Scientic Reports | (2021) 11:11808 |
Dierential eects of heat‑not‑burn
and conventional cigarettes
on coronary ow, myocardial
and vascular function
Ignatios Ikonomidis1*, Dimitrios Vlastos2, Gavriela Kostelli1, Kallirhoe Kourea1,
Konstantinos Katogiannis1, Maria Tsoumani3, John Parissis1, Ioanna Andreadou3 &
We compared the eects of Heat‑not‑Burn cigarette (HNBC) to those of tobacco cigarette (Tcig), on
myocardial, coronary and arterial function as well as on oxidative stress and platelet activation in
75 smokers. In the acute study, 50 smokers were randomised into smoking a single Tcig or a HNBC
and after 60 min were crossed‑over to the alternate smoking. For chronic phase, 50 smokers were
switched to HNBC and were compared with an external group of 25 Tcig smokers before and after
1 month. Exhaled carbon monoxide (CO), pulse wave velocity (PWV), malondialdehyde (MDA) and
thromboxane B2 (TxB2) were assessed in the acute and chronic study. Global longitudinal strain (GLS),
myocardial work index (GWI), wasted myocardial work (GWW), coronary ow reserve (CFR), total
arterial compliance (TAC) and ow‑mediated dilation (FMD) were assessed in the chronic study. Acute
HNBC smoking caused a smaller increase of PWV than Tcig (change 1.1 vs 0.54 m/s, p < 0.05) without
change in CO and biomarkers in contrast to Tcig. Compared to Tcig, switching to HNBC for 1‑month
improved CO, FMD, CFR, TAC, GLS, GWW, MDA, TxB2 (dierences 10.42 ppm, 4.3%, 0.98, 1.8 mL/
mmHg, 2.35%, 19.72 mmHg%, 0.38 nmol/L and 45 pg/mL respectively, p < 0.05). HNBCs exert a less
detrimental eect on vascular and cardiac function than tobacco cigarettes.
Trial registration Registered on https:// clini caltr ials. gov/ (NCT03452124, 02/03/2018).
Smoking constitutes a major modiable risk factor for cardiovascular disease 1. e pathophysiological mecha-
nisms underlying its deleterious eects include arterial elasticity 2 and myocardial deformation impairment 3,
endothelial dysfunction 4, oxidative stress accentuation 2, and platelet activation enhancement 5. Carotid-femoral
pulse wave velocity (cfPWV) measurement allows a reproducible, non-invasive quantication of arterial stiness
6 with diagnostic 7 and prognostic value 8. In addition, total arterial compliance (TAC) represents a pulsatile
component of le ventricular (LV) aerload and quanties the compliance of the entire arterial system, with
signicant physiological and prognostic implications 9. Flow mediated dilation (FMD) of the brachial artery 10
evaluates endothelial function and predicts cardiovascular mortality. Every 1% improvement in FMD has been
associated with 13% reduction of the risk for cardiovascular events 11. Coronary ow reserve (CFR) as assessed
by echocardiography is marker of coronary microcirculatory function with predictive value for adverse cardio-
vascular outcome 12. Importantly, CFR may predict mortality and risk for myocardial infarction (MI) in patients
with normal or near-normal coronary arteries and preserved regional and global le ventricular function both at
rest and during stress 13. In this respect, smoking has been demonstrated to reduce CFR by 21% in asymptomatic
smokers without coronary artery disease 14. Global longitudinal strain (GLS) computation by speckle tracking
echocardiography is a standardized modality of myocardial deformation assessment 15. Myocardial work index
(MW) is a novel index of the ventricular-arterial interaction which combines longitudinal myocardial defor-
mation with dynamic non-invasive LV pressure measurements creating pressure–strain loops. us, this index
minimizes pitfalls of myocardial strain examination that derive from its load-dependent nature 16. Regarding
oxidative stress burden, plasma malondialdehyde (MDA) 17 and protein carbonyls (PC) 18 concentration serve
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as widely used biomarkers, while plasma thromboxane B2 (TxB2) levels are associated with increased platelet
More than 5600 chemical components have been identied in traditional tobacco cigarettes, many of which
harmfully aect the cardiovascular system 20. In this respect, the development of potential reduced exposure
products (PREPs) has been suggested as a means to reduce the adverse eects associated with the use of tobacco
products 21. Although rst generation PREP use was, indeed, associated with reduced biomarkers of exposure
(BoE) levels, neither oxidative stress burden nor markers of inammation were improved 22. Heat-not-burn
cigarette (HNBC) has been developed as a new-generation non-combustible PREP 23. Early evidence suggests
that its use results in a signicantly reduced BoE levels in comparison with traditional combustible cigarettes
24–31; however, this has not been consistently associated with cardiovascular function benets. On the one hand,
evidence has arisen that HNBC acutely impairs oxidative stress equilibrium, platelet activation, endothelial
function, and peripheral blood pressure to a lesser extent than traditional smoking 32. On the other hand, in rat
models, exposure to HNBC aerosol was as detrimental to FMD as cigarette smoke 33. us, the eects of HNBC
use on cardiovascular function have not been fully dened.
e aim of this study was to investigate the eects of HNBC on endothelial function, arterial stiness,
myocardial deformation, oxidative stress, and platelet activation both in an acute context and aer 1month of
switching to HNBC smoking, in comparison with traditional tobacco cigarette.
