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Effect of smoking on endothelium-independent vasodilatation
Atherosclerosis 240 (2015) 330e332
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Short communication
Effect of smoking on endothelium-independent vasodilatation Gaetano A. Lanza*, Francesco R. Spera, Angelo Villano, Giulio Russo, Antonino Di Franco, Priscilla Lamendola, Filippo Crea Cattolica del Sacro Cuore, Roma, Italy Institute of Cardiology e Department of Cardiovascular Sciences, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2015 Received in revised form 25 March 2015 Accepted 28 March 2015 Available online 1 April 2015
Background: Smoking induces an impairment of endothelium-dependent vasodilatation. In this study we assessed whether smoking also causes an impairment of endothelium-independent vasodilatation. Methods: We studied 2 groups of young healthy subjects: 1) 12 medical students (24.5 ± 0.9 years; 6 male) without cardiovascular risk factors (CVRFs), except smoking (10 cigarettes/day); 2) 12 matched controls (24.5 ± 1.1 years; 6 male) without any CVRF. Nitrate-mediated dilatation (NMD) of the brachial artery was assessed in response to the random administration of 4 different doses (10, 20, 30 and 40 mg) of sublingual nitroglycerin (NTG). Flow-mediated dilatation (FMD) was also assessed. Results: The increasing doses of NTG determined a progressive increase of NMD in both groups, but the doseeresponse curve was significantly lower in smokers compared to controls (p < 0.001). FMD was also lower in smokers, compared to controls (6.12 þ 0.6 vs. 8.06 þ 0.9%, p < 0.001). Conclusions: Our data show that smoking induces an early impairment of endothelium-independent arterial dilatation. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nitrate-mediated dilatation Endothelial function Smoking
Smoking has been shown to cause an impairment of endothelium-dependent arterial dilatation [1e8]. Whether smoking also induces an impairment of endothelium-independent vasodilator function remains instead questionable. Some studies, indeed, found a reduced arterial dilator response to short-acting nitrates in smokers [1,2], whereas a normal nitrate mediated dilatation (NMD) was found in others [3e8]. In previous studies, however, a single high dose of nitrates was often used to assess NMD [1e8], which may not be ideal to reveal NMD abnormalities. In smokers, indeed, maximal arterial dilatation might not be impaired and might be achieved with high doses, whereas an impaired NMD might be revealed by the administration of low doses of nitrates. Aim of this study was to investigate whether smoking is associated with early abnormalities in endothelium-independent vasodilatation in young healthy subjects as assessed by the arterial response to low doses of sublingual nitroglycerin (NTG).
1. Methods 1.1. Study population We studied 12 young healthy medical students of our University who smoked 10 or more cigarettes per day, but had no other cardiovascular risk factors (CVRFs), as established by clinical history, medical visit and standard laboratory tests. A group of 12 age- and sex-matched healthy medical students without any CVRF, including smoking, were studied as controls. All individuals had normal blood pressure (<130/80 mmHg), and glucose (<110 mg/dL) and cholesterol (<200 mg/dL) plasma levels; furthermore, they had no family history of cardiovascular disease, were not involved in physical activities and were not taking any drugs. Women were studied starting from the third day after the last menstrual cycle. Subjects gave written informed consensus to participate in the study. The protocol was approved by our Institutional board review.
