F reference cigarettes were purchased from the University of Kentucky (https://www.coresta.org/university-kentucky-reference-cigarette). The Kentucky reference cigarette 3R4F is widely used for the analysis of mainstream smoke constituents as a comparator for toxicological in vitro and in vivo toxicological studies (29, 39, 69, 100, 115, 171), providing the opportunities to compare toxicological testing across laboratories (https://www.coresta.org/university-kentucky-reference-cigarette). Mainstream CS from 3R4F cigarettes was generated on 30-port rotary smoking machines (SM2000, PMI R&D) in accordance with the Health Canada intense smoking protocol [Health_Canada (1999), which is based on ISO standard 3308 (68)]: 55-mL puff volume, 2-s puff duration, 1 puff/30 s, and 100% blockage of ventilation holes (https://www.canada.ca/en/health-canada/services/health-concerns/reports-publications/tobacco/tobacco-products-information-regulations.html); the 3R4F puff count was 10–11 per cigarette (average, 10.4 ± 0.3). The smoking machine and chamber layout for the sham and 3R4F exposure conditions are depicted in Fig. 2. The sham group was exposed to fresh air generated through a smoking machine without cigarette sticks.

Fig. 2.

Fig. 2.Schematic diagram of the generation and delivery of 3R4F cigarette smoke in the whole body exposure chamber. Smoking machine (SM) and chamber layout for the sham and 3R4F groups.

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The carrier, base, and test formulations were first prepared by adding each ingredient and diluting to the final mass composition (Supplementary Table S1). The prepared mix (inhalation formulation) was stored away from light, at a controlled temperature of 2–8°C, but under uncontrolled humidity conditions. The blended flavor mix is shared in the Supplementary Table S2. The test liquid formulation was freshly prepared every 3 wk following initial stability testing. PG and VG were quantified using capillary gas chromatography (GC, 7890A/7890B series, Agilent Technologies, Santa Clara, CA) with a DB-WAXETR column (Agilent Technologies) using a flame ionization detector (FID) and n-heptadecane as internal standard. Nicotine was quantified by GC-FID using DB-5 column (Agilent Technologies) and isoquinoline as internal standard. Guaiacol was quantified using GC (Agilent 7890B) equipped with 5977A mass selective detector (MSD) with a DB-WAX UI column and data acquired in SIM mode. The carrier, base, and test aerosols were generated by using a capillary aerosol generator (CAG; Fig. 3). The CAG was developed by Philip Morris and further refined by Virginia Commonwealth University (53, 60). It was previously shown to deliver aerosols in a consistent manner and at similar particle-size distribution and concentrations as a prototype e-cigarette (169). The CAG temperature was set at 250–275°C to match the temperature of the heated coil during puffing of e-cigarettes (41). The generator was fitted with a diffuser and compressed air to prevent aerosol backflow. Condensation aerosol was created when output from the generator was mixed with dilution air at ambient temperature. The aerosol was further diluted with filtered air to achieve the target concentrations in the test atmosphere and delivered via glass tubing to the exposure chamber (Fig. 3). In this study, aerosols generated from carrier, base, and test will be named as “E-vapor aerosols.”

Fig. 3.

Fig. 3.Schematic diagram of the generation and delivery of E-vapor aerosols in the whole body exposure chamber. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring. Capillary aerosol generator (CAG) and chamber layout for carrier (PG/VG group), base (PG/VG/N), and test (PG/VG/N/F) groups.

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Animals and Inhalation Exposure

All procedures involving animals were performed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and licensed by the Agri-Food & Veterinary Authority of Singapore, with approval from an Institutional Animal Care and Use Committee (IACUC protocol #15044) and in compliance with the National Advisory Committee for Laboratory Animal Research’s Guidelines on the Care and Use of Animals for Scientific Purposes (NACLAR, 2004). Female ApoE−/− mice (B6.129P2-ApoEtm1/Unc N11), bred under specific pathogen-free conditions, were obtained from Taconic Biosciences (Rensselaer, NY). The health status of the animals was verified by using the health-check certificate provided by the breeder. The mice were maintained and exposed under specific hygienic conditions with filtered conditioned fresh air at 22 ± 2°C and 55 ± 15% humidity. Additional details of animal housing, randomization, and acclimatization have been previously published (13, 91, 122, 123).

The mice were whole body exposed to diluted mainstream smoke from 3R4F cigarettes (achieved concentration, 574.5 µg TPM/L, equivalent to 35.9 µg nicotine/L), base aerosol (nicotine-matched to 3R4F, 36.4 µg nicotine/L; PG-matched to carrier, 171.2 ± 16.7 µg/L; VG-matched to carrier, 543.5 ± 68.2 µg/L), test aerosol (nicotine-matched to 3R4F, 36.7 µg nicotine/L; PG-matched to carrier, 172.9 ± 19.6 µg/L; VG-matched to carrier, 546.2 ± 74.0 µg/L), carrier aerosol (without nicotine; PG-matched to carrier, 179.1 ± 21.9 µg/L; VG-matched to carrier, 577.2 ± 64.0 µg/L), or filtered air (sham group) for 3 h/day, 5 days/wk, for up to 6 mo (Supplementary Fig. S1, C and D). Intermittent exposure to fresh filtered air for 60 min after the 1st and 2nd h of exposure was provided to avoid excessive carboxyhemoglobin (COHb) buildup in the 3R4F group (Fig. 1B).

Analysis of Biomarkers of Exposure in Blood, Plasma, and Urine

COHb analysis.

Blood COHb concentrations were determined twice during the study, at months 2 and 5 of exposure. Carboxyhemoglobin (CoHb) in heparinized whole blood was determined through the spectrophotometric measurement using the COBAS B221 blood gas analyzer (Roche, Basel, Switzerland) as previously described (13, 91, 122, 123). Blood was collected from the facial vein under anesthesia within 15 min postexposure.

Plasma nicotine, cotinine, and PG measurement.

For plasma collection, blood was placed on ice after collection and processed. Aliquoted plasma was transferred to storage at ≤ −70°C. Plasma PG, nicotine, and cotinine levels were measured by ABF (Planegg, Germany) by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with minor modifications (106, 134).

Urine collection for analysis of nicotine and total metabolites.

After 1 mo of exposure, urine was collected during exposure by placement of individual mice into exposure cages with a raised bottom grid and in the 18-h period following the exposure in a urine metabolic cage. Urine collected during exposure, urine from the 18-h overnight collection, and water from rinsing of the cage (~100 µL of water) were pooled per animal, aliquoted, and stored at ≤ −70°C. Analysis of nicotine metabolites (trans-3′-hydroxycotinine, norcotinine, cotinine, nicotine-N′-oxide, and nornicotine) in urine was performed at ABF using LC-MS/MS after 1,3-diethyl-2-thiobarbituric acid derivatization.

The same samples were analyzed for analysis of other urinary non-nicotine biomarkers, such as hydroxypropyl mercapturic acid (3HPMA), S-phenylmercapturic acid (SPMA), 2-cyanoethylmercapturic acid (CEMA), N-(2-hydroxypropyl)methacrylamide (HPMA), and N-acetyl-S-(1-hydroxymethyl-2-propenyl)-l-cysteine and N-acetyl-S-(2-hydroxy-3-butenyl)-l-cysteine (MHBMA1/2) by ABF.

Analysis of biomarkers of oxidative stress and inflammation.

After 4 mo of exposure, urine samples were analyzed at ABF for biomarkers of oxidative stress and inflammation, including malondialdehyde (MDA), 2,3-dinor-8-iso-prostanglandin F2α, 2,3-dinor-thromboxane B2 (2,3-d-TXB2), 8-iso-prostaglandin F2α (PGF2α), tetranor-prostaglandin E-M, leukotriene-E4 (LTE4), and hydroxyeicosatetraenoic acid (HETE).

Hematology and lipoprotein profile.

Blood samples for hematological assessment were collected at 3 and 6 mo of exposure under terminal pentobarbital sodium anesthesia from the retro-orbital venous plexus. Approximately 120 μL of blood was collected from each animal in EDTA tubes, which were immediately inverted 10 times and placed on a roller until analysis. Before analysis, blood was diluted fivefold with isotonic buffer, and the diluted samples were used for duplicate analyses. Red blood cell parameters such as hematocrit and hemoglobin levels and erythrocyte and reticulocyte counts were determined by using a Sysmex XT-2000i analyzer (Sysmex, Kobe, Japan).

Serum lipid profiles and triglyceride, lipoprotein, cholesterol, and cholesterol chylomicron levels were determined by LipoSEARCH (Skylight Biotech, Akita, Japan). For serum collection, the blood was transferred directly to a serum separator tube. After centrifugation, the serum was transferred to a microfuge tube and frozen at ≤−70°C for storage or shipment.

Atherosclerosis Plaque Assessment

Aortic arch planimetry.

The aortic arch of the allocated animals was collected after 3 and 6 mo of exposure. The aortic arch was flushed with saline, microdissected, cut longitudinally, and then pinned onto a rubber surface. The arch was imaged, stained with oil red O, and reimaged to generate both unstained and stained images. The Visiopharm image analysis software (version 6.6.1.252; Visiopharm, Hoersholm, Denmark) was used to determine the plaque area. With the use of software macros, the perimeter of the aortic arch and the stained regions were outlined, and these borders were confirmed and manually refined. The final relative plaque area, as a percentage, was calculated by dividing the plaque area by the total area of evaluation.

MicroCT scans of the thoracic aorta.

Additionally, a microcomputed tomography (microCT) method was used to determine the volumetric and area information of the atherosclerotic lesions on the thoracic aorta. The animal body was trimmed with the ribcage exposed. The mice were perfused with cold phosphate-buffered saline, followed by 4% formaldehyde (10% formalin in neutral-buffered phosphate solution) at room temperature. The liver, gastrointestinal tract, and forelimbs were removed. The spine was cut below the level of the kidneys to obtain the torso along with the animal’s head. This section was immersed in a fixative at room temperature and shipped to Scanco Medical (Wangen-Brüttisellen, Switzerland) for microCT. The volume and surface area of the atherosclerotic plaques at the thoracic aorta were measured after 3 and 6 mo of exposure.