Study population. e mean age of the 50 participants was 48 ± 5years, 53% were female, and reported
smoking 27 ± 9 cigarettes per day per individual with a smoking history of 38 ± 18 pack-years. Tobacco smokers,
used as controls, had a mean age of 46 ± 14years, 53% were female and reported smoking 26 ± 8 cigarettes per
day per individual with a smoking history of 37 ± 19 pack-years. All subjects had similar baseline characteristics
in terms of arterial stiness, myocardial deformation, oxidative stress, and platelet activation status (Table1).
During the chronic study, only 3 of the 50 (6%) participants reported simultaneous use of IQOS with tradi-
tional cigarettes (< 5 per day). e remaining 47 participants successfully refrained from traditional cigarette
smoking and used HNBC exclusively during the 1-month follow-up period (17 ± 6 heets per day per individual).
No side-eects were reported. In the control group, smokers used 28 ± 8 Tcig per day per individual during one
Acute study. Vascular function. During the acute study, PWV was increased aer HBNC and Tcig smok-
ing compared to baseline (Table2, p = 0.04 and p = 0.005 respectively). However, Tcig smoking resulted in a
greater increase of PWV than HNBC pung (mean change 1.11m/s; 95% CI 0.35–1.18, p = 0.005 vs 0.54m/s;
95% CI 0.05–1.04, p = 0.03 respectively, dierence = 0.57m/s; 95% CI 0.005–1.131, p = 0.04) (Table2, Fig.1A,
Supplementary Fig.1). Furthermore, compared to baseline, Tcig smoking caused an increase in brachial systolic
blood pressure and heart rate (p = 0.03 and p = 0.02, respectively) while HNBC pung showed no signicant
changes on the above indices (p = 0.14 and p = 0.77) (Supplementary Fig.2).
Oxidative stress and platelet activation. In the acute setting, Tcig caused a signicant MDA increase, in contrast
with the lack of increase of MDA levels aer HNBC pung (baseline: 1.34 ± 0.72 vs Tcig: 2.56 ± 0.85, p = 0.03,
vs HNBC: 1.28 ± 0.95nmol/L, p = 0.55) (Table2, Fig.1B, Supplementary Fig.3). us, MDA levels were found
lower aer HBNC than aer Tcig (p = 0.02). Additionally, the MDA increase caused by Tcig was signicantly
correlated with the respective increase in PWV (r = 0.825, p < 0.001).
PC levels were unchanged during the acute phase of the study (p for the overall model, p = 0.54, Table2).
Compared to baseline, acute Tcig smoking signicantly increased TxB2 concentration; TxB2 levels did
not change signicantly following HNBC pung (baseline: 378 ± 103 vs Tcig: 398 ± 105, p = 0.02, vs HNBC:
Table 1. Characteristics of the study cohort. SBP systolic blood pressure, DBP diastolic blood pressure, BMI
body mass index, BP blood pressure, CO exhaled carbon monoxide, CRP-hs C-reactive protein highly sensitive,
values are mean ± SD.
N = 50 Control
N = 25 p-value
Age (years) 48 ± 9 46 ± 11 0.7
Sex (female) 27 (53%) 14 (53%) 0.6
SBP (mmHg) 125 ± 15 121 ± 13 0.7
DBP (mmHg) 79 ± 10 76 ± 10 0.6
Heart rate (bpm) 66 ± 9 67 ± 8 0.8
BMI (kg/m2)27.2 ± 5.1 27.3 ± 5.2 0.9
CO (ppm) 14.9 ± 7.4 15.8 ± 4.9 0.7
Pack-years 38 ± 18 37 ± 19 0.3
LDL-cholesterol (mg/dl) 114 ± 22 118 ± 19 0.5
CRP-hs (mg/dl) 1.8 ± 0.4 1.9 ± 0.5 0.4
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362 ± 113pg/ml, p = 0.16). us, TXB2 levels were found lower aer HBNC than aer Tcig (p = 0.005) (Table2,
Fig.1C, Supplementary Fig.4).
CO exposure. In the acute phase, compared to baseline, exhaled CO levels were signicantly elevated aer
Tcig smoking but were not aected by HNBC pung (CO base: 14.9 ± 7.4 vs CO Tcig: 17.5 ± 7.8, p < 0.001, vs
CO HNBC: 14.2 ± 7.3ppm, p = 0.1) us, CO levels were found lower aer HBNC than aer Tcig (p < 0.001)
Chronic study. Vascular function. ere was a signicant interaction between changes of FMD, CFR,
TAC, central SBP and smoking status (HNBC or Tcig) at 1month (F = 5.6, p = 0.01; F = 7.1, p = 0.001; F = 6.8,
p = 0.001, and F = 5.4, p = 0.03, respectively).
FMD and CFR were both signicantly elevated within 1month of switching to HNBC compared to tobacco
smokers (dierence in FMD = 4.3%; 95% CI 1.23–7.51, p = 0.009; dierence in CFR = 0.98; 95% CI 0.23–1.80,
p = 0.02) (Table3, Figs.2A,B and 3, Supplementary Figs.5, 6).
Central SBP was significantly reduced in the HNBC group than in tobacco smoking group (differ-
ence = 10.4mmHg-1; 95% CI 3.05–17.88, p = 0.02). TAC was signicantly increased in the HNBC group than in
tobacco smoking group (dierence = 1.8mL/mmHg; 95% CI 0.3–3.5, p = 0.04). Compared to baseline, PWV, HR,
brachial SBP and DBP values were not reduced aer 1month of using HNBC (p > 0.05, Table3, Supplementary
Fig.7). Compared to baseline, no signicant changes were observed in all vascular markers within 1month in
the control group of tobacco smokers (p > 0.05, Table3).
