Animal

Animal experiments were conducted in accordance with the National Institutes of Health, Guide for the Care and Use of Laboratory Animals under protocols (IACUC# 108714) approved by the Institutional Animal Care and Use Committee at the University of Toledo. Adult male and female C57BL/6 mice at 2–3 months of age were used for this study. All mice were reared under a 12 h dark/light cycle, fed with standard chow, and provided with water ad libitum. These conditions were utilized for the entire duration of the experiment.

E-cigarette Exposure Protocol

A whole body smoking exposure system from SCIREQ was used for the experiment. This specialized computer-operated machinery generates e-cigarette vapor using a controlled power supply (as shown in Fig. 1) and is housed in an exhaust hood. Male and female mice were held in the restraint chamber connected to the e-cigarette vaping system. The whole system is placed in an exhaust hood. Control mice were held in a separate chamber that exposed to room air only. The e-liquid consists of 50% propylene glycol, 50% glycerin, and 24 mg/ml nicotine as described previously31. The e-liquid filled in a chamber with an atomizer was vaporized at a frequency of 1 puff per minute and duration of 10 seconds per puff. Generated vapor was delivered to the mouse restraint chamber using an air pump and subsequently cleared out by a second pump. Both pumps as well as the vaping trigger were controlled by the InExposure computer software. Total exposure time was 3 hours per day with a 10 min break every hour for 14 consecutive days. Mice were acclimated for 30 min in the restraint chamber before the first exposure to e-cigarette vapor.

Blood Samples and Organ Collection

Blood was collected from the heart with a syringe after animals were euthanized with xylazine at the last day of e-cigarette exposure. The plasma fraction was obtained by centrifugation and stored at −80 °C. Following blood collection, the heart was collected and weighed. One half of the heart was flash frozen and stored at −80 °C for use in biochemical assessments of cardiac toxicity. The other half of the heart was fixed in 4% formaldehyde solution and processed for morphometric analysis of tissue injury as we previously published32,33,34.

Cardiovascular Function Assessments

Echocardiographic measurements was performed once at baseline (one day prior to e-cigarette exposure) and repeated at 14 days after e-cigarette exposure. The left ventricle diastolic dimension, systolic dimension, wall thickness, and heart rate were measured using an Acuson Sequoia C512 (Siemens) ultrasound machine as previously described35,36,37. Briefly, animals were anesthetized with 2% isoflurane delivered on 100% Oxygen. The chest area was treated with Nair to remove the hair. An ultrasonic probe was used to assess the left ventricle size. The end diastolic dimension (EDD) and end systolic dimension (ESD) were measured using M-mode on short axis position. The diastolic volume and systolic volume were estimated using the following formula: Diastolic Volume = 4/3*π*(EDD*1/2)3; Systolic Volume = 4/3*π*(ESD*1/2)3, assuming that the left ventricle shape is spherical. Ejection fraction (EF%) was calculated as: EF% = (Diastolic volume-Systolic Volume)/Diastolic volume * 100%. The aorta dimension was measured by positioning the probe to the aorta area in long axis.

Western Blot Analysis

Tissue homogenates were prepared by placing left ventricle tissue in ice-cold RIPA lysis buffer (pH 7.0) from Santa Cruz Biotechnology (Santa Cruz, CA; SC-24948) containing protease inhibitors and phosphatase inhibitors. About 40 µg homogenates were analyzed using Western blot. The primary antibodies used in western blot analyses were: Rabbit anti-CD31, 1:500 dilution (Abcam Inc., Cat. No.: ab28364); Rabbit anti-α-smooth muscle actin, 1:500 dilution (Abcam Inc., Cat. No.: ab5694); Rabbit anti-GAPDH antibody, 1:1000 dilution (Santa Cruz Inc., Cat. No.: sc-25778). Secondary antibodies included goat anti-rabbit IgG-HRP (Santa Cruz Inc., Cat. No.: sc-2030); goat anti-mouse IgG-HRP (Santa Cruz Inc., Cat. No.: sc-2031). The Western blot images were cropped for presentation in each figure, and the uncropped Western blot images were provided in the Supplementary Info File.