Cardiovascular Tissue Collection and Processing

On the scheduled necropsy date and approximately 16–24 h after the last exposure, nine mice per group were anesthetized with 100 mg/kg pentobarbital sodium before exsanguination and perfusion with 0.9% saline. Cardiovascular tissues, dedicated for transcriptomics analysis, including the heart left ventricle and thoracic aorta, were collected after 3 and 6 mo of exposure and stored at ≤−70°C until molecular analysis (Fig. 1A). Heart tissues were collected and further dissected into atrial and ventricular parts. The atrial part was used for plaque composition and plaque vulnerability analysis, and the ventricular part was used for morphometric and histopathological analysis.

Heart Ventricle Histopathology and Morphometry

After 3 and 6 mo of exposure, left ventricles were collected and fixed in 4% formaldehyde (pH 7.4). Step-serial sections of the heart ventricle were stained with hematoxylin and eosin and Masson’s trichrome stain for morphometric and histopathological evaluation. The slides were scanned (ScanScope, Aperio Technologies, Vista, CA), and morphometric analysis was performed by using the Visiopharm stereology software. The muscle-to-lumen ratio was determined by stereological quantification of left ventricular muscle area and left ventricular lumen area (superimposed point grid) on 13–15 step-serial sections. Left ventricular thickness was measured in accordance with a randomized strategy on five step-serial sections. Histopathological end points, including fibrosis, hemorrhage, mineralization, inflammatory cell infiltration, and myocyte degeneration, were evaluated by LPT Laboratory of Pharmacology and Toxicology (Hamburg, Germany; https://www.lpt-pharm-tox.de/).

Assessment of Aortic Root Atherosclerotic Plaque Composition

Plaque composition was evaluated on Masson’s trichrome-stained slides by LPT Laboratory of Pharmacology and Toxicology after 3 and 6 mo of exposure. Five continuous levels (~36 µm apart) that best showed the three aortic valves were selected for analysis (Supplementary Fig. S2). Plaque composition end points included cholesterol clefts, chondroid tissue, foamy macrophages, fibrous tissue, lipid/eosinophilic fibers, necrotic tissue, and ossification. Quantification of the tissue types within plaques was performed by superimposing a point grid onto the histological slides, with each point corresponding to a defined size. Whenever a point hit the structure of interest, it was tallied by using a counter. The tissue types were quantified in relation to total plaque size.

Transcriptomics Analysis

Thoracic aorta (6–10/group) and left ventricle (9–10/group) samples were randomized to ensure balanced assignment of the experimental groups across the RNA extraction batches and Affymetrix hybridization fluidics stations (Thermo Fisher Scientific, Waltham, MA). The left ventricle was selected because of its acute sensitivity to toxicants and because it is less affected by pulmonary hypertension than the right ventricle (87). Total RNA was isolated from tissues by using miRNeasy Mini Kit (Qiagen, Hilden, Germany). RNA concentration and quality were assessed by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. The acceptance criterion for downstream processing of the RNA samples was an RNA integrity number > 6. The mean (range) RNA integrity numbers for the thoracic aorta and heart left ventricle were 8.3 (7.0–9.4) and 8.9 (7.3–10.0), respectively. For mRNA analysis, 100 ng of total RNA was reverse-transcribed to cDNA by using the Affymetrix HT 3′ IVT PLUS kit (Thermo Fisher Scientific). cDNA was labeled, amplified to cRNA, and fragmented. The cRNA was hybridized to a GeneChip Mouse Genome 430 2.0 Array (Thermo Fisher Scientific). The arrays were washed and stained on an FS450 DX GeneChip Fluidics Station (Thermo Fisher Scientific) in accordance with the FS450_0001 protocol and then scanned by using a 3000 7G GeneChip scanner (Thermo Fisher Scientific). The data were deposited in the ArrayExpress database (https://www.ebi.ac.uk/arrayexpress/) under accession number (E-MTAB-8578).

Raw data files were processed with custom computable document format files from a brain array (mouse4302mmentrezg v16.0) and normalized by frozen robust microarray analysis (104). Following quality control procedures (pseudo-images, normalized unscaled standard error plot, relative log expression plots, and median absolute value relative log expression) (40), raw P values were generated for the control versus treatment comparisons by using the limma package in R (140) and adjusted by Benjamini-Hochberg false discovery rate (FDR) multiple test correction (8). FDR-adjusted P values < 0.05 were considered significant.

Gene-Set Analysis

Gene-set analysis (GSA) was conducted with the c2.cp gene-set collection from mSigDB (version 5.0) (89). Two GSA approaches, Camera/Q1 (174) and Roast/Q2 (173), and over-representation analysis (160) were applied and jointly evaluated. Q1 tests for the significance of genes in the set versus those not in the set, whereas Q2 tests for a significant difference between the conditions. With this, Q2 is more appropriate in the context of comparative toxicity assessment (e.g., to reveal a significant effect of exposure on a given gene set), whereas Q1 can prioritize gene sets that dominate these responses. P values were adjusted by Benjamini-Hochberg FDR multiple test correction (8). FDR-adjusted P values < 0.05 were considered significant.

Ingenuity pathway analysis.

Analyses of differentially expressed genes (FDR-adjusted P values < 0.05) were conducted by using Qiagen’s Ingenuity Pathway Analysis software (IPA). Core analysis followed by comparative analysis were performed to interpret the impact of CS and E-vapor aerosols. Differential gene expression data were submitted to IPA to assess the impact of each treatment on the biological processes and functions that could be affected in the context of CVDs. Biological functions, pathways, and upstream regulators were analyzed in IPA measuring Z-score, which represents the statistical measure of the match between the expected relationship direction and observed gene expression. Results with a Z-scores >2 or <−2 were considered statistically significant (78).

Cardiac and Vascular Ultrasound Analysis

Transthoracic echocardiography.

Mice were anesthetized with isoflurane (2–5% in oxygen; 1 L/min) and placed supine on a soft electric warming pad. The temperature was continuously monitored and maintained at 37°C. Echocardiographic variables were assessed by using a high-resolution micro-ultrasound system (Vevo 3100, FujiFilm VisualSonics, Tokyo, Japan). Measurements were obtained at the midpapillary level in the parasternal short-axis view in grayscale M-mode and B-mode images. Short-axis M-mode measurements were obtained perpendicular to the midventricular level and confirmed by two-dimensional echocardiography. Conventional measurements of the left ventricle included end-diastolic and end-systolic diameters, anterior and posterior wall thicknesses, fractional shortening, end-systolic and end-diastolic volumes, ejection fraction, and left ventricular mass. Corrected left ventricular mass was calculated considering the specific gravity of cardiac muscle and an intrinsic correction factor. Left ventricle internal diastolic diameter and anterior and posterior wall thicknesses were measured in the short-axis M-mode view by using the left ventricle trace mode. The transmitral inflow pattern was measured in the apical four-chamber view to assess left ventricular diastolic function. The transmitral waveform was used to measure the following parameters: isovolumic relaxation time (IVRT), E and A myocardial diastolic velocities, isovolumic contraction time (IVCT), and aortic ejection time (AET). Three left ventricular trace measurements were averaged for each animal. Myocardial performance index (MPI) and reflecting systolic and diastolic performance was calculated as follows: (isovolumic contraction time + isovolumic relaxation time)/aortic ejection time. All images were analyzed by two expert operators with a blinded approach by using the VisualSonics Software.

Tissue-Doppler imaging.

Tissue-Doppler imaging is a sensitive tool for assessing both regional and global left ventricle function in mice and has been shown to correlate with the hemodynamic indexes of left ventricle systolic function (116). Tissue-Doppler images were acquired in the parasternal short-axis view at the midventricular level at a rate of 483 frames/s and depth of 1 cm. The Nyquist limit of the velocity was 15 cm/s, with a pulse repetition frequency of 2.5 kHz.

Cardiac electrical activity.

PR, QRS, and QT intervals were manually measured (by using the distance tool) from the echocardiography traces. Two independent measurements were acquired and averaged for each animal. This approach provided an indirect and rough estimate of atrioventricular and intraventricular conduction in the different experimental groups (26).

Pulse wave velocity.

abdominal aorta.

Longitudinal B-mode images were obtained by placing the ultrasound probe above the abdominal aorta, with the region of interest located in the focal zone of the transducer. Electrocardiography and respiration signals were acquired during imaging by using the Advancing Physiological Monitoring Unit provided with the imaging station. Abdominal imaging data were acquired at 21 MHz by using the EKV imaging mode (frame rate, 700 frames/s). A dedicated VisualSonics software was employed to assess pulse propagation velocity (PPV). To this end, longitudinal B-mode images of the abdominal aorta were used to calculate vessel diameter values (26). Diameter measurements were characterized by an appropriate temporal resolution. A single-beat diameter waveform was obtained by applying edge detection and contour tracking techniques on these images.

carotid artery.

Pulse wave velocity (PWV) was also measured by using a linear array transducer and color Doppler probe from the level of the aorta to the bifurcation of the common carotid artery at the internal and external branches. Electrocardiogram and Doppler signals were then simultaneously recorded at a sweep speed of 200 mm/s for several cardiac cycles, and the data were stored for subsequent offline analysis. The distance between the points for probe applanation over the aorta and carotid bifurcation was measured in millimeters by using an on-screen digital caliper. The time intervals between the R wave of the electrocardiogram and the foot of the Doppler carotid and aortic waveforms were measured, and the pulse-transit time from the carotid to the aorta was calculated by subtracting the mean R wave-to-carotid foot time interval from the R-wave-to-aortic foot time interval. PWV was then calculated as the distance (in mm) divided by the R wave-to-aortic foot time interval minus the R wave-to-carotid foot time interval (in ms).

Statistical Analysis

Unless otherwise indicated, data are expressed as means ± SE. Pairwise comparisons between groups were performed, and unadjusted P values are stated. For continuous variables, if the data of the two groups being compared did not exhibit strong deviation from the normal distribution (as assessed by performing a Shapiro-Wilk test at the 5% level on the standardized residuals of both groups), a two-sample t-test accounting for variance heterogeneity was performed. Otherwise, an exact Mann-Whitney-Wilcoxon two-sample test was used. For body weight comparisons, and because the sample size per group was greater than 30, only the t-test was performed. For score variables (histopathology), the Cochran-Mantel-Haenszel test was used, and the Fisher exact test was used for incidence variables. All analyses were performed with the SAS system 9.2. Results were considered to be significantly different in a specific comparison if P < 0.05.