Myocardial function. ere was a signicant interaction between changes of GLS, PWV/GLS, GWI, GWW,
GWE, and smoking status at 1month (HNBC or Tcig) (p < 0.05 for all comparisons).
GLS was improved in the HNBC compared to the control group at follow-up (dierence = 2.35%; 95% CI
0.23–4.48, p = 0.03) (Table3, Supplementary Fig.8).
PWV/GLS ratio, as a marker of ventricular-arterial interaction, was found to be improved within 1month
of switching to HNBC compared to conventional tobacco smoking (p = 0.03) (Table3).
Moreover, GWI and GWW were both reduced within one month of HNBC use compared to con-
ventional tobacco use (difference in GWI = 152mmHg%; 95% CI 81.74–224.05, p = 0.001; difference in
GWW = 19.72mmHg%; 95% CI 4.35–35.08 p = 0.014) (Table3, Fig.2C). e increase in TAC was signicantly
correlated with the decrease in GWI (r = 0.344, p = 0.03). Compared to baseline, all myocardial deformation
indices did not change signicantly in the control group of tobacco smokers at 1month (p > 0.05).
Oxidative stress and platelet activation. ere was a signicant interaction between changes of MDA, PC, TXB2
and smoking status (HNBC or Tcig) at 1month (F = 7, p = 0.01; F = 4.8, p = 0.04; and F = 6.8, p = 0.01 respectively).
MDA and PC concentration signicantly decreased in subjects switching to HNBC compared to tobacco
smokers (dierence MDA = 0.38nmol/l; 95% CI 0.10–0.66, p = 0.009; PC = 7.73nmol/mg protein; 95% CI
0.19–15.28, p = 0.04) (Table4, Supplementary Fig.9). Additionally, replacement of Tcig by HNBC caused a TxB2
reduction than tobacco smoking (dierence = 45pg/ml; 95% CI 5.28–86.31, p = 0.03) (Table2, Supplementary
Fig.10). None of the aforementioned parameters changed in the control group at 1month compared to baseline
(p > 0.05 Table4).
At follow-up, the measured decrease in plasma MDA levels was signicantly positively correlated with the
increase in FMD (r = 0.51, p = 0.03).
Table 2. Comparative acute eects of heat-not-burn cigarettes versus tobacco cigarette on arterial stiness,
oxidative stress, platelet activation, and exposure to CO in the acute study (n = 50). *p < 0.05 of the overall
model by ANOVA for the within subject eects of the tobacco products. † p < 0.05 for the comparison of
variables between baseline and aer use of tobacco products by post hoc analysis using Bonferroni correction.
PWV carotid-femoral pulse wave velocity, CO carbon monoxide, HNBC heat-not-burn cigarette, MDA
malondialdehyde, TCig tobacco cigarette, TxB2 thromboxane B2, values are mean±SD.
Baseline Sham HNBC TCig
PWV (m/s) 9.7 ± 1.3 9.9 ± 1.7 10.2 ± 1.7†10.8 ± 2.4*†
cSBP (mmHg) 121 ± 16 120 ± 15 119 ± 17.6 120 ± 17
Heart rate (bpm) 66 ± 9 66 ± 9 66 ± 8 70 ± 10*†
SBP (mmHg) 125 ± 15 125 ± 16 128 ± 19 130 ± 19*†
DBP (mmHg) 79 ± 10 78 ± 10 80 ± 11 82 ± 10
MDA (nmol/l) 1.34 ± 0.72 1.35 ± 0.83 1.28 ± 0.95 2.56 ± 0.85*†
PC (nmol/mg protein) 15.7 ± 5.8 14.5 ± 4.9 12.8 ± 5.2 13.9 ± 5.6
TxB2 (ng/ml) 378 ± 103 375 ± 105 362 ± 113 398 ± 105*†
CO (ppm) 14.9 ± 7.4 14.4 ± 3.8 14.1 ± 7.3 17.5 ± 7.8*†
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Figure1. Acute eects of heat-not-burn pung versus tobacco cigarette smoking. Comparison between the
acute eects of heat-not-burn versus tobacco cigarette smoking on (A) arterial stiness, (B) oxidative stress
burden, and (C) platelet activation status. HBNC showed a smaller increase of PWV than tobacco cigarette.
All biomarkers are impaired following Tcig smoking, in contrast with HNBC pung. PWV carotid-femoral
pulse wave velocity, HNBC heat-not-burn cigarette, MDA malondialdehyde, Tcig tobacco cigarette, TxB2
thromboxane B2, SE Standard Error.
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CO exposure. ere was a signicant interaction between changes of CO and smoking status (HNBC or
Tcig) at 1month (F = 8.5, p = 0.001). Compared to baseline, CO measurements were reduced aer switching to
HNBC (p < 0.001), while remaining unchanged in controls subjects (p = 0.2) (dierence in CO between groups:
10.42ppm; 95% CI 3.07–17.76, p = 0.007) (Table4).
In the acute crossover phase of this study, it was demonstrated that a single HNBC pung resulted in a smaller
increase of pulse wave velocity compared to tobacco cigarette smoking and was not associated with further
impairment of oxidative stress and platelet activation or increased exposure to CO, compared to baseline. Con-
versely, acute smoking of a tobacco cigarette had detrimental eects on the examined markers of vascular func-
tion, oxidative stress, and platelet activation. Furthermore, in the chronic phase of this study, switching from
Tcig to HNBC for 1month was associated with improvement in endothelial function, coronary ow reserve,
arterial compliance, and myocardial work, as well as with reduction of oxidative stress burden, platelet activation,
and exposure to CO. ese changes were not evident in the parallel control group of subjects who continued
smoking tobacco cigarettes for a month. Interestingly, the reduction in oxidative stress burden aer switching
to HNBC was associated with the respective improvement of endothelial function, while the increase in arterial
compliance was associated with the concomitant reduction of myocardial work.