1.2. Nitrate-mediated dilatation Cattolica del Sacro * Corresponding author. Istituto di Cardiologia, Universita Cuore, Largo A. Gemelli, 8, 00168 Roma, Italy. E-mail address: [email protected] (G.A. Lanza). http://dx.doi.org/10.1016/j.atherosclerosis.2015.03.041 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
NMD was assessed by the same expert operator in the early afternoon, with subjects in a fasting state for 8 h; subjects were invited to refrain from heavy exercise during the period of the
G.A. Lanza et al. / Atherosclerosis 240 (2015) 330e332
study, and smokers also to not smoke in the 2 h preceding the tests. Vasodilatation of the brachial artery in response to 4 different doses of sublingual nitroglycerin (NTG, 10, 20, 30 and 40 mg) was assessed in 4 different consecutive days with a previously described method [9,10]. Briefly, before each test the subject remains supine for at least 10 min in a quiet and heated room (temperature of 22e24 C). The right brachial artery is displayed with a high-resolution ultrasound system (Acuson Sequoia, Siemens®) equipped with a software and a vascular high-frequency probe (10 MHz) to view two-dimensional and color-Doppler images. The operator selects the arterial segment to be analyzed, and the system software automatically identifies the inner edges of the vessel and traces the artery walls using their different echogenicity respect to the lumen of the vessel. Then the brachial artery diameter is automatically measured every second during the whole duration of the test [11] without any intervention of the operator and with the probe maintained in a fixed position by a mechanical support. After acquisition of baseline images of the right brachial artery for 1 min, a dose of sublingual NTG is administered and the arterial diameter is monitored continuously until achievement of maximal dilatation and for other 5 min thereafter. The basal diameter is calculated as the average of measurements collected during the first minute and NMD is automatically calculated as the maximum percentage change of the brachial artery diameter after NTG administration compared to baseline. The doses were obtained by progressive dilution of NTG vials (5 mg) with the aid of a graded pipette and were administered in a random sequence and in a double-blind way (subject and operator). In preliminary tests, intra-operator variability of NMD measurements (with NTG 30 mg) was found to be low (8.8%). 1.3. Flow-mediated dilatation On the fifth day, endothelium-dependent dilatation was assessed by measuring flow mediated dilatation (FMD) of the right brachial artery, using the same methods applied for NMD assessment [9e11]. Shortly, after obtaining basal measurement of the arterial diameter, a sphygmomanometer cuff, positioned 1 cm under the antecubital fossa, was inflated to 250 mmHg to cause forearm ischemia, and released after 5 min to elicit forearm reactive hyperemia. FMD was calculated as the maximum percent change of the brachial artery diameter during hyperemia compared to the basal diameter. Intra-operator variability of FMD measurements at our Center was found to be low (10.5%).
331
Italia Inc, Firenze, Italy) was used for analyses. 2. Results There were no significant differences in basal characteristics between the 2 groups (Table 1). Smokers smoked an average of 13.7 ± 2.2 cigarettes per day (range 10e17). Basal diameter of the brachial artery was 3.31 ± 0.54 mm in smokers and 3.24 ± 0.63 mm in controls (p ¼ 0.68), and remained unchanged at the measurements performed before each test; similarly, basal heart rate and blood pressure did not show significant changes in the 2 groups throughout the different dilator tests (data not shown). NMD in response to the various doses of NTG is summarized in Table 1, and illustrated in Fig. 1. The increasing doses of NTG determined a progressive significant (p < 0.001) increasing arterial dilatation in both groups, but the dilator response was significantly lower in smokers compared to controls (p < 0.001). Between-group comparisons for each NTG dose showed that only the difference at 30 mg achieved statistical significance (p < 0.01 after Bonferroni correction and adjustment for possible confounding variables). FMD was significantly reduced in smokers compared to controls (6.12 þ 0.6% vs. 8.06 þ 0.9%, p < 0.001). Sex did not significantly affect the differences in NMD and FMD between smokers and controls (p ¼ 0.53 and p ¼ 0.413 for groupegender interaction, respectively). 3. Discussion The novel finding of this study is that, by using low NTG doses, it is possible to demonstrate that smoking is associated with an early impairment of endothelium-independent arterial dilatation in young healthy subjects without any other CVRFs. While an impairment of endothelium-dependent vasodilatation has consistently been found in smokers, controversial results were reported about endothelium-independent vasodilatation [1e8]. Celermajer et al. [1] found a lower NMD in smokers, as compared with controls, but the same group failed to demonstrate a significant independent relation between smoking and NMD in another study [12]. A limitation of most previous studies was that a single high dose of NTG (usually 300e400 mg) was given to assess NMD, which might have made difficult to identify an abnormal response in most cases. A high dose of nitrates, indeed, in most cases might achieve maximal arterial dilatation even when a reduced NMD is present, which might instead be unmasked by a lower dilator effect in response to low NTG doses as compared to healthy controls.