RESULTS

Characterization of E-Vapor Aerosols and 3R4F CS Exposure

The concentrations of TPM, PG, and VG generally reflected consistent aerosol delivery to the chambers during the 6-mo inhalation study (Table 2). The average TPM concentration was within 10% of the target concentration (560 µg/L) for 3R4F smoke. Similarly, aerosol from the base and test formulations delivered average nicotine concentrations that were within 10% of those of the 3R4F group. In alignment with the smoke chemistry, PG was not detected in 3R4F CS, less than the limit of detection (LOD). VG was present only in small quantities in 3R4F smoke (53.5 µg/L), corresponding to ~10% of the concentrations in the carrier, base, and test aerosols (Table 2). Additionally, in line with smoke chemistry, CO was present at an elevated level (608.3 ppm) in 3R4F CS (Table 2). The mass compositions of PG, VG, and nicotine in the E-vapor aerosols are shown in Supplementary Table S1.

Table 2. Characterization of test atmosphere containing E-vapor aerosols (carrier, base, and test) and 3R4F CS

Sham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
Nicotine, µg/L<LOD35.905 (SD 2.78)<LOD36.414 (SD 2.19)36.729 (SD 2.74)
Total particulate matter, µg/L<LOD574.520 (SD 16.58)1119.603 (SD 73.57)1131.362 (SD 81.85)1112.291 (SD 108.56)
Carbon monoxide, ppm<LOD608.398 (SD 22.85)NANANA
Vegetable glycerin, µg/L<LOD53.513 (SD 6.44)577.278 (SD 64.02)543.559 (SD 68.29)546.254 (SD 74.05)
Propylene glycol, µg/L<LOD<LOD179.164 (SD 21.99)171.293 (SD 16.77)172.977 (SD 19.66)
Guaiacol, ng/L<LODNA<LOD<LOD4.094 (SD 0.18)
Mass median aerodynamic diameter, µmNA0.815 (SD 0.07)0.955 (SD 0.10)0.922 (SD 0.11)1.012 (SD 0.17)
Geometric SDNA1.277 (SD 0.07)1.393 (SD 0.05)1.358 (SD 0.04)1.493 (SD 0.10)

Values are means (SD). CS, cigarette smoke; LOD, limit of detection; NA, not analyzed. LOD for nicotine is 0.03 μg/L. LOD for propylene glycol is 0.75 and 0.73 μg/L for sham and 3R4F chambers, respectively. LOD for vegetable glycerin is 0.91 μg/L. Guaiacol is a flavor marker in atmosphere. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring.

To verify the aerosolization and presence of flavoring in the test atmosphere during the 6-mo exposure, the test atmosphere was analyzed for the presence of guaiacol. Guaiacol levels in sham, carrier, and base aerosols were below the LOD (Table 2). Guaiacol was only detected above the quantification limit in the test aerosol (3R4F CS: 4.09 ng/L). The mass median aerodynamic diameters were similar in 3R4F CS and the three E-vapor aerosols and ranged from 0.8 to 1 µm, indicating that they were all in the respirable range (Table 2). The particle size distribution of the aerosols were in respirable range with a particle size ≤ 2 μm and a geometric standard deviation of 1–1.5 (113), corresponding to a predicted respirability of 80% in rodents (6) (Table 2).

Reduced Levels of Carbonyl Compounds and Tobacco-Specific Nitrosamines in the E-Vapor Aerosols

When compared with 3R4F CS, E-vapor aerosols from the carrier, base, and test formulations contained much lower levels of carbonyls (Table 3). Acrolein and crotonaldehyde in E-vapor aerosols were at baseline levels (relative to sham), below the LOD. The levels of formaldehyde, acetaldehyde, and propionaldehyde were only marginally higher in the carrier, base, and test aerosols than in the sham atmosphere. Consistent with the smoke chemistry, all five carbonyls were at considerably elevated levels in the 3R4F CS test atmosphere. At equal nicotine concentrations in the base and test aerosol-exposed groups, the concentrations of formaldehyde, acetaldehyde, and propionaldehyde (after deducting the concentrations in the sham atmosphere) were approximately 35–60-, 2,000–3,000-, and 500-fold lower, respectively, than the levels in 3R4F CS. Similar to the carbonyls, the levels of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) in the E-vapor aerosols were close to the sham levels (Table 3).

Table 3. Quantification of carbonyls and TSNAs in the test atmospheres containing CS and E-vapor aerosols

Sham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
Carbonyls
    Formaldehyde, µg/L0.017 (SD 0.01)0.665 (SD 0.08)0.028 (SD 0.01)0.036 (SD 0.03)0.028 (SD 0.01)
    Acetaldehyde, µg/L0.009 (SD 0.00)30.686 (SD 1.90)0.012 (SD 0.00)0.022 (SD 0.01)0.020 (SD 0.01)
    Propionaldehyde, µg/L0.001 (SD 0.00)3.471 (SD 0.62)0.007 (SD 0.00)0.007 (SD 0.00)0.007 (SD 0.00)
    Crotonaldehyde, µg/L<LOD2.844 (SD 0.75)<LOD<LOD<LOD
    Acrolein, µg/L0.001 (SD 0.00)3.980 (SD 0.45)0.001 (SD 0.00)0.001 (SD 0.00)0.001 (SD 0.00)
TSNAs
    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone, ng/L<LOD7.838 (SD 0.35)<LOD<LOD<LOD
    N-Nitrosonornicotine, ng/L<LOD8.097 (SD 0.48)<LOD<LOD<LOD

Values are means (SD). CS, cigarette smoke; TSNA, tobacco-specific nitrosamine; PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; LOD, limit of detection; NA, not applicable. LOD for nicotine is 0.03 μg/L. LOD for propylene glycol is 0.75 and 0.73 μg/L for sham and 3R4F chambers, respectively. LOD for vegetable glycerin is 0.91 μg/L. Guaiacol is a flavor marker in atmosphere.

Nicotine Uptake and Control of Exposure

The actual uptake of nicotine and PG in the 3R4F CS, base, and test groups was measured in blood and urine samples (Fig. 4). Plasma from mice exposed to nicotine (3R4F, base, and test) contained increased concentrations of nicotine and cotinine after 1 and 4 mo of exposure (Fig. 4, A and B). Similarly, the absolute levels of total nicotine metabolites (in µmol) measured in urine were in line with the nicotine concentrations in the 3R4F, base, and test groups atmospheres (after 1 mo: 0.66, 0.72, and 0.81 µmol, respectively; after 4 mo: 0.67, 0.79, and 0.79 µmol, respectively) (Fig. 4C). As expected, groups exposed to E-vapor aerosols (carrier, base, and test) had higher plasma levels of PG than the 3R4F CS group (Fig. 4D). COHb concentration (as a biomarker of combustion and CS exposure) was significantly increased in the 3R4F CS group, whereas the levels of COHb in the carrier-, base-, and test aerosol-exposed animals were close to those in the sham-exposed animals (<5%) (Fig. 4E).

Fig. 4.

Fig. 4.Test atmosphere exposure and nicotine biomarkers. Levels of propylene glycol, nicotine, and nicotine metabolites in the 3R4F cigarette smoke (CS)- and E-vapor aerosol-exposed mice. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; M, months. A: concentration of nicotine in plasma. B: concentration of cotinine in plasma. C: absolute levels of total nicotine metabolites in urine: trans-3′-hydroxycotinine, cotinine, norcotinine, nicotine, nicotine-1′-N-oxide, and nornicotine. D: concentration of propylene glycol in plasma. E: percentage of COHb in blood. n = 8. +P < 0.05, significant vs. sham; cP < 0.05, significant vs. carrier; tP < 0.05, significant vs. test; and bP < 0.05, significant vs. base.

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Reduced Levels of Harmful and Potentially Harmful Constituent Biomarkers in Urine in Mice Exposed to E-Vapor Aerosols

Mice exposed to the carrier, base, and test aerosols showed significantly lower levels of selected HPHC biomarkers in urine than CS-exposed mice (Table 4). The urinary levels of SPMA, a biomarker of benzene exposure, were significantly increased only in the 3R4F group, whereas the carrier, base, and test groups had background levels similar to that in the sham group. This was similar to observations for urinary levels of CEMA, a biomarker of acrylonitrile exposure, and MHBMA1 and MHBMA2, biomarkers of 1,3-butadiene. Unlike other HPHC biomarkers, HPMA, the acrolein exposure marker, showed substantial background levels in the sham control group, reflecting exposure to exogenous acrolein as well as metabolism of endogenous acrolein (120, 122, 181, 182). However, HPMA concentrations were still significantly higher in the 3R4F group than in the carrier, base, or test groups (Table 4).

Table 4. Urinary biomarkers of exposure

Marker of Exposure in Urine, ng/mLSham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
SPMA1.984 (SD 0.74)185.230 (SD 37.07)b,t,+1.834 (SD 0.70)0.936 (SD 0.47)c,+0.784 (SD 0.19)c,+
CEMA4.781 (SD 1.58)557.028 (SD 118.96)b,t,+5.746 (SD 1.50)3.886 (SD 1.09)c4.681 (SD 1.21)
HPMA4330.075 (SD 1633.65)8667.750 (SD 1290.77)b,t,+5325.625 (SD 1770.79)3624.275 (SD 1224.38)c4294.575 (SD 1314.31)
MHBMA10.264 (SD 0.44)279.228 (SD 94.73)b,t,+1.012 (SD 1.22)1.462 (SD 1.52)+0.771 (SD 1.10)
MHBMA21.267 (SD 0.95)81.119 (SD 6.98)b,t,+1.561 (SD 0.86)0.777 (SD 0.75)1.125 (SD 0.77)

Values are means (SD); n = 8. Sham values are considered as background levels. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; SPMA, S-phenylmercapturic acid; CEMA, 2-cyanoethylmercapturic acid; HPMA, N-(2-hydroxypropyl)methacrylamide; MHBMA1, N-acetyl-S-(1-hydroxymethyl-2-propenyl)-l-cysteine; MHBMA2, N-acetyl-S-(2-hydroxy-3-butenyl)-l-cysteine.

+P < 0.05, significant vs. sham;

cP < 0.05, significant vs. carrier;

tP < 0.05, significant vs. test; and

bP < 0.05, significant vs. base.