Arterial elasticity is impaired in the context of both acute and chronic smoking, as assessed by carotid to
femoral PWV 2,34. e use of electronic cigarettes has shown less detrimental eects on arterial elasticity com-
pared to smoking tobacco cigarettes 2. Increased arterial stiness has been shown to be reversible, albeit aer a
considerable period of abstinence from smoking, spanning from 2 to 10years 2,34–37. To our knowledge, this is
Table 3. Arterial elasticity, myocardial deformation, endothelial function, and ventricular-arterial coupling
progression aer 1month of switching from tobacco cigarette smoking to heat-not-burn product pung
(n = 50) compared to tobacco cigarette smokers (n = 25). *p < 0.05 for interaction with group, derived from post
hoc analysis by ANOVA. HNBC heat-not-burn cigarette, TCig tobacco cigarette, CFR coronary ow reserve,
PWV carotid-femoral pulse wave velocity, FMD ow-mediated dilation, GCW constructive myocardial work,
GLS global longitudinal strain, GWE myocardial work eciency, GWI global myocardial work index, GWW
wasted myocardial work, values are mean±SD.
Group Baseline One month p-value
Body weight (kg) HNBC 76.9 ± 2.4 77.1 ± 1.9 0.7
Tcig 80.9 ± 3.4 80.5 ± 3.5 0.8
PWV (m/s) HNBC 9.7 ± 1.3 10.1 ± 1.5 0.3
Tcig 9.7 ± 1.6 10.2 ± 2.3 0.4
Central SBP (mmHg) HNBC 121 ± 16 112 ± 17* 0.02
Tcig 121 ± 16 123 ± 15 0.3
TAC (ml/mmHg) HNBC 19.1 ± 4.2 20.9 ± 5.2* 0.03
Tcig 17.6 ± 8.4 17.5 ± 7.6 0.2
Heart rate (bpm) HNBC 66 ± 9 65 ± 8 0.4
Tcig 67 ± 8 68 ± 8 0.8
SBP (mmHg) HNBC 125 ± 15 126 ± 16 0.9
Tcig 121 ± 13 122 ± 13 0.9
DBP (mmHg) HNBC 79 ± 10 78 ± 9 0.7
Tcig 76 ± 10 76 ± 10 0.9
GLS (%) HNBC − 19.9 ± 2.3 − 20.9 ± 2.5* 0.002
Tcig − 19.7 ± 1.3 − 20 ± 0.7 0.3
FMD (%) HNBC 7.8 ± 4.3 12.1 ± 4.2* 0.01
Tcig 7.9 ± 3.9 8.3 ± 3.5 0.6
CFR HNBC 2.4 ± 0.6 3.5 ± 0.8* < 0.001
Tcig 2.5 ± 0.2 2.6 ± 0.2 0.2
PWV/GLS HNBC − 0.5 ± 0.1 − 0.48 ± 0.08* < 0.001
Tcig − 0.49 ± 0.1 − 0.51 ± 0.1 0.4
GWI (mmHg%) HNBC 1949 ± 315 1828 ± 320* 0.01
Tcig 1954 ± 290 1986 ± 290 0.1
GCW (mmHg%) HNBC 2214 ± 339 2156 ± 385 0.1
Tcig 2202 ± 379 2180 ± 356 0.3
GWW (mmHg%) HNBC 80 ± 55 65 ± 37* < 0.001
Tcig 74 ± 33 78 ± 42 0.09
GWE (%) HNBC 95.8 ± 2.3 96.4 ± 1.8 0.1
Tcig 95.5 ± 2 95 ± 2 0.5
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Figure2. Progression of coronary ow reserve, ow mediated dilation, and myocardial work within 1month
of follow-up. Replacement of Tcig smoking with HNBC pung for 1-month results in coronary and peripheral
endothelial function improvement, along with wasted myocardial work reduction. CFR coronary ow reserve,
FMD ow-mediated dilation, GWW global wasted work, SE Standard Error.
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the rst study to demonstrate that acute HNBC pung is associated with a smaller increase of PWV compared
to tobacco smoking. HNBC pung increases nicotine blood levels. Nicotine activates the sympathetic nerv-
ous system by acting via splanchnic nerves to the adrenal medulla and stimulates the release of epinephrine 38.
Acute elevation of nicotine levels has been linked to endothelial dysfunction 39 leading to reduced nitric oxide
release and thus contributes in addition to increased sympathetic activity to increased vascular tone. ese
mechanisms likely underlie the increase of PWV aer acute HNBC pung. Conversely, the deleterious acute
eects of smoking a tobacco cigarette on arterial stiness were in line with the respective oxidative stress burden
accentuation, underlining their interplay in our acute study 2,40. TAC constitutes an index of arterial compliance
of the entire arterial tree and entails physiologic and prognostic signicance 9. To our knowledge, this is the rst
study to highlight that replacement of Tcig with a HNBC results in improved TAC and central SBP, suggesting
a less detrimental eect of this novel smoking product on le ventricular aerload. e discrepancy between
Figure3. Coronary ow of le anterior descending artery at rest (le panel) and aer adenosine infusion (right
panel) for calculation of coronary ow reserve. Coronary ow of le anterior descending artery at rest (le
panel) and aer adenosine infusion (right panel) for calculation of coronary ow reserve at baseline (A) and
aer switching to HNBC for one month (B). e coronary ow reserve increased from 2.5 to 3.1 aer 1month
of switching from tobacco cigarette to HNBC.