1.4. Statistical analysis Assuming a maximal NMD of 15% (SD, 2.5%) in controls, 8 patients per group were needed to have 80% power to detect as significant (at 0.05 level) a 25% difference in maximal dilatation between groups; 12 subjects per group were enrolled to take multiple comparisons into account. Continuous and discrete variables were compared by unpaired t-test or Fisher exact test, respectively. A generalized linear model with a repeated measure design was applied to compare the NTG doseeresponse curves. Direct comparisons between NMD values for each NTG dose were done by t-test, with results being corrected for multiple (four) comparisons by Bonferroni rule. Results were always adjusted for brachial artery diameter and systolic blood pressure. The influence of sex on the differences between the two groups in FMD and NMD was assessed by including sex as an independent variable in the respective statistical models. A p value <0.05 was considered as statistically significant. The SPSS 20.0 statistical software (SPSS
Table 1 Main clinical characteristics of subjects enrolled in the study and nitrate-mediated results. Smokers (n ¼ 12) Age (years) 24.5 ± 0.9 Sex (M/F) 6/6 BMI (Kg/m2) 22.6 ± 2.3 Cigarettes/day 13.7 ± 2.2 Systolic BP 122.5 ± 7.2 Diastolic BP 79.6 ± 7.8 Heart rate 70.2 ± 9.2 Nitrate-mediated dilatation NTG 10 mg (%) 5.0 ± 1.5 NTG 20 mg (%) 9.5 ± 2.8 NTG 30 mg (%) 12.3 ± 2.8 NTG 40 mg (%) 13.7 ± 1.9
Controls (n ¼ 12)
p
24.5 ± 1.1 6/6 21.9 ± 2.9 e 122.9 ± 7.8 79.8 ± 8.2 74.6 ± 6.5
1.0 1.0 0.47 e 0.89 0.94 0.19
6.0 ± 1.0 11.5 ± 2.9 16.4 ± 3.6 16.3 ± 3.3
>0.05a >0.05a <0.01a >0.05a
BMI ¼ body mass index; BP ¼ blood pressure. NTG ¼ nitroglycerin. a With Bonferroni correction for multiple comparisons.
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G.A. Lanza et al. / Atherosclerosis 240 (2015) 330e332
impaired endothelial NO production. It should be acknowledged that a limitation of our study resides in the small number of subjects enrolled; thus, the ideal dose of NTG to assess NMD needs to be better established in larger studies.
Disclosures None.
References
Fig. 1. Dilatation of the brachial artery following the administration of increasing doses of sublingual nitroglycerin (nitrate-mediated dilatation, NMD). *p < 0.01 vs. controls after Bonferroni correction and adjustment for differences in basal artery diameter, heart rate and systolic blood pressure.