E-Vapor Aerosol Exposure Exerts Smaller Effects on Oxidative Stress and Inflammation Markers than 3R4F CS Exposure

Biomarkers of oxidative stress and inflammation.

Selected biomarkers of oxidative stress were quantified in urine after 4 mo of exposure. No significant changes in urinary 2,3-d-TXB2, 11-dh-TXB2, and HETE levels were observed (data could be accessed at https://www.intervals.science/studies#/search/). The levels of MDA and PGF2α, the products of lipid peroxidation and radical-mediated oxidation of arachidonic acid, were significantly higher in the urine of 3R4F CS-exposed mice than in sham-exposed mice (P < 0.05) (Fig. 5). The level of 2,3-di-PGF2α, a β-oxidation product of prostaglandin F2α, was also higher in the urine samples of 3R4F CS-exposed mice than in sham-exposed mice (+2.4 fold) (Fig. 5, A–C). Overall, urinary MDA, PGF2α, and 2,3-di-PGF2α levels were comparable between animals exposed to E-vapor aerosols and the sham control.

Fig. 5.

Fig. 5.Effect of E-vapor aerosols and 3R4F cigarette smoke exposure on urinary biomarkers of oxidative stress and inflammation at 4 mo of exposure. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring. Absolute values of malondialdehyde (MDA; A), 2,3-di-PGF2α (B), 8-iso-prostaglandin F2α (PGF2α; C), t-PGE-M (D), and tetranor-prostaglandin E-M, leukotriene-E4 (LTE4; E) are shown. n = 8. +P < 0.05, significant vs. sham; cP < 0.05, significant vs. carrier; tP < 0.05, significant vs. test; and bP < 0.05, significant vs. base.

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The levels of urinary t-PGE-M and LTE4 were significantly higher in 3R4F CS-exposed mice than in sham-exposed mice (2.2- and 11.4-fold, respectively; P < 0.05). No significant elevations in these biomarkers were noted in animals exposed to E-vapor aerosols. The levels of urinary t-PGE-M and LTE4 were significantly lower in the base (−45 and −61.3%) and test (−46.4 and −100%) groups than in 3R4F CS-exposed mice (P < 0.05; Fig. 5, D and E).

E-Vapor Aerosol Exposure Exerts Smaller Effects on Hematological Parameters and Lipids Levels than 3R4F CS Exposure

Relative to sham exposure, 3R4F CS exposure caused a modest but statistically significant increase in the following hematological parameters at both 3 and 6 mo postexposure: erythrocyte count, hematocrit level (+22.9 and +20.1% after 3 and 6 mo), mean corpuscular hemoglobin level (+8.3 and +6.7% after 3 and 6 mo), hemoglobin level (+23.2 and +19.8% after 3 and 6 mo), and reticulocyte count (+14 and +28.7% after 3 and 6 mo). No statistically significant changes were observed in animals exposed to E-vapor aerosols from the carrier, base, or test formulations (Fig. 6, A–D).

Fig. 6.

Fig. 6.Effect of E-vapor aerosols and 3R4F cigarette smoke (CS) exposure on hematological parameters at 3 mo (3M) and 6 mo (6M) of exposure. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring. Erythrocytes (A), hematocrit (B), hemoglobin (C), and reticulocytes (D) were counted or measured in blood. Erythrocytes and reticulocytes are reported as number of cells per liter. n = 11–12. +P < 0.05, significant vs. sham; cP < 0.05, significant vs. carrier; tP < 0.05, significant vs. test; and bP < 0.05, significant vs. base.

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Relative to the sham exposure, platelet and basophil counts also increased significantly in response to 3R4F CS exposure at 6 mo (Table 5), whereas exposure to E-vapor aerosols did not cause significant changes in these parameters. No significant treatment-related differences were noted in white blood cell counts (eosinophils, lymphocytes, monocytes, and neutrophils) (Table 5).

Table 5. Effect of E-vapor aerosols and 3R4F CS on white blood cells and platelets in ApoE−/− mice at 3 and 6 mo

Sham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
Platelet, 109/L
    3 mo1041.04 (SD 153.85)1055.71 (SD 297.97)978.25 (SD 238.35)1164.08 (SD 316.87)1066.38 (SD 195.28)
    6 mo1044.00 (SD 359.68)1435.83 (SD 271.68)b,+1205.27 (SD 322.53)1138.83 (SD 190.99)1250.29 (SD 272.23)
Basophil count count, %
    3 mo0.57 (SD 0.79)0.18 (SD 0.33)t0.13 (SD 0.16)0.20 (SD 0.33)0.37 (SD 0.27)c
    6 mo0.25 (SD 0.38)1.35 (SD 1.23)b,+0.45 (SD 0.76)0.36 (SD 0.44)0.76 (SD 0.97)
Eosinophil count (relative), %
    3 mo0.19 (SD 0.18)0.12 (SD 0.16)0.14 (SD 0.16)0.08 (SD 0.14)0.10 (SD 0.19)
    6 mo0.35 (SD 0.51)0.38 (SD 0.30)0.17 (SD 0.23)0.43 (SD 0.39)0.25 (SD 0.19)
Lymphocyte count (relative), %
    3 mo83.00 (SD 3.16)84.53 (SD 5.22)80.05 (SD 7.73)85.91 (SD 5.13)c83.09 (SD 5.79)
    6 mo76.40 (SD 8.61)76.04 (SD 7.76)77.46 (SD 13.13)76.79 (SD 6.46)77.78 (SD 6.50)
Monocyte count (relative), %
    3 mo4.84 (SD 3.67)5.79 (SD 3.95)b5.95 (SD 4.74)3.22 (SD 3.64)6.34 (SD 6.19)
    6 mo8.51 (SD 5.41)7.88 (SD 6.02)8.19 (SD 8.90)7.39 (SD 5.37)6.93 (SD 5.07)
Neutrophil count (relative), %
    3 mo11.40 (SD 2.83)9.39 (SD 3.99)13.74 (SD 5.66)10.59 (SD 3.57)10.10 (SD 3.52)
    6 mo14.49 (SD 6.36)14.32 (SD 5.00)13.73 (SD 5.91)15.03 (SD 3.81)14.28 (SD 3.90)

Values are means (SD); n = 11–12. First value indicates the mean platelet count, reported as number of cells per liter. Relative concentrations of white blood cells in the blood are reported as the percentage of total cells (%). CS, cigarette smoke; ApoE−/−, apolipoprotein E-deficient; PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring.

+P < 0.05, significant vs. sham;

cP < 0.05, significant vs. carrier;

tP < 0.05, significant vs. test; and

bP < 0.05, significant vs. base.

Analysis of blood lipids and lipid particles showed that 3R4F CS exposure caused a significant increase in the levels of total cholesterol and chylomicron cholesterol (Table 6). When compared with the sham group, the 3R4F CS group had an increased level of total cholesterol at 3 mo (P < 0.05) and 6 mo of exposure. Similarly, chylomicron cholesterol levels had increased significantly after 3 and 6 mo in the CS 3R4F group (P < 0.05).

Table 6. Effect of E-vapor aerosols and 3R4F CS on blood lipoprotein cholesterol in ApoE−/− mice at 3 and 6 mo

Sham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
Total cholesterol, mg/dL
 3 mo336.6 (SD 83.63)431.8 (SD 61.03)b,t,+342.7 (SD 58.37)351.9 (SD 34.03)364.7 (SD 96.37)
 6 mo416.0 (SD 53.70)451.8 (SD 48.72)b,t387.3 (SD 27.02)349.5 (SD 56.51)+364.7 (SD 53.20)+
Chylomicrons cholesterol, mg/dL
 3 mo19.2 (SD 9.07)36.1 (SD 4.31)b,t,+20.7 (SD 3.72)24.9 (SD 4.31)c22.5 (SD 9.01)
 6 mo42.5 (SD 7.30)55.2 (SD 5.71)b,t,+42.1 (SD 6.17)33.9 (SD 10.14)c,+34.9 (SD 8.68)c,+
HDL cholesterol, mg/dL
 3 mo17.8 (SD 3.43)15.7 (SD 4.18)15.7 (SD 2.76)16.6 (SD 2.76)17.5 (SD 3.81)
 6 mo19.8 (SD 4.74)20.4 (SD 4.96)19.5 (SD 3.74)20.6 (SD 5.45)20.7 (SD 4.61)
LDL cholesterol, mg/dL
 3 mo92.7 (SD 43.82)88.0 (SD 10.71)86.0 (SD 19.87)84.8 (SD 9.06)91.7 (SD 18.77)
 6 mo104.1 (SD 10.80)97.7 (SD 12.13)96.3 (SD 14.68)92.3 (SD 11.81)+97.7 (SD 13.77)
VLDL cholesterol, mg/dL
 3 mo206.9 (SD 63.35)292.0 (SD 44.70)b,t,+220.4 (SD 40.49)225.6 (SD 25.77)233.2 (SD 74.64)
 6 mo249.6 (SD 35.56)278.5 (SD 35.20)b,t229.4 (SD 19.48)202.7 (SD 39.81)+211.4 (SD 39.31)+

Values are means (SD); n = 10. First value indicates the mean total cholesterol, chylomicron, HDL cholesterol, LDL cholesterol, and VLDL cholesterol levels. CS, cigarette smoke; ApoE−/−, apolipoprotein E-deficient;PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring.

+P < 0.05, significant vs. sham;

cP < 0.05, significant vs. carrier;

tP < 0.05, significant vs. test; and

bP < 0.05, significant vs. base.

E-Vapor Aerosol Exposure Exerts Smaller Effects on Atherosclerotic Plaque Surface and Volume than 3R4F CS Exposure

Planimetric analysis of the aortic arch revealed atherosclerotic progression in all groups over the 6-mo exposure period (Fig. 7, A and B). Mice exposed to 3R4F CS showed a significant (P < 0.05) increase in aortic arch area covered by atherosclerotic plaque, relative to sham-exposed mice (2.1-fold increase after 3 mo and 1.4-fold increase after 6 mo; P < 0.05). No significant difference in plaque surface area was observed between mice exposed to E-vapor aerosols and sham-exposed mice (Fig. 7ab). In comparison with 3R4F CS-exposed mice, mice exposed to the carrier, base, and test aerosols showed smaller plaque areas. In comparison to the 3R4F group, the plaque surface area decreased by 49.9 and 25.2, 44.9 and 27.3, and 47.5 and 25.7% in the carrier, base, and test groups, respectively, at 3 and 6 mo of exposure.