Table 4. Progression of exposure to CO, oxidative stress burden, and platelet activation aer 1month of
switching from tobacco cigarette smoking to heat-not-burn product pung. *p < 0.05 for interaction with
group, derived from post hoc analysis by ANOVA. CO carbon monoxide, MDA malondialdehyde, PC protein
carbonyls, TxB2 thromboxane B2values are mean ± SD.
Group Baseline Follow-up p-value
CO (ppm) HNBC 14.9 ± 7.4 6.7 ± 6.4* < 0.001
Tcig 15.8 ± 4.9 17.4 ± 4.8 0.3
MDA (nmol/l) HNBC 1.34 ± 0.72 1.11 ± 0.95* 0.01
Control 1.43 ± 0.9 1.45 ± 0.83 0.3
PC (nmol/mg protein) HNBC 15.7 ± 5.8 9 ± 4.4* 0.04
Control 16.1 ± 8.8 17.2 ± 3.3 0.3
TxB2 (pg/ml) HNBC 378 ± 103 323 ± 137* 0.01
Control 417 ± 24 407 ± 16 0.4
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lack of improvement of PWV and increase of TAC in our study may be explained by the dierent vascular beds
these indices represent. On the one hand, PWV assesses the aortoiliac segment of the arterial tree 41; on the
other hand, TAC incorporates the eects of the large, central, elastic arteries as well as the small, peripheral,
muscular arteries 9. us, improved arterial compliance of the peripheral arterial system could improve TAC,
without aecting PWV.
Augmentation of oxidative stress is a key pathophysiological mediator of cardiovascular harm caused by acute
and chronic smoking 2,34. MDA is the product of cellular membrane phospholipids peroxidation and thus a valid
and reproducible marker of lipid peroxidation 17. PCs represent an irreversible form of protein modication that
are formed early during oxidative stress conditions, are relatively stable in contrast to lipid peroxidation products,
are not a result of one specic oxidant, and thus they are considered a sensitive and reproducible marker of total
protein oxidation 18. In our previous studies, both MDA and PCs were increased in smokers and their levels were
reduced either aer nicotine replacement treatment or varenicline use in medically-assisted smoking cessation
programs 34 or aer switching to electronic cigarette smoking. 2,42. us, plasma concentrations of MDA 17 and
PC 18 are valid markers of oxidative stress burden related to tobacco smoking. Recently, evidence has arisen that
acute HNBC pung is less detrimental to oxidative stress equilibrium compared with Tcig smoking, as assessed
by Nox2-derived peptide, vitamin E, and HBA levels 32. Similarly, our study demonstrated that MDA levels
were not elevated acutely aer using an HNBC, in contrast with Tcig use. Furthermore, in our chronic study, we
demonstrated reduced MDA and PC levels within 1month of replacing Tcig with HNBC.
Increased platelet activation plays a signicant role in promoting atherosclerosis and thrombotic complica-
tions, and is enhanced by Tcig smoking 5. A recent study suggests that HNBC invokes platelet activation to a
lesser degree than Tcig, as evident by comparatively lesser increase in soluble CD40 ligand and soluble P-selectin,
as markers of platelet activation 32. Plasma TxB2 is a valid marker of platelet activation 19. In agreement with the
aforementioned study 32, in our acute study, we did not detect signicant changes in plasma TxB2 acutely aer
HNBC pung compared to baseline. Conversely, we observed the increasedTXB2 levels aer Tcig smoking.
Further expanding on this nding, plasma TxB2 levels were signicantly reduced aer 1month of HNBC use
while no changes were observed in the parallel group of tobacco smokers in the same period.
Exhaled CO levels constitute a widely used marker of acute smoking status 43. In addition, inhaled CO has
been associated with endothelial damage and pathological capillary permeability 34. A recent study investigating
the BoE footprint of HNBC highlighted that exhaled CO levels within 5days of switching from Tcig to HNBC
are equal to those measured aer total abstinence from tobacco products 24. Similarly, in our study, acute HNBC
use did not increase exhaled CO levels, while CO levels were signicantly reduced at the 1-month follow-up
examination, approaching though the levels previously reported for passive-smokers 24,43. is nding suggests
that some of the HNBC users were substantially exposed to passive smoking in their family and/or working
environment despite adherence to HNBC use during the chronic study.
Endothelial dysfunction is a prominent end-point of the pathophysiological cascades activated by Tcig smok-
ing 4. In this regard, FMD and CFR evaluation reecting endothelial and coronary microcirculatory function
respectively, possess unequivocal diagnostic and prognostic value 10,12. Early investigation of the interaction
between HNBC and endothelial function has not produced consistent results. On the one hand, acute HNBC
pung has been shown to impair FMD to a lesser extent than Tcig 32. On the other hand, an animal model study
recognised HNBC and Tcig as equally detrimental on endothelial function 33. Our study found a signicant
increase in FMD aer switching to HBNC use for 1month, in accordance with recently published evidence of
FMD improvement aer replacing TCig smoking with electronic-cigarette vaping 44. Interestingly, FMD improve-
ment was positively correlated with MDA reduction at 1month, suggesting the contribution of reduced oxidative
stress to improvement of endothelial function. Furthermore, the current study is the rst to our knowledge that
has demonstrated a signicant improvement in CFR within 1month of switching to HNBC. Studies strongly
support the independent prognostic value of a reduced CFR for adverse cardiovascular outcome 13.