Maruhashi et al. [13] very recently used 75 mg of NTG to assess NMD in 436 subjects, showing that several CVRFs, including smoking, were associated with reduced NMD. Smoking, however, failed to be associated with NMD in multivariable analyses. Our data show that an accurate assessment of NMD can more reliably be obtained using much lower doses of NTG. Specifically, in our study a dose of 30 mg seemed the most appropriate to identify a different response to NTG in young smokers, suggesting that such a dose should probably be preferred when assessing NMD in clinical studies. It should be noticed that the significant dilator effect of such low doses of NTG does not seem to have been observed in previous studies [13,14]. Some methodological differences between these studies and ours can, at least in part, account for these discrepancies. In previous studies NTG was given as sublingual tablets or spray, whereas a saline solution was used in our study, with possible differences in drug availability; methods to assess NMD in several previous studies were potentially more operatordependent, whereas a fully automated system was used in our study; finally, mixed populations of subjects and patients were usually included in previous studies, whereas a very selected group of young subjects, without any other CVRF, was enrolled in this study. The reduced NMD in smokers suggests an impairment of the response of vascular smooth muscle cells to the direct effect of NTG, possibly due to abnormalities in intracellular pathways [15,16], although a reduced release of NO from NTG and/or an increased inactivation of NTG-derived NO [17,18] cannot be excluded. Finally, it is worth noting that the reduced NMD in smokers may complicate the interpretation of the impaired endotheliumdependent vasodilatation. Indeed, a reduced FMD might be caused, at least in part, by a reduced response of smooth muscle cells to a normal endothelial release of NO rather than to an
[1] D.S. Celermajer, K.E. Sorensen, D. Georgakopoulos, et al., Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-dependent dilation in healthy young adults, Circulation 88 (1993) 2149e2155. €chinger, J. Minners, Long-term cigarette smoking impairs [2] A.M. Zeiher, V. Scha endothelium-dependent coronary arterial vasodilator function, Circulation 92 (1995) 1094e1100. [3] G.E. McVeigh, L. Lemay, D. Morgan, et al., Effects of long-term cigarette smoking on endothelium-dependent responses in humans, Am. J. Cardiol. 78 (1996) 668e672. [4] J. Lekakis, C. Papamichael, C. Vemmos, et al., Effect of acute cigarette smoking on endothelium-dependent brachial artery dilatation in healthy individuals, Am. J. Cardiol. 79 (1997) 529e531. [5] K. Ozaki, T. Hori, T. Ishibashi, et al., Effects of chronic cigarette smoking on endothelial function in young men, J. Cardiol. 56 (2010) 307e313. [6] S. Thorne, M.J. Mullen, P. Clarkson, A.E. Donald, J.E. Deanfield, Early endothelial dysfunction in adults at risk from atherosclerosis: different responses to Larginine, J. Am. Coll. Cardiol. 32 (1998) 110e116. [7] P. Poredos, M. Orehek, E. Tratnik, Smoking is associated with dose-related increase of intima-media thickness and endothelial dysfunction, Angiology 50 (1999) 201e208. [8] A.M. Esen, I. Barutcu, M. Acar, et al., Effect of smoking on endothelial function and wall thickness of brachial artery, Circ. J. 68 (2004) 1123e1126. [9] L. Ghiadoni, F. Faita, M. Salvetti, et al., Assessment of flow-mediated dilation reproducibility: a nationwide multicenter study, J. Hypertens. 30 (2012) 1399e1405. [10] R. Nerla, P. Tarzia, A. Sestito, et al., Effect of bariatric surgery on peripheral flow-mediated dilation and coronary microvascular function, Nutr. Metab. Cardiovasc Dis. 22 (2012) 626e634. [11] V. Gemignani, F. Faita, L. Ghiadoni, et al., A system for real-time measurement of the brachial artery diameter in B-mode ultrasound images, IEEE Trans. Med. Imaging 26 (2007) 393e404. [12] M.R. Adams, J. Robinson, R. McCredie, et al., Smooth muscle dysfunction occurs independently of impaired endothelium-dependent dilation in adults at risk of atherosclerosis, J. Am. Coll. Cardiol. 32 (1998) 123e127. [13] T. Maruhashi, J. Soga, N. Fujimura, et al., Nitroglycerine-induced vasodilation for assessment of vascular function: a comparison with flow-mediated vasodilation, Arterioscler. Thromb. Vasc. Biol. 33 (2013) 1401e1408. [14] O.T. Raitakiri, J.P. Seale, D.S. Celermajer, Impaired vascular resposnes to nitroglycerin in subjects with coronary atherosclerosis, Am. J. Cardiol. 