Fig. 7.

Fig. 7.Effect of E-vapor aerosols and 3R4F cigarette smoke exposure on atherosclerotic plaque area and volume in apolipoprotein E-deficient mice. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; M, month. A: representative images of atherosclerotic lesions in the aortic arch, with measurements acquired by planimetry at 6 mo. B: relative atherosclerotic plaque surface area in the aortic arch, evaluated by planimetry (n = 19–20). C: representative images of atherosclerotic lesions in the thoracic aorta acquired by CT. D: relative atherosclerotic plaque volume evaluated by micro-CT (n = 15–16). E: relative atherosclerotic plaque surface evaluated by micro-CT (n = 15–16). +P < 0.05, significant vs. sham; cP < 0.05, significant vs. carrier; tP < 0.05, significant vs. test; and bP < 0.05, significant vs. base.

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Additionally, quantitative microCT investigation was performed on the thoracic aorta of a separate cohort of mice (n = 16) (Fig. 7C). Consistent with the planimetry findings, the atherosclerotic plaque surface and volume were greater in 3R4F CS-exposed mice than in sham-exposed mice after 6 mo of exposure (Fig. 7, D and E). No significant difference in plaque surface area or volume were observed among mice exposed to E-vapor aerosols (Fig. 7E). Mice exposed to E-vapor aerosols presented lower plaque surface area than 3R4F CS-exposed mice (37.7 and 26.7% in carrier, 35.7 and 20.1% in base, and 39.8 and 29.3% in test, respectively, after 3 and 6 mo of exposure) (Fig. 7). Similarly, a lower plaque volume was observed in mice exposed to E-vapor aerosols than in 3R4F CS-exposed mice (36.1 and 37.2% in carrier, 38.7 and 28.9% in base, and 47.2 and 43.4% in test aerosols (P < 0.05) at 3 and 6 mo of exposure) (Fig. 7D).

E-Vapor Aerosol and 3R4F CS Exposures Exert No Effects on Plaque Composition, Heart Weight, or Heart Morphology

Our morphological and histopathological assessment found no significant differences in the effects of exposure to E-vapor aerosols or 3R4F CS on atherosclerosis lesions of the aortic root (Table 7) or heart ventricle histopathology (Table 8). Histological evaluation of the aortic root, which focused on degeneration of the lamina media, endothelial discontinuation, presence of inflammatory infiltrates, fibrosis of the lamina media, and thrombus formation, did not show significant changes in response to 3R4F CS or E-vapor aerosols. In the heart ventricle, evaluation of fibrosis, hemorrhage, mineralization, inflammatory cell infiltration, and myocyte degeneration revealed no significant effects of exposure (Table 8). The absence of effects on the heart structure by all types of exposure was supported by heart weight measurements, heart weight-to-tibia length ratio (Supplementary Table S3), and heart ventricular diameter and mass assessed by echocardiography (Supplementary Fig. S3).

Table 7. Histopathology scores of the aortic root in the 3R4F CS and E-vapor aerosols groups at 3 and 6 mo

Sham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
Degeneration, lamina media
    3 mo1.00 (SD 0.85)0.667 (SD 0.78)1.000 (SD 0.95)0.417 (SD 0.51)0.583 (SD 0.67)
    6 mo0.000 (SD 0.00)0.250 (SD 0.62)0.083 (SD 0.29)0.083 (SD 0.29)0.083 (SD 0.29)
Endothelial discontinuation
    3 mo0.667 (SD 0.98)0.667 (SD 0.89)0.333 (SD 0.49)0.500 (SD 0.67)0.417 (SD 0.67)
    6 mo0.333 (SD 0.49)0.583 (SD 0.67)t0.083 (SD 0.29)0.833 (SD 0.58)c,+0.083 (SD 0.29)b
Mixed inflammatory infiltrates, anulus fibrosus
    3 mo2.500 (SD 0.80)2.167 (SD 0.94)2.667 (SD 0.78)2.333 (SD 0.78)2.167 (SD 0.72)
    6 mo1.500 (SD 0.67)1.833 (SD 0.94)1.333 (SD 0.49)1.750 (SD 0.97)1.750 (SD 0.75)
Mixed inflammatory infiltrates, lamina media
    3 mo1.833 (SD 0.94)1.333 (SD 0.89)1.667 (SD 0.65)1.333 (SD 0.65)1.333 (SD 0.78)
    6 mo0.167 (SD 0.39)0.500 (SD 0.67)0.083 (SD 0.29)0.333 (SD 0.65)0.417 (SD 0.67)
Plaque formation
    3 mo3.333 (SD 0.78)3.250 (SD 1.14)3.000 (SD 0.74)2.833 (SD 0.72)3.000 (SD 0.74)
    6 mo3.500 (SD 0.52)3.917 (SD 0.51)3.750 (SD 0.45)3.667 (SD 0.65)3.833 (SD 0.58)
Thrombus
    3 mo0.000 (SD 0.00)0.000 (SD 0.00)0.000 (SD 0.00)0.000 (SD 0.00)0.000 (SD 0.00)
    6 mo0.000 (SD 0.00)0.000 (SD 0.00)0.000 (SD 0.00)0.000 (SD 0.00)0.000 (SD 0.00)

Values are means (SD); n = 12. CS, cigarette smoke; PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring.

+P < 0.05, significant vs. sham;

cP < 0.05, significant vs. carrier;

tP < 0.05, significant vs. test; and

bP < 0.05, significant vs. base.

Table 8. Histopathology scores of the left ventricle in the 3R4F CS and E-vapor aerosols groups at 3 and 6 mo

Sham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
Degeneration, myocytes
    3 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)
    6 mo0.1667 (SD 0.39)0.1667 (SD 0.58)0.0833 (SD 0.29)0.6667 (SD 1.15)0.0833 (SD 0.29)
Dilatation, ventricle
    3 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)
    6 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)
Fibrosis, myocardium
    3 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0833 (SD 0.29)
    6 mo0.4167 (SD 0.79)0.0833 (SD 0.29)0.0000 (SD 0.00)0.5833 (SD 1.38)0.2500 (SD 0.62)
Fibrosis, pericardium/subepicardia
    3 mo0.0000 (SD 0.00)0.1667 (SD 0.58)0.0833 (SD 0.29)0.0000 (SD 0.00)0.0000 (SD 0.00)
    6 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)
Inflammatory cell infiltration, mixed, myocardium
    3 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)
    6 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.1667 (SD 0.58)0.5000 (SD 1.17)0.0000 (SD 0.00)
Inflammatory cell infiltration, mononuclear, myocardium
    3 mo0.2500 (SD 0.45)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0833 (SD 0.29)0.0000 (SD 0.00)
    6 mo0.0833 (SD 0.29)0.0833 (SD 0.29)0.0833 (SD 0.29)0.0833 (SD 0.29)0.1667 (SD 0.39)
Plaque, myocardial blood vessels
    3 mo0.0833 (SD 0.29)0.1667 (SD 0.39)0.2500 (SD 0.45)0.1667 (SD 0.39)0.3333 (SD 0.65)
    6 mo0.0000 (SD 0.00)0.0833 (SD 0.29)0.0000 (SD 0.00)0.2500 (SD 0.62)0.0000 (SD 0.00)
Thrombus, myocardial blood vessels
    3 mo0.0833 (SD 0.29)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)
    6 mo0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)0.0000 (SD 0.00)

Values are means (SD); n = 12. CS, cigarette smoke; PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring.

E-Vapor Aerosol Exposure Exerts Smaller Effects on Systolic and Diastolic Heart Function than 3R4F CS Exposure

Systodiastolic function of the heart ventricle was assessed by transthoracic echocardiography. Data derived from the ECG trace did not show significant differences among the experimental groups (Table 9). Analysis of the systodiastolic performance demonstrated that relative to sham exposure, exposure to 3R4F CS caused significant (P < 0.05) impairment of ejection fraction (−15.5%), fractional shortening (−20.1%), and cardiac output (−18.7 mL/min) after 6 mo exposure (P < 0.05; Fig. 9). In mice exposed to E-vapor aerosols at similar levels of nicotine to that in CS, E-vapor aerosol exposure caused no significant changes relative to sham for ejection fraction, fractional shortening, and cardiac output (Fig. 8, A–C).

Table 9. In vivo ECG measurements (PR-RR-QT interval) in response to 3R4F CS and E-vapor aerosols exposure at 2, 4, and 6 mo

Sham3R4FCarrier (PG/VG)Base (PG/VG/N)Test (PG/VG/N/F)
PR interval
    EKG trace, ms
        2 mo37.07 (SD 4.94)40.32 (SD 4.46)36.93 (SD 4.12)37.60 (SD 7.46)37.88 (SD 8.71)
        4 mo40.50 (SD 9.82)43.80 (SD 9.17)42.78 (SD 6.17)42.83 (SD 8.33)41.93 (SD 11.37)
        6 mo32.26 (SD 10.86)41.42 (SD 3.36)42.19 (SD 10.59)45.42 (SD 6.68)48.13 (SD 6.19)
QRS interval
    EKG trace, ms
        2 mo33.73 (SD 6.10)31.93 (SD 4.10)31.18 (SD 5.16)31.73 (SD 4.11)32.30 (SD 4.69)
        4 mo34.13 (SD 7.69)28.47 (SD 7.29)34.75 (SD 5.19)31.83 (SD 7.49)30.77 (SD 8.81)
        6 mo22.50 (SD 5.72)26.92 (SD 9.57)27.83 (SD 5.23)30.58 (SD 5.15)32.50 (SD 5.30)
QT interval
    EKG trace, ms
        2 mo88.53 (SD 9.80)87.28 (SD 6.77)82.27 (SD 7.90)78.27 (SD 11.62)76.12 (SD 12.60)
        4 mo83.64 (SD 6.19)78.20 (SD 8.43)79.87 (SD 8.72)77.75 (SD 7.21)75.33 (SD 9.29)
        6 mo84.29 (SD 7.94)82.50 (SD 8.34)79.93 (SD 9.18)81.83 (SD 8.81)84.38 (SD 4.42)

Values are means (SD); n = 2–10. CS, cigarette smoke; PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring.

Fig. 8.