Novel markers of ventricular arterial-interaction incorporate measurements of aerload in the investigation of
myocardial function using speckle tracking echocardiography; the shortcomings of ejection fraction assessment
and the diagnostic pitfalls arising from the load-dependent nature of global longitudinal strain estimation are
overcome. us, a non-invasive and more clinically feasible method of myocardial work estimation is provided by
constructing longitudinal myocardial strain-pressure curves using speckle tracking imaging 16. In our study myo-
cardial eciency was improved aer switching to HNBC for one month, as shown by the respective improvement
of the GLS 11 and the reduction of the myocardial work index (GWI), global wasted myocardial work (GWW)
and the increase of the global myocardial work eciency (GWE) as assessed by the respective strain-pressure
curves. Interestingly, improved GWI was associated with increasing TAC values us, the reduction of GWI aer
switching to HNBC may be attributed to the reduction of LV aerload as indicated by the concomitant reduction
of central SBP and increase of TAC at one month. Furthermore, the observed improvement of the ratio PWV/
GLS aer one month of HNBC use also suggests an improvement in ventricular-arterial interaction 11. us, in
our study we observed that the improvement of vascular function and oxidative stress, likely on the grounds of
reduced CO exposure, evolved in parallel with the improvement of myocardial work eciency aer switching
from the traditional tobacco smoking to HNBC for one month. Alternatively, a potential reduction in the blood
nicotine levels aer switching to HNBC from Tcig smoking may have also accounted for the improvement of
cardiac and vascular markers in our chronic study.
In an elegant study by Frati etal. 45, the use of unsupervised machine learning techniques identied dierent
clusters of individuals within a larger study cohort with a similar within each cluster but dierent among clusters
responses to HNBC or e-cigarette smoking for various vascular and biochemical markers. e identication of
clusters of subjects with favorable or unfavorable responses to novel smoking products for surrogate markers of
cardiovascular function may facilitate the denition of the potential individual specic features of cardiovascular
safety of these smoking products.
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Design. Our study was an acute independent, randomised, cross-over trial followed by a chronic case control
According to our initial Study design, (ClinicalTrials.gov, NCT03452124) the primary outcome for the acute
study was the eect of HNBC in comparison to Tcig on PWV, while primary outcome for the chronic study was
the eect of use of HNBC for one month in comparison to Tcig smoking for the same period on LV deforma-
tion. e study was approved by the Attikon University Hospital scientic ethics committee (Approval number:
2874/06-12-17), funded by the Hellenic Society of Lipidology of Atherosclerosis and Vascular Disease. e
study was conducted according to the Declaration of Helsinki and written informed consent was provided by
the participants. Our study was registered on https:// clini caltr ials. gov/, NCT03452124, 02/03/2018.
Study population. Out of 95 screened smokers attending the Attikon University Hospital smoking ces-
sation unit, 50 current smokers (age: 48 ± 5years, 53% female, 27 ± 9 cigarettes/day, 29 ± 9 pack-years) and 25
controls smokers (age: 46 ± 14years, 53% female, 26 ± 8 cigarettes/day, 30 ± 8 pack-years) with no intention to
quit smoking were included in our study (Fig.4). Smoking status was veried by way of self-reported smoking
burden per day and exhaled carbon monoxide (eCO) concentration measurement [parts per million (ppm),
Bedfont Scientic, Maidstone, Kent UK] (exclusion criteria: < 5 cigarettes per day end exhaled CO < 10ppm).
Exclusion criteria included history of cardiovascular disease, hepatic or renal failure, active neoplasia, alcohol
abuse, psychiatric illness, pregnancy, breastfeeding, cigar smoking, or the presence of any additional risk fac-
tor for cardiovascular disease (dyslipidaemia: total cholesterol > 200mg/dl or the use of cholesterol-lowering
agents; hypertension: blood pressure > 140/90mmHg or use of anti-hypertensive drugs; diabetes mellitus: fast-
ing plasma glucose > 125mg/dl or use of antidiabetic drugs).
Study protocol. Our study included an acute and a chronic phase. e acute phase of the study entailed
an initial sham smoking session of inhaling on a non-lighted cigarette for 7min, simulating the mean duration
Figure4. Flow chart of the study population. HNBC heat-not-burn cigarette, TCig tobacco cigarette.
Scientic Reports | (2021) 11:11808 |
of a traditional cigarette smoking. In the acute phase, the participants were thereaer randomised into either
a tobacco cigarette (Tcig) smoking session, using a single mainstream Tcig [Marlboro Red, Papastratos-Philip
Morris International (PMI), Athens, Greece], or a single HEETS stick (PMI, amber avour) pung session
(HNBC) using a commercially available HNBC (IQOS, PMI). Randomization was performed by an attending
research nurse using a table of random numbers as reproduced from the online randomization soware http://
www. graph pad. com/ quick calcs/ index. cfm, as previously published 45. Proper use of IQOS device was demon-
strated by a qualied research nurse, who also served as a supervisor during the HNBC pung session. Aer
a washout period of 60min, the subjects were crossed over to the alternative session (Tcig or HNBC, Fig.5).
Vascular studies and blood sampling were completed during 20-min at baseline before initiation of smoking and
within the wash-out period. e decision about the washout period duration was based on evidence that the
acute eects of a smoking on arterial function resolve within 60min 41.
