Acute and Long-term Effects of E-Cigarette Aerosol Inhalation on Biomarkers of Endothelial Function and Vascular Reactivity
Phase: EARLY_PHASE1
Conditions: Endothelial Dysfunction, Biochemical Markers
Sponsor: University of Pennsylvania

MRI and Biological Markers of Acute E-Cigarette Exposure in Smokers and Vapers 
NCT 03479203 
September 15, 2017 

To determine the acute effects of e-cig aerosol inhalation we will quantify surrogate markers of EDF at the micro- 
and macro-vascular level before and after a nicotine-free e-cig aerosol in healthy subjects without history of 
cardiovascular disease.   
 
In order to isolate and assess the acute effects of inhaled aerosol on surrogate MRI markers of EDF (endothelial 
dysfunction), nicotine-naïve (i.e. non-smokers) will undergo an e-cig aerosol inhalation challenge using a com-
mercially available, disposable, non-flavored nicotine-free e-cig (ECO series, epuffer.com). 
 
All MR procedures will be performed on a 3T Siemens TIM Trio imager (Siemens Medical Solutions) 
using standard RF coils (8-channel extremity coil for cuff paradigm studies at the location of the femoro-popliteal 
artery and vein, and a combination of two body matrix and 12-channel head coils for PWV (pulse wave velocity) 
of the aorta and neurovascular reactivity in the superior sagittal sinus (SSS) in conjunction with the custom-
designed pulse sequences. Subjects will be asked to fast for eight hours, have abstained from taking vasoactive 
medications (including over-the-counter medications) as well as vigorous physical activity. Each visit will com-
prise two blood draws and two qMRI (quantitative magnetic resonance imaging) examinations, one before and 
one after an e-cig challenge to assess the changes in the functional and mechanical metrics described in the 
previous section. The e-cig challenge will consist of 16 two-second-long puffs (equivalent to smoking one con-
ventional cigarette). Smoking will be directly supervised to prevent superficial smoking or hyperventilation by the 
study subjects. The challenge will last approximately 20 minutes. A second blood draw will be taken six hours 
after the e-cig challenge. From the blood samples collected, inflammatory biomarkers will be assessed. 
 
Blood will be drawn from the antecubital vein of the subject’s left or right arm. At least 2 mL of plasma 
will be isolated and kept frozen after centrifuging blood for 15 min.  
 
The qMRI protocol will consist of three parts (cuff paradigm, PWV and breath-hold index). The patient 
set-up for PWV and breath-hold index (BHI) will be combined, i.e. the patient will enter the scanner head-first, 
supine, with head and body matrix coils in place. Vascular reactivity in the femoral artery and vein will be quan-
tified first. After the cuff-induced hyperemia portion of the protocol the patient will be prepared for the second 
part designed to measure aortic PWV and BHI.  
 
 
 
 
 
 
 
Data and Statistical Analysis 
The fractional changes in functional and mechanical qMRI parameters between pre- and post-nicotine challenge 
will be determined. For each parameter paired two-tailed t-tests will be performed with p<0.05 indicating statis-
tical significance. The data will allow us to determine which of the qMRI metrics (peripheral vascular reactivity, 
central arterial stiffness or neurovascular reactivity), are most sensitive to e-cig aerosol inhalation. We will also 
further conduct multivariate regression analysis on the within-subject changes controlling for other subject-spe-
cific covariates.   
 
To quantify the changes in the levels of inflammatory biomarkers in serum collected from study participants 
before and after e-cigarette aerosol exposure and compare these metrics with qMRI-derived biomarkers. 
 
Blood drawn from the antecubital vein by a phlebotomist from study participants in will be collected into tubes 
containing clot activator. Serum will be separated within 30 minutes by centrifugation and stored at −70°C prior 
to analysis. Inflammatory biomarkers will be detected via enzyme-linked immunosorbent assay (ELISA) tech-
niques from blood draws #1 and 2. MCP-1 and MIP-1 concentrations will be measured using commercially avail-
able ELISA kits (R&D Systems, Abingdon, UK); sICAM, sRAGE, IFN-g and TNF-α with RayBiotech kits 

(RayBiotech, Inc., Norcross, GA 30092); HMGB1 will be assayed by ELISA kits supplied by IBL International 
(IBL GmbH).   
Analyses 
Descriptive statistics will initially be performed to determine the characteristics of the various distributions and to 
identify outliers by displaying the data in the form of box and whisker plots and by computing means and standard 
deviations. Acute changes from baseline (blood draws pre- and post e-cig aerosol challenge) will be evaluated 
by paired t-tests for each of the variables listed above.  To compare the qMRI metrics with inflammatory indices, 
Spearman correlations will be performed.  We expect moderate inter-modality correlations (R2 ~ 0.5) for individ-
ual qMRI measures. We then use linear regression to create a classification model using the eight qMRI indices 
jointly. We expect R2 > 80%, substantially greater than each individual parameter alone, indicating that inhaled 
e-cig aerosol promotes systemic EDF.