Masson’s Trichrome Staining for Fibrosis

Left ventricle sections fixed in 4% formaldehyde buffer solution (pH 7.2) were paraffin-embedded and cut into a thickness of 4 μm onto a microscopy slide. The tissue sections were deparaffinized with xylene and rehydrated by sequential incubations in ethanol and water followed by Masson’s Trichrome staining. The whole-slide scanning was performed on these slides using an Olympus whole-slide scanning system. Cardiac fibrosis was calculated and presented as percentage area of blue staining versus red staining using a computer aided morphometry as previously described37,38.

Immuno-Fluorescent Staining of CD31 and CD34

Paraffinized heart tissue sections (4 µm in thickness) were steamed for 15 min in the Trilogy solution from Sigma-Aldrich (Cat. No. 920 P) for deparaffinization and antigen retrieval. Afterwards, the slides were blocked with 1% BSA in TBS-T solution followed by incubating overnight at 4 °C with anti-CD31 antibody from Abcam (Cat. No. ab28364, 1:50 dilution) or anti-CD34 antibody from R&D (Cat. No. AF6518). Alexa fluor 594 conjugated anti-rabbit antibody from ThermoFisher) (Cat. No. A-11012, 1;100 dilution) was used to visualize the CD31 immunostaining, and Alexa fluor 594 conjugated anti-sheep antibody from Abcam (Cat. No. ab96937) was used to visualize CD34. For co-immunostaining of CD31 and CD34, the secondary antibody for CD31 was switched to Alexa fluor 488 conjugated anti-rabbit antibody from Abcam (Cat. No. ab150073). Fluorescent images of CD31 and CD34 staining were taken using a Leica confocal microscope. In each slide, 5 images were randomly taken from different areas of the slide. Images were analyzed using FiJi ImageJ software to calculate the percentage of CD31 positive areas.

Measurement of Cotinine

An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was used to measure the plasma cotinine concentration as previously described39. Plasma samples were spiked with 0.05% aqueous formic acid containing a deuterated internal standard of cotinine, extracted with a solid phase resin, and analyzed using LC-MS/MS via multiple reaction monitoring (MRM) to quantitate cotinine. Identification of samples were blinded to the LC-MS experimenter. Briefly, 15 µl of plasma was transferred to a 1.5 mL microcentrifuge tube followed by addition of dH 2 O (200 µL, 0.05% formic acid [FA]) containing cotinine-d3 (250 nM), followed by dilution with ammonium acetate (10 mM, 200 µL). The mixture was applied to a biotage WCX solid phase extraction column. The column was washed successively with ammonium acetate (500 µL at 50 mM), isopropyl alcohol (125 µL) and dichloromethane (500 µL). Analytes were eluted with a mixture of dH 2 O:isopropyl alcohol (85:15) +0.1% FA (125 µL) followed by LC-MS analysis via a Shimadzu Nexera XR UPLC coupled with a Shimadzu 8050 triple quadrupole mass spectrometer. The instrument was optimized for MRM transitions using analytical grade standards of cotinine and cotinine-d3, (Sigma Aldrich and Cambridge Isotopes). The total ion chromatograms (TIC) for each MRM event were integrated to determine the area under the curve (AUC). Quantitation was carried out using a calibration curve.

ELISA Measurement of plasma VEGF

The Plasma VEGF was measured using a commercial available ELISA kit from R&D System (Cat. No. MMV00). Plasma samples were first diluted 5 times with diluent buffer provided from the kit, then a 50 µL of the diluted samples or the serially diluted standards of VEGF were added to each well of a 96-well plate. The antibody incubation and washing procedures were performed following the manufacturer’s protocol. Plasma concentration of VEGF was quantified against the estamblmished standard curve.

Statistical Analyses

Data are presented as Mean ± Standard Error of the Mean (SEM). Data were analyzed using Student t-test or One-way ANOVA where appropriate.