For the chronic phase, all participants of the acute phase, were instructed to replace Tcig smoking with HNBC
pung for 1month and were compared with an external group of 25 Tcig smokers, with no intention to quit
smoking, (2:1 ratio) before and aer 1month.
Exhaled CO, arterial elasticity and blood sampling for oxidative stress and platelet activation, which were
assessed at baseline, aer each smoking session of the acute phase and at the chronic phase. Endothelial function,
coronary ow reserve and myocardial deformation were assessed at baseline and aer 1month, in the chronic
study. Arterial elasticity, myocardial, endothelial function examinations were executed by a single, blinded-
to-treatment and to values of measured biomarkers, operator. In the acute study the time elapsed between
smoking (Tcig or HNBC) and assessment of vascular markers was approximately 10min. For the chronic study,
participants were instructed to abstain from smoking (Tcig or HNBC) in the morning before the vascular and
echocardiography assessment. HNBC adherence was assessed by asking the participants to answer a question-
naire regarding the daily use of HNBC sticks as well as by measurement of the exhaled CO in each participant
during a clinic visit at days 15 and 30 of the chronic study. e HNBC sticks were provided to the participants by
the investigators at baseline and at day 15 during a clinic visit and aer the participant had returned the empty
boxes of the used HNBC sticks.
Endothelial function. Brachial artery FMD was ultrasonically assessed in line with published methodol-
ogy recommendations 17 and expressed as the percent increase of the baseline arterial diameter. Intra-observer
variability of the brachial artery diameter was 0.1 ± 0.12mm.
Coronary ow reserve. CFR was measured by transthoracic Doppler echocardiography, analysing colour-
guided pulse-wave Doppler signals derived from long axis apical projections. e maximal velocity end veloc-
ity–time integral in the distal le anterior descending artery were recorded at rest and following intravenous
adenosine administration, according to published methodology 46 and quantied as the ratio of hyperaemic to
resting maximal diastolic velocity. Coronary ow reserve assessment of le anterior descending artery was fea-
sible in all subjects of the study, though in 3 out of 75 participants, we had to use intravenous contrast (Sonovue,
Bracco, Italy) to improve Doppler imaging of the coronary ow.
Arterial stiness. Carotid-to-femoral PWV was estimated according to a previous published methodology
(Complior, Alam Medical, Vincennes, France) 40, by computing the ratio of the distance between the carotid and
femoral pulse palpation site to the pulse wave transit time (m/s). e Cointra-observer variability was 6%, in
Figure5. Acute phase protocol. Following an initial sham smoking session, subjects were randomised to either
a heat-not-burn or traditional cigarette smoking session; following a washout period of 60min, they were
crossed-over to the alternative smoking session. Each session was followed by a vascular stiness examination
and blood-sampling for oxidative stress and platelet activation assessment. Exam examination, HNBC heat-not-
burn cigarette, TCig tobacco cigarette.
Scientic Reports | (2021) 11:11808 |
accordance with existing evidence in similar studies 34. Total arterial compliance (TAC) was evaluated utilising
the stroke volume (SV) to pulse pressure (PP) ratio (SV/PP), based on the two-element Windkessel model 9; SV
measurements were derived from two-dimensional echocardiography.
In the acute and chronic study assessment of vascular markers (FMD, PWV and PP) was performed in a
Myocardial deformation. Myocardial deformation was assessed by way of 2-dimensional strain measure-
ment, with speckle-tracking analysis by dedicated soware (Echopac 203, GE Horten Norway). LV apical 2-,
3-, and 4-chamber views at ≥ 50 frames per second frame rate were acquired and the global longitudinal strain
(GLS) was calculated from the respective apical views, applying previously published methodology 17,34. PWV/
GLS ratio (− m/s%) was calculated as a marker of ventricular arterial interaction 36.
Myocardial work. Myocardial work (MW) was estimated by combining echo-derived le ventricular (LV)
strain with brachial blood pressure to construct LV strain-pressure curves non-invasively, following recently
published methodology 16. Brachial cu systolic pressure measurements provide the peak systolic LV pressure
value, which is combined with the input of valvular timing events that dene isovolumetric and ejection phases,
allowing the construction of an LV pressure curve. is is combined with LV strain data into a pressure-strain
loop (PSL), the area within which represents MW. Global MW index (GWI) is dened as the work within the LV
PSL from mitral valve closure to mitral valve opening, while constructive MW (GCW) is the component of MW
that contributes to LV ejection. On the contrary, wasted MW (GWW) is the work wasted as myocyte lengthen-
ing during systole, which does not contribute to LV ejection. MW eciency (GWE) is dened as the ratio of
GCW to the sum of GCW and GWW [GWE = GCW/(GCW + GWW)].
Oxidative stress. A commercially available spectrophotometry kit (Oxford Biomedical Research, Roches-
ter Hills, Mich, colorimetric assay for lipid peroxidation; measurement range 1–20nmol/l) was used to deter-
mine plasma MDA levels. Plasma PC levels were measured by spectrophotometrical assessment of 2,4-dinitro-
phenylhydrazine PC derivatives, as previously published 40, and results were expressed as nmol/mg protein. For
MDA and PC, the intra-assay variability was 3.39% and 4.52%, respectively and the inter-assay variability was
4.75% and 5.93%, respectively.
In the chronic study two-dimensional echocardiography preceded the coronary ow reserve assessment,
aer completion of vascular studies.
Platelet activation. A commercially available ELISA kit was used to measure blood levels of romboxane
B2 (romboxane B2 EIA Kit Cayman Ann Arbor MI USA) with an assay range 1.6–1000pg/ml.