Fig. 8.Echocardiographic evaluation of left ventricular function in response to E-vapor aerosols and 3R4F cigarette smoke (CS) exposure. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; M, months. A–D: effect of CS and E-vapor aerosols on systolic function: percentage ejection fraction (A), fractional shortening (B), cardiac output (C), and isovolumic contraction time (D) at 3 and 6 mo after exposure to E-vapor carrier, base, or test aerosol or 3R4F CS (n = 10–12). E: isovolumic relaxation time as a parameter of diastolic dysfunction. F: myocardial performance index as an index of global systodiastolic dysfunction, assessed in the 4-chamber Doppler mode. G: myocardial performance index as an index of global systodiastolic dysfunction, assessed by tissue-Doppler imaging.

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We also assessed, both by conventional and tissue-Doppler imaging, the impact of E-vapor aerosols and 3R4F CS on isovolumic relaxation time, reliable indexes of diastolic function, and isovolumic contraction time, an additional parameter of systolic performance. After 6 mo of exposure, the isovolumic contraction and relaxation times in CS-exposed mice had increased significantly by ~25.9 and 37.2% in comparison with the values recorded in sham-exposed mice (Fig. 8, D and E). In comparison with sham exposure, E-vapor aerosol exposure caused no change in isovolumic contraction time. However, mice exposed to the base and test aerosols showed an increase in isovolumic relaxation time (34.3 and 23%, respectively; P < 0.05) relative to sham-exposed mice after 6 mo of exposure (Fig. 8, D and E).

To assess the global impact of E-vapor aerosols and 3R4F CS exposure on heart function, we derived the MPI from four-chamber Doppler and tissue-Doppler imaging data. Four-chamber Doppler ultrasound analysis demonstrated that in comparison with sham exposure, 3R4F CS exposure caused a significant increase in MPI after 2 (by 18.8%; P < 0.05), 4 (by 20.3%; P < 0.05), and 6 (by 50.5%; P < 0.05) mo of exposure. Similarly, tissue-Doppler analysis confirmed a significant increase in MPI at 6 mo of exposure in the 3R4F CS group, relative to the sham group. To a lesser extent, it was observed that exposure to the E-vapor aerosols had a slight impact on the MPI. Tissue-Doppler analysis revealed a slight but significant (P < 0.05) increase in MPI in the base and test groups relative to the carrier and sham groups at 6 mo (Fig. 8, F and G).

E-Vapor Aerosol Exposure Exerts a Smaller Effect on PWV and Arterial Stiffness than 3R4F CS Exposure

To determine the impact of exposure on arterial stiffness, we measured the PPV and PWV at the abdominal aorta and carotid artery (Fig. 9A). Relative to the sham group, the 3R4F, base, and test groups showed a significant increase (P < 0.05) in pulse propagation velocity in the abdominal aorta (at 4 mo, 14.6, 7.7., and 7.5%, respectively; and at 6 mo, 33.8, 24.3, and 25.5%, respectively) (Fig. 9B). In comparison with the PPV in the 3R4F CS group, that in the base- and test-exposed groups was significantly lower (6 and 6.2% after 4 mo and 7 and 6.1% after 6 mo, respectively; P < 0.05) (Fig. 9B).

Fig. 9.

Fig. 9.Effect of E-vapor aerosols and 3R4F cigarette smoke on pulse wave velocity (PWV) and pulse propagation velocity (PPV) in apolipoprotein E-deficient mice. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; M, months. A: schematic representation of PWV and PPV acquisition area. B: abdominal aorta PPV at 2, 4, and 6 mo of exposure (n = 8–12). C: carotid artery PWV at 6 mo (n = 10–12). +P < 0.05, significant vs. sham; cP < 0.05, significant vs. carrier; tP < 0.05, significant vs. test; and bP < 0.05, significant vs. base.

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Similarly, a significant increase in PWV relative to the sham-group levels was observed in the 3R4F CS group (32.1%; P < 0.05) and in groups exposed to nicotine-containing aerosols (22.1% in the base group and 21.4% in the test group; P < 0.05) at 6 mo postexposure (Fig. 9C). In comparison with 3R4F CS-exposed mice, mice exposed to the base and test aerosols showed a significantly lower PWV (reduction of 7.5 and 8.1%, respectively; P < 0.05).

E-Vapor Aerosol Exposure Exerts Less Molecular Dysregulation in Cardiovascular Tissues than 3R4F Exposure

Exposure to 3R4F CS resulted in a time-dependent increase in the number of differentially expressed genes in the left heart ventricle and thoracic aorta (FDR adjusted; P < 0.05) (Figs. 10 and 11). After 3 mo of exposure to 3R4F CS, 278 genes were significantly dysregulated in the heart ventricle. After 6 mo of exposure to 3R4F CS, the number of differentially expressed genes increased to 330 (125 genes upregulated and 205 downregulated) in the left heart ventricle and 43 (20 genes upregulated and 23 downregulated) in the thoracic aorta (Figs. 10 and 11). In contrast, no statistically significantly differentially expressed genes were detected at any time point in left heart ventricle and thoracic aorta tissues from mice exposed to the carrier, base, or test aerosols.

Fig. 10.

Fig. 10.Molecular dysregulation in the heart ventricle, assessed through a systems toxicology approach. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; M, months. A: volcano plot representing the changes in gene expression in the heart ventricle after exposure to E-vapor aerosols (carrier, base, or test) or 3R4F cigarette smoke (at 3 and 6 mo) (n = 10). Yellow, significantly upregulated; cyan, significantly downregulated genes; FDR, false discovery rate (P < 0.05). B: gene-set analysis (GSA) of the c2.cp collection of mSigDB (89). The average fold change (FC) of the gene sets is represented by color scale. Significant enrichment (FDR-adjusted P value < 0.05) for 3 different methods is indicated: Camera/Q1 (*–), roast/Q2 (-*-), and ORA (over-representation analysis) (–*). Gene sets with significant Q1 and Q2 enrichment for any contrast are shown. C: heatmap representing the mapping of the differentially expressed genes on “extracellular matrix organization” mechanisms. D: heatmap representing the mapping of differentially expressed genes on Reactome “phase 1: functionalization of compounds.” E: heatmap representing the mapping of differentially expressed genes on Reactome “interferon signaling.” Overexpressed genes are shown in orange, and repressed genes are shown in blue. *P < 0.01 and xP < 0.05, significance for each log2 change; NA, not analyzed.

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Fig. 11.

Fig. 11.Molecular dysregulation in the thoracic aorta, assessed through a systems toxicology approach. PG, propylene glycol; VG, vegetable glycerol; N, nicotine; F, flavoring; M, months. A: volcano plot representing the changes in gene expression groups in the thoracic aorta after exposure to E-vapor aerosols (carrier, base, or test) or 3R4F cigarette smoke (at 3 and 6 mo) (n = 6–10) (total, n = 139). Yellow, significantly upregulated genes; cyan, significantly downregulated genes (FDR P < 0.05). B: gene-set analysis (GSA). Average fold change (FC) of the gene sets is represented by color scale. Significant enrichment (FDR-adjusted P value < 0.05) for 3 different methods is indicated: Camera/Q1 (*–), roast/Q2 (-*-), and ORA (over-representation analysis) (–*). C: heatmap representing the mapping of differentially expressed genes on PID (Pathway Interaction Database) “circadian pathways.” Orange, overexpressed genes; blue, repressed genes; NA, not analyzed.

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Analysis of gene sets.

3R4F CS exposure substantially dysregulated the genes involved in collagen formation and extracellular matrix organization, integrin signaling, inflammatory signaling, and xenobiotic and mitochondrial responses (Fig. 10B). In particular, mechanisms related to extracellular matrix organization, extracellular matrix receptor interaction, collagen formation, the Naba collagen gene set, the Naba basement membrane gene set, and related integrin components (such as the integrin 1 pathway) were significantly downregulated (Q1, Q2, and over-representation analysis) after 3 and 6 mo of exposure (Fig. 10B). Mechanisms such as metabolism of xenobiotics by cytochrome-P450 enzymes, glutathione conjugation, and ABC family protein-mediated transport were significantly upregulated in the 3R4F group at 3 and 6 mo (Q1–Q2). Similarly, mitochondrial tRNA amino acetylation and pyruvate metabolism were (Q1–Q2) activated after 3 and 6 mo of exposure to 3R4F CS, whereas the BioCarta monocyte pathway gene set, interferon-γ signaling, cytokine signaling in the immune system, and BioCarta ccr5 pathway gene set were downregulated. In contrast, very minimal effects on gene sets were observed in the heart ventricle in response to exposure to the carrier, base, or test aerosol (Fig. 10B). Mapping of differentially expressed genes to extracellular matrix organization, xenobiotic phase 1 response, and interferon signaling are further explained in Supplementary Material 1 and visualized in Fig. 10B.

In the thoracic aorta, significant gene dysregulation was observed in response to 3R4F CS exposure for 6 mo, particularly in genes related to circadian pathways, circadian rhythms in mammals, and circadian expression (Fig. 11, B and C). Mapping of differentially expressed genes to circadian pathways are further explained in Supplementary Material 1 and Fig. 11C).

Pathway analysis.

To complement the GSA, IPA was used to identify exposure-affected primary biological functions, pathways, and upstream regulators in the heart ventricle. 3R4F CS exposure significantly affected processes related to cell assembly, organization, and function of the heart ventricle after 3 and 6 mo (Fig. 12). Biological functions such as cytoplasm organization, cytoskeleton organization, neuron development, microtubule dynamics, formation of cellular protrusions, and cell-to-cell contact were significantly altered in response to 3R4F CS exposure for 3 and 6 mo (Z-scores < −2). In parallel, mechanisms related to cell movements, such as lymphocyte movement, cell invasion, spread, and migration, were predicted to be negatively regulated, whereas organismal death and weight loss were predicted to be positively regulated in response to 3R4F CS exposure at 3 and 6 mo. None of these biological functions or processes was identified in the animals exposed to E-vapor aerosols. Overall, IPA indicated that 3R4F CS exposure altered the cardiac transcriptome, but exposure to E-vapor aerosols did not (Fig. 12).

Fig. 12.