87 (2001) 217e219. [15] T.J. Verbeuren, F.H. Jordaens, C.E. Van Hove, et al., Release and vascular activity of endothelium-derived relaxing factor in atherosclerotic rabbit aorta, Eur. J. Pharmacol. 191 (1990) 173e184. [16] K.E. Sorensen, I.B. Kristensen, D.S. Celermajer, Atherosclerosis in the human brachial artery, J. Am. Coll. Cardiol. 29 (1997) 318e322. [17] L. Raij, E.G. DeMaster, E.A. Jaimes, Cigarette smoke-induced endothelium dysfunction: role of superoxide anion, J. Hypertens. 19 (2001) 891e897. [18] J.D. Morrow, B. Frei, A.W. Longmire, et al., Increase in circulating products of lipid peroxidation (F2-isoprostanes) in smokers. Smoking as a cause of oxidative damage, N. Engl. J. Med. 332 (1995) 1198e1203.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Short communication
Effect of smoking on endothelium-independent vasodilatation Gaetano A. Lanza*, Francesco R. Spera, Angelo Villano, Giulio Russo, Antonino Di Franco, Priscilla Lamendola, Filippo Crea Cattolica del Sacro Cuore, Roma, Italy Institute of Cardiology e Department of Cardiovascular Sciences, Universita
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 February 2015 Received in revised form 25 March 2015 Accepted 28 March 2015 Available online 1 April 2015
Background: Smoking induces an impairment of endothelium-dependent vasodilatation. In this study we assessed whether smoking also causes an impairment of endothelium-independent vasodilatation. Methods: We studied 2 groups of young healthy subjects: 1) 12 medical students (24.5 ± 0.9 years; 6 male) without cardiovascular risk factors (CVRFs), except smoking (10 cigarettes/day); 2) 12 matched controls (24.5 ± 1.1 years; 6 male) without any CVRF. Nitrate-mediated dilatation (NMD) of the brachial artery was assessed in response to the random administration of 4 different doses (10, 20, 30 and 40 mg) of sublingual nitroglycerin (NTG). Flow-mediated dilatation (FMD) was also assessed. Results: The increasing doses of NTG determined a progressive increase of NMD in both groups, but the doseeresponse curve was significantly lower in smokers compared to controls (p < 0.001). FMD was also lower in smokers, compared to controls (6.12 þ 0.6 vs. 8.06 þ 0.9%, p < 0.001). Conclusions: Our data show that smoking induces an early impairment of endothelium-independent arterial dilatation. © 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Nitrate-mediated dilatation Endothelial function Smoking
Smoking has been shown to cause an impairment of endothelium-dependent arterial dilatation [1e8]. Whether smoking also induces an impairment of endothelium-independent vasodilator function remains instead questionable. Some studies, indeed, found a reduced arterial dilator response to short-acting nitrates in smokers [1,2], whereas a normal nitrate mediated dilatation (NMD) was found in others [3e8]. In previous studies, however, a single high dose of nitrates was often used to assess NMD [1e8], which may not be ideal to reveal NMD abnormalities. In smokers, indeed, maximal arterial dilatation might not be impaired and might be achieved with high doses, whereas an impaired NMD might be revealed by the administration of low doses of nitrates. Aim of this study was to investigate whether smoking is associated with early abnormalities in endothelium-independent vasodilatation in young healthy subjects as assessed by the arterial response to low doses of sublingual nitroglycerin (NTG).
1. Methods 1.1. Study population We studied 12 young healthy medical students of our University who smoked 10 or more cigarettes per day, but had no other cardiovascular risk factors (CVRFs), as established by clinical history, medical visit and standard laboratory tests. A group of 12 age- and sex-matched healthy medical students without any CVRF, including smoking, were studied as controls. All individuals had normal blood pressure (<130/80 mmHg), and glucose (<110 mg/dL) and cholesterol (<200 mg/dL) plasma levels; furthermore, they had no family history of cardiovascular disease, were not involved in physical activities and were not taking any drugs. Women were studied starting from the third day after the last menstrual cycle. Subjects gave written informed consensus to participate in the study. The protocol was approved by our Institutional board review.