Statistical analysis. We used the soware STATA v.11 and SPSS v.22 in order to procced to data analysis.
We used Shapiro–Wilk test to examine if values had normal distribution, and Levene test for evaluating data
homoscedasticity, as it was previously published 34. Non-parametric variables were transformed into rank for
analysis. Two tailed tests with p < 0.05 were used during data acquisition. ANOVA (general linear model, SPSS
22, SPSS Inc, Chicago, Ill) for repeated measurements was applied for analysis of the examined indices of vas-
cular, myocardial, and endothelial function, as well as biochemical markers (at baseline, aer each smoking
session, and at the 1-month follow-up examination) with the parameter of time used as a within-subject factor.
e Greenhouse–Geisser correction was used when the sphericity assumption, as assessed by Mauchly’s test,
was not met. We used parametric (Pearson r) and non-parametric (Spearman rho) correlation coecients to
examine cross-sectional associations. Multiple comparisons of baseline values of study markers to values aer
sham, Tcig or HNBC in the acute study were performed with Bonferroni correction. A p-value of < 0.05 was
considered statistically signicant. e ordering of intervention (begin with HBNC versus begin with Tcig) and
the brachial systolic blood pressure (SBP) were included as covariates in the model and the respective p-value for
their interaction with the overall model was calculated. T-test was performed to compare the absolute or percent
changes of the parameters evaluated in the chronic phase of the study. Inter- and intra-observer variabilities (%)
of vascular, myocardial function, and biochemical markers were calculated as the SD of the dierences between
the rst and second measurements, and expressed as a percentage of the average value in 30 healthy volunteers.
Power analysis. For the acute study, we planned a study of a continuous response variable from matched
pairs of study subjects (HNBC and Tcig users). In a pilot study of ten smokers randomised to HNBC or Tcig and
then crossed-over to the alternate smoking (Tcig or HNBC) the dierence in the PWV of matched pairs aer
HNBC and Tcig smoking was normally distributed with standard deviation 1.3 and the true dierence in the
mean PWV of the matched pairs aer HBNC and Tcig smoking was 0.54m/s. us, we needed to study 47 pairs
of subjects to be able to reject the null hypothesis that this response dierence in PWV aer HNBC and Tcig is
zero with probability (power) 0.8. e Type I error probability associated with this test of this null hypothesis
For the chronic study, we planned a study of a continuous response variable from independent control (Tcig
smokers) and experimental subjects (HNBC users) with 0.5 control(s) per experimental subject. In a pilot study
of 10 HNBC and 5 Tcig smokers the response for the GLS within each subject group was normally distributed
with a standard deviation of 3.1% and the true dierence of GLS means between the HNBC and Tcig smok-
ers was 2.3%. us, we needed to study at least 50 experimental and 25 control subjects to be able to reject the
null hypothesis that the population means of the HNBC and Tcig smoking groups are equal with a probability
Scientic Reports | (2021) 11:11808 |
(power) of 0.8. e Type I error probability associated with this test for this null hypothesis is 0.05, as previously
Ethical approval. It was approved by the Attikon University Hospital scientic ethics committee (Approval
number: 2874/06-12-17), registered on https:// clini caltr ials. gov/ (NCT03452124, 25/02/2018). e study was
conducted according to the Declaration of Helsinki and written informed consent was provided by the partici-
Our study was a single centre study. Its design does not permit to explore denite causative associations among
changes in oxidative stress, vascular and myocardial function markers. We should also acknowledge as a study
limitation the lack of measurement of cotinine blood or urine levels to appraise for the actual number of products
smoked by our study participants and to clarify whether dierences in circulating nicotine levels in addition to
dierences in CO production aer HNBC and Tcig smoking mediate the observed dierences of cardiovascular
markers in our study. Long term follow-up is needed to assess whether the observed improvement in vascular
and myocardial function, aer switching to HBNC, is associated with reduced cardiovascular events. Cluster
analysis using unsupervised machine learning techniques to identify subgroups of participants with favourable
or unfavourable responses to HNBC use for the measured vascular, cardiac and biochemical markers was not
Acute HNBC pung showed a less detriment eect on arterial elasticity compared to Tcig and did not cause a
further augmentation of oxidative stress burden, platelet activation, and exposure to CO compared to baseline,
in contrast to acute smoking of tobacco cigarette. Switching from Tcig to HNBC for one month resulted in
improved endothelial function, oxidative stress burden as well as in reduction of platelet activity and exposure
to CO, while caused an improvement in coronary ow reserve and myocardial work eciency compared with
e datasets generated during and/or analysed during the current study are available from the corresponding
author on reasonable request. Anonymised data are only available upon request from the authors.
Received: 21 April 2020; Accepted: 17 May 2021
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I.I. contributed to the study design, data analysis and writing of the manuscript. D.V. contributed to data analysis
and writing of the manuscript. G.K. contributed to patient recruitment, and randomization. Ka.K. contrib-
uted to patient recruitment and study design. Ko.K. performed echocardiographic studies and respective data
analysis. M.T. and I.A. performed the bio markers measurement and analysis. D.A. and J.P. critically reviewed
Scientic Reports | (2021) 11:11808 |
the manuscript and contributed to the scientic analysis of the results. All authors discussed the results and
contributed to the nal manuscript.
e study was funded by Special Account for research Grants (11645) of the National and Kapodistrian University
of Athens without any direct or indirect support from Tobacco Company.
e authors declare no competing interests.
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 021- 91245-9.
Correspondence and requests for materials should be addressed to I.I.
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