Fig. 12.Ingenuity Pathway Analysis (IPA) of transcriptomics data from the heart ventricle. IPA findings representing the biological processes (A), canonical pathways (B), top 10 upstream regulators (C), and top 3 chemical upstream regulators (D) significantly impacted in the 3R4F, carrier, base, and test groups (n = 9–10). M, months. *P < 0.05; ***P < 0.01. Blue, predicated as downregulated; orange, predicated as upregulated. The intensity of coloring varies with Z-score value. Z-scores > 2 are indicated by ▲; Z-scores < −2 are indicated by ▼.

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Canonical pathway analysis.

Exposure to 3R4F CS significantly altered biological signaling in the heart ventricle, including canonical pathways related to GP6 signaling, ILK signaling, actin nucleation by the ARP-WASP complex, thrombin signaling, and CXCR4 signaling after 3 mo. It also caused additional alterations in HMGB1 signaling, sphingosine-1-phosphate signaling, Tec kinase signaling, and regulation of actin-based motility by Rho after 6 mo (Z-scores < −2) (Fig. 12B). While the majority of enriched pathways appeared to be downregulated, RhoGDI signaling was predicted to be activated in response to 3R4F CS exposure after 3 and 6 mo (Z-score > 2). IPA of canonical pathways confirmed a nonsignificant effect of exposure to carrier, base, or test aerosol exposures at any time point (Fig. 12B).

Upstream regulator analysis.

Upstream regulator analysis identifies transcription factors or genes responsible for a downstream effect on gene expression (96). In the present study, IPA identified Tgfb1, F2, and IL2 as the top three upstream regulators inhibited by exposure to 3R4F CS for 3 and 6 mo. α-Catenin appeared among the top 10 upstream regulators as overexpressed following 3R4F CS exposure. None of these upstream regulators was significantly affected following exposure to aerosols from the nicotine-free carrier, base, or from test formulations at nicotine-matched concentrations (Fig. 12C). The upstream regulator analysis also identifies chemicals or drugs that cause similar downstream effects. The downstream effects that were observed following 3R4F CS exposure in the present study corresponded to activation of sirolimus (mTOR inhibitor), U0126 (MAPK inhibitor), and wortmannin [phosphatidylinositol 3 kinase (PI3K) inhibitor] (80, 132) (Fig. 12D).

DISCUSSION

In the present study, we assessed and compared the effects of exposure to E-vapor aerosols and 3R4F CS at nicotine-matched concentrations on the cardiovascular system of ApoE−/− mice. Exposure to E-vapor aerosols from carrier, base, and test formulations had a significantly lower biological effect on the cardiovascular system of mice than 3R4F CS. Mice in the E-vapor aerosol groups were exposed at approximately twofold higher TPM levels than those in the 3R4F group; even then, the resulting biological responses in the E-vapor aerosols groups were substantially lower than those in the CS group. Exposure to smoke from 3R4F reference cigarettes affected multiple molecular mechanisms, increased atherosclerotic plaque surface and volume in the aortic arch, and caused systolic and diastolic dysfunction in the heart ventricle.

Daily monitoring of aerosol components (PG, VG, and nicotine) indicated that the aerosol was generated and delivered to the inhalation chambers in a consistent manner, with similar mean nicotine concentrations in the 3R4F, base, and test atmospheres. Based on our study exposure regimen and the body surface area conversion factor of 12.3 (17), assuming 0.03 L/min volume, 25-g body weight, and complete retention of nicotine in mice, the estimated delivered dose was 193 µg nicotine/day and corresponded to the human equivalent nicotine dose of 37.5 mg/day. This confirms that the study exposure regimen was within the relevant levels of daily nicotine exposure for e-cigarette consumers (27, 139). Successful aerosolization and delivery of flavoring to the exposure chamber (test formulation) was confirmed by the presence of a representative flavor compound, guaiacol, in the test atmosphere. Particle size and distribution in the dispersed aerosols were similar across the exposure groups, indicating equal respirable efficiencies and uptake in the upper respiratory tract of the mice (6, 113).

The high concentrations of CO, carbonyl compounds, and tobacco-specific nitrosamines measured in the 3R4F CS are consistent with the smoke chemistry of this reference cigarette (130). The major concentrations of CO are reflected by the levels of COHb in humans and experimental animals (66, 159). High levels of COHb in the 3R4F CS-exposed mice confirmed appropriate exposure to CS. Similarly, the measured levels of metabolites of tobacco-specific nitrosamines indicated HPHC uptake from CS exposure, but these compounds were not detectable in E-vapor aerosol-exposed mice. These findings are consistent with previous reports on lower levels or absence of tobacco-specific nitrosamines in e-cigarette aerosols compared with smoke from cigarettes (35, 75, 76) and significantly lower levels of tobacco-specific nitrosamine metabolites in the urine of e-cigarette users (135).

Carbonyl compounds such as acrolein, acetaldehyde, and formaldehyde are considered the most significant cardiovascular and pulmonary toxicants in CS (25, 57). At equal nicotine concentrations in the base and test aerosol-exposed groups, the concentrations of formaldehyde and acetaldehyde were drastically (35–60- and 2,000–3,000-fold) lower than those in 3R4F CS in this study. E-vapor aerosols generated with CAG led to comparable findings as published data (47, 48, 102). Carbonyl compounds generated in E-vapor aerosols might be the result of the puffing regimen or device setup (102, 151), voltage supplied or temperature of the heater coil (33, 47, 48, 138), or chemical composition of the e-cigarette liquids (20). In this study, we used the capillary aerosol generator, with a controlled temperature and e-liquid formulations, producing example E-vapor aerosol that contain lower concentrations of carbonyl compounds, tobacco-specific nitrosamines, and carbon monoxide than CS.

Exposure to 3R4F CS had a significant structural impact on vascular walls, resulting in increased atherosclerotic plaque area and volume. These effects were not observed in mice exposed to the E-vapor aerosols at comparable nicotine levels, which suggests a low impact of nicotine or flavoring on atherosclerotic plaque progression. In the thoracic aorta and heart ventricle, we observed dysregulation of genes related to circadian rhythm, collagen formation, cell-extracellular matrix interaction, and extracellular matrix receptor interactions following exposure to CS. Alterations in the extracellular matrix of the heart ventricle can result in decreased tensile strength, increased heart dilatation, and heart failure (22, 52, 73, 81, 156, 166, 172). The absence of significant transcriptional changes following exposure to E-vapor aerosols is likely related to the substantially reduced levels of HPHCs such as polycyclic aromatic hydrocarbons, carbonyl compounds, and CO, consistent with the findings on other electronic nicotine delivery devices (20, 33, 47, 76, 102, 138, 151). Transcriptional effects on the cardiovascular system were only observed in CS-exposed mice, suggesting that nicotine at a level of 35 µg/L (exposure at comparable levels in base and test aerosol-exposed mice) is not responsible for the transcriptional dysregulation of the cardiovascular system. Our results are in accordance with previous in vitro and in vivo studies demonstrating a lower transcriptomic effect of E-vapor aerosol exposure in comparison to CS exposure (56, 67, 85). 3R4F CS-exposed mice showed additional dysregulation of hematological parameters and lipids, which perhaps influenced the increase in atherosclerotic plaque area. Increases in hemoglobin, red blood cell counts, and hematocrit are typical CS-related hematological changes in ApoE-deficient mice, which were also observed in previous studies (120, 122). While in the present study we observed a CS-related increase in the platelet count, Phillips et al. (122) demonstrated an age-related increase in platelet counts in ApoE-deficient mice. No direct exposure effects were observed for white blood cell counts (WBC), which is unlike the cigarette smoke exposure effects reported for this clinical end point in human smokers (7, 15, 59, 131). Concerning the platelet counts in long-term smokers, conflicting results have been reported. A higher platelet count but a lower total platelet mass was seen in male smokers compared with nonsmokers (45), whereas in another study, smoking was associated with significantly lower platelet counts in female smokers and with a nonsignificant trend toward higher platelet counts in male smokers (49). In addition, a smoking-related increase in the mean Platelet volume-to-platelet ratio and a decrease in the platelet-to-lymphocyte ratio have been reported (51). Given the interstudy variation of these hematological effects of CS exposure in the ApoE-deficient mouse model, which somehow reflect the heterogeneity in the human clinical findings as well, it appears that these parameters are to be interpreted with caution.

Chylomicron and cholesterol particle accumulation may suggest an alteration of lipid clearance in response to 3R4F exposure (23, 99, 107, 110, 118, 124, 154). Here we used the genetically engineered ApoE-deficient mouse model as a disease-prone model that is sensitive to cigarette smoke-related atherogenic effects. Exposure to cigarette smoke further increases the already elevated lipoprotein levels. As demonstrated by Boue and colleagues (13, 122), we also observed a slight increases in various plasma lipid/lipoprotein molecules following exposure to 3R4F CS. Apart from the different lipoprotein profile proportions in ApoE-deficient mice and in humans, cholesterol transport and metabolism are sufficiently similar in the two species, suggesting that induced disturbances in plasma lipoprotein metabolism through gene manipulation would lead to atherosclerosis in mice that is comparable with the human disease in some important aspects (43, 44, 94). In human the accumulation of chylomicron and very low-density lipoprotein remnants was observed in association with the development of premature atherosclerosis (108, 112). While the mechanisms are not fully elucidated, it seems that CS exposure could alter the lipid integrity (12). Kunimoto et al. and others (79, 176, 177) have shown that smoking increases oxLDL and causes aortic lipids accumulation, thereby favoring atherosclerosis development. CS through prooxidant mechanisms may also favor chylomicron and cholesterol particles oxidation, which could lead to their accumulation in blood, causing endothelium alteration and atherosclerosis development (4, 142, 175). We did not observe an effect on lipid metabolism in mice exposed to the carrier, base, or test aerosol, which may suggests that PG/VG, nicotine, and flavoring have lower hematological and oxidative effects than CS exposure. Inflammation and oxidative stress are involved in the pathogenesis of atherosclerosis and heart failure (11, 42, 133, 137, 155). Smoking is a major risk factor for atherosclerosis progression and heart failure, closely linked with increased oxidative stress, inflammation, DNA damage, and decreased antioxidant activity (16, 70, 97, 125). Our analysis highlights an increase in the biomarkers of oxidative stress and inflammation in mice exposed to 3R4F CS. By contrast, we identified lower levels of oxidative stress markers in mice exposed to E-vapor aerosols.

Ultrasound analysis of the vascular wall and heart ventricle revealed alterations in vascular and cardiac function in response to 3R4F CS exposure. PWV and PPV in the carotid artery and abdominal aorta were altered in response to CS exposure, which suggested changes in the dilatative capacity of the vascular wall. The impact of CS exposure on arterial stiffness and endothelial dysfunction was also reported by others (98, 128, 163). Mahmud and Feely (98) highlighted that smoking a single cigarette causes an acute increase in arterial stiffness in smokers and nonsmokers, suggesting that the threshold of the arterial wall is very low. In the present study, no significant changes were observed for the PWV and PPV in the carrier (PG/VG) group. Smaller increases in pulse wave velocity and pulse propagation velocity than those induced by 3R4F CS exposure were observed in mice exposed to base and test aerosols, without additional effect of flavor, and a significantly smaller impact relative to that of 3R4F CS, indicating that nicotine was probably the reason for the observed increase following exposure to E-vapor aerosols. Our results are aligned with reports of nicotine increasing pulse wave velocity in the abdominal aorta and in the thoracic aorta in mice (34, 115, 165). In healthy nonsmokers, oral nicotine administration (2 mg) elicited a significant increase in PWV in the carotid-femoral artery (2). In e-cigarette inhalation studies in humans, an increase in PWV has been reported to be attributable to nicotine (3 mg/mL and 1.2% nicotine) (19, 64). From a mechanistic point of view, nicotine has been shown to affect endothelial nitric oxide synthase expression, nitric oxide availability, and smooth-muscle-1α-adrenergic receptors activation; altogether, the alteration of these mechanisms could contribute to an impaired relaxation and could lead to an increase of arterial stiffness (92, 103). In the context of CVDs, PWV is considered as a robust mortality risk factor (157). Vlachopoulos et al. (161) demonstrated that an increase in aortic PWV by 1 m/s corresponds to an age-, sex-, and risk factor-adjusted increase of 15% in the risk of cardiovascular and all-cause mortality. Long-term effects of e-cigarette use on arterial stiffness in humans have not yet been published; most studies until today have investigated the acute effects of e-cigarette aerosol exposure and found increases in PWV similar to those elicited from the smoke of a cigarette (19, 38, 162). In one study, however, switching from cigarette use to e-cigarette use for the duration of 1 mo significantly improved the arterial stiffness parameter “augmentation index corrected for heart rate” (64). Whether the significant reduction in PWV seen after the 6-mo exposure to E-vapor aerosol in the ApoE−/− mouse model might correspond to a reduced health risk in long-term e-cigarette users compared with continued smoking cannot be predicted at this stage (35, 36).

Systolic parameters such as ejection fraction, functional shortening, cardiac output, and isovolumic contraction and relaxation times (diastolic parameters) were notably affected by 3R4F CS exposure, which is consistent with published reports on the impact of smoking on human cardiac function (3, 72, 86, 90). In previous studies, mice exposed to CS for 32 wk exhibited impaired systolic and diastolic functions (147), and rats exposed for 5 wk exhibited significant alterations in fractional shortening (50, 71). Greater left ventricle mass, higher left ventricle mass-to-volume ratio, and higher prevalence of left ventricle hypertrophy have also been reported in human smokers (109). We did not observe left ventricle hypertrophy in response to CS exposure through morphometric heart weight and ultrasound measurements, which suggests that left ventricle dysfunction could be related to functional alterations. The absence of the structural effect on the heart ventricle is different from findings in our previous study where we observed a slight but significant impact on heart weight and left ventricle thickness (146). Calcium is a major factor in regulating muscle cell contraction by binding to troponin and activating the sliding of the thick and thin actin filaments, developing pressure within the ventricle, and resulting in the ejection of blood. Dysregulation of intracellular calcium homeostasis contributes to both systolic and diastolic dysfunction by interfering with ryanodine receptors, the sarcoplasmic reticulum calcium-ATPase 2a pathway, and the sodium-potassium pump (21, 37, 55, 95). The systolic and diastolic alterations observed in this study suggest changes in calcium release, availability, binding, and efflux (3, 30–32, 82, 143, 144). The results of our molecular upstream analysis suggested the activation of kinase inhibitors in response to 3R4F CS exposure. PI3K inhibition has been shown to be related to a reduction in L-type calcium channels on the cell surface, resulting in impaired cardiac contraction and function in vivo (46, 117). Hu et al. (21) demonstrated that ablation of α- and β-PI3K isoforms in the heart leads to T-tubule disorganization and loss. Our transcriptomics analysis highlighted alterations in cytoskeleton organization and microtubule dynamics, downregulation of PI3K complex signaling, and upregulation of the wortmannin-sensitive upstream regulator, suggesting that an alteration in PI3K/P38 MAPK signaling in response to 3R4F CS exposure could be involved in the left ventricle dysfunction. A slight increase in isovolumic relaxation time was observed in mice exposed to nicotine-containing aerosols, indicating an effect of nicotine on calcium clearance (61). The aggregation of systolic and diastolic parameters under the MPI confirmed an adverse effect of 3R4F CS exposure; in animals exposed to e-cigarette aerosols, the MPI value was close to that seen in sham-exposed animals.

The impact of modified risk tobacco product aerosols on the cardiovascular system was also evaluated in a previous ApoE−/− mouse study (120, 122). In both studies, this one and previous study (122), the study-design, exposure conditions were comparable (Supplementary Fig. S4A). In both studies, a similar molecular response was observed in response to 3R4F CS after 6 mo of exposure. The transcriptomic response to 3R4F CS exposure for 6 mo demonstrated highly correlated fold changes (Pearson correlation coefficient of 0.78) and shared 47% differentially expressed genes (Supplementary Fig. S4, B–D). In both studies, very low to no biological responses were observed following exposure to E-vapor aerosols or THS 2.2 aerosol, respectively.

The sole use of female mice and the duration of exposure in this study could be considered as possible limitations. In our study, we exclusively used female ApoE−/− mice to examine the impact of CS and E-vapor aerosols on cardiorespiratory disease end points. Female mice were chosen because of their sensitivity toward respiratory disease end points that we addressed in parallel to cardiovascular disease end points. In the context of respiratory disease and more particularly COPD, it has been shown that women present more severe COPD with early onset disease (<60 yr) and greater susceptibility to COPD with lower tobacco smoke exposure compared with men (5, 28, 141). A similar trend in increased COPD susceptibility and severity has been observed in mice. Van Winkle et al. (158) have demonstrated that female mice show more significant proximal airway injury than male mice after acute injection of naphthalene, an important constituent of side-stream cigarette smoke. Concerning the impact of sex differences on the progression of CVD, available literature does not indicate a significant effect of sex on atherosclerotic plaque size. In fact, female mice had a similar lesion area as males (119, 179). The use of only female ApoE−/− mice gave us the opportunity to investigate CVD and COPD end points and while reducing the number of mice and duration of the study to a minimum, in line with the 3R principles (https://nc3rs.org.uk/the-3rs).

Although it may appear that the duration of exposure 6 mo study is too short and does not represent the human exposure context, a 6 mo exposure paradigm represents 20–25% of the animal’s life considering that the life span of mice is around 24–30 mo (115). If we compare this ratio to humans, considering an average life expectancy of 80 yr, 20–25% would correspond to chronic CS or E-vapor aerosol exposure for approximately 16 to 20 yr. Moreover, molecular and functional investigations by our group and others have demonstrated that in the cardiovascular system of ApoE−/− mice, increased alterations (over and above the genetically driven background changes) such as atherosclerosis progression, molecular dysregulation, and increased PPV can be observed early between 1 and 3 mo of CS exposure and that specific end points may reach their saturation already at ages of 5 to 10 mo (3 to 8 mo of exposure) so that little or no statistically significant exposure effects can be distinguished thereafter (24, 74, 91, 123, 164). In consequence, for such end points that lose statistical significance in their signal-to-background ratio, there would be no benefit in extending the duration of exposure.

In summary, at equal nicotine concentrations in the base and test aerosol-exposed groups, we observed a significantly reduced impact of inhaled E-vapor aerosols on the cardiovascular system of ApoE−/− mice relative to CS. Exposure to 3R4F CS altered the systolic and diastolic functions of the heart, accelerated atherosclerotic progression, altered lipid profiles, and caused alterations of the heart ventricle as well as aorta transcriptomes in this mouse model of cardiovascular disease, whereas these effects were smaller or completely absent in mice exposed to E-vapor aerosols.

DISCLOSURES

The testing facility was Philip Morris International (Singapore and Neuchâtel). This work involved E-vapor formulation (MarkTen, manufactured by Nu-Mark, a subsidiary of Altria). All authors, except A. Buettner and W. K. Schlage, are employees of Altria Client Services, LLC or Philip Morris International (PMI) Research and Development. W. K. Schlage is contracted and paid by PMI. A. Buettner is an employee of Histovia, GmbH, which was contracted and paid by PMI to perform the histopathological analysis.

AUTHOR CONTRIBUTIONS

K.M.L., J.Z., B.P., A.S., A. Kuczaj, K.L., P.V., M.C.P., and J.H. conceived and designed research; E.T.W., T. Lee, S.K.W., and T. Low performed experiments; J.S., B.T., E.G., P.L., A.B., Y.X., F.M., A.S., N.V.I., and J.H. analyzed data; J.S., E.T.W., and B.T. interpreted results of experiments; J.S. and B.T. prepared figures; J.S., W.K.S., A. Kuczaj, K.L., M.C.P., and J.H. drafted manuscript; J.S., E.T.W., B.T., S.K.W., T. Low, K.M.L., J.Z., A. Kumar, W.K.S., A. Kuczaj, K.L., P.V., M.C.P., and J.H. edited and revised manuscript; J.S., E.T.W., B.T., T. Lee, S.K.W., T. Low, K.M.L., J.Z., A. Kumar, W.K.S., E.G., B.P., P.L., A.B., Y.X., F.M., A.S., A. Kuczaj, N.V.I., K.L., P.V., M.C.P., and J.H. approved final version of manuscript.

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AUTHOR NOTES

  • *J. Szostak and E. T. Wong contributed equally to this work.

  • Address for reprint requests and other correspondence: J. Szostak, PMI R&D, Philip Morris Products S.A. Quai Jeanrenaud 5, CH-2000 Neuchâtel, Switzerland (e-mail: ).
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