1.2. Nitrate-mediated dilatation Cattolica del Sacro * Corresponding author. Istituto di Cardiologia, Universita Cuore, Largo A. Gemelli, 8, 00168 Roma, Italy. E-mail address: [email protected] (G.A. Lanza). http://dx.doi.org/10.1016/j.atherosclerosis.2015.03.041 0021-9150/© 2015 Elsevier Ireland Ltd. All rights reserved.
NMD was assessed by the same expert operator in the early afternoon, with subjects in a fasting state for 8 h; subjects were invited to refrain from heavy exercise during the period of the
G.A. Lanza et al. / Atherosclerosis 240 (2015) 330e332
study, and smokers also to not smoke in the 2 h preceding the tests. Vasodilatation of the brachial artery in response to 4 different doses of sublingual nitroglycerin (NTG, 10, 20, 30 and 40 mg) was assessed in 4 different consecutive days with a previously described method [9,10]. Briefly, before each test the subject remains supine for at least 10 min in a quiet and heated room (temperature of 22e24 C). The right brachial artery is displayed with a high-resolution ultrasound system (Acuson Sequoia, Siemens®) equipped with a software and a vascular high-frequency probe (10 MHz) to view two-dimensional and color-Doppler images. The operator selects the arterial segment to be analyzed, and the system software automatically identifies the inner edges of the vessel and traces the artery walls using their different echogenicity respect to the lumen of the vessel. Then the brachial artery diameter is automatically measured every second during the whole duration of the test [11] without any intervention of the operator and with the probe maintained in a fixed position by a mechanical support. After acquisition of baseline images of the right brachial artery for 1 min, a dose of sublingual NTG is administered and the arterial diameter is monitored continuously until achievement of maximal dilatation and for other 5 min thereafter. The basal diameter is calculated as the average of measurements collected during the first minute and NMD is automatically calculated as the maximum percentage change of the brachial artery diameter after NTG administration compared to baseline. The doses were obtained by progressive dilution of NTG vials (5 mg) with the aid of a graded pipette and were administered in a random sequence and in a double-blind way (subject and operator). In preliminary tests, intra-operator variability of NMD measurements (with NTG 30 mg) was found to be low (8.8%). 1.3. Flow-mediated dilatation On the fifth day, endothelium-dependent dilatation was assessed by measuring flow mediated dilatation (FMD) of the right brachial artery, using the same methods applied for NMD assessment [9e11]. Shortly, after obtaining basal measurement of the arterial diameter, a sphygmomanometer cuff, positioned 1 cm under the antecubital fossa, was inflated to 250 mmHg to cause forearm ischemia, and released after 5 min to elicit forearm reactive hyperemia. FMD was calculated as the maximum percent change of the brachial artery diameter during hyperemia compared to the basal diameter. Intra-operator variability of FMD measurements at our Center was found to be low (10.5%).
331
Italia Inc, Firenze, Italy) was used for analyses. 2. Results There were no significant differences in basal characteristics between the 2 groups (Table 1). Smokers smoked an average of 13.7 ± 2.2 cigarettes per day (range 10e17). Basal diameter of the brachial artery was 3.31 ± 0.54 mm in smokers and 3.24 ± 0.63 mm in controls (p ¼ 0.68), and remained unchanged at the measurements performed before each test; similarly, basal heart rate and blood pressure did not show significant changes in the 2 groups throughout the different dilator tests (data not shown). NMD in response to the various doses of NTG is summarized in Table 1, and illustrated in Fig. 1. The increasing doses of NTG determined a progressive significant (p < 0.001) increasing arterial dilatation in both groups, but the dilator response was significantly lower in smokers compared to controls (p < 0.001). Between-group comparisons for each NTG dose showed that only the difference at 30 mg achieved statistical significance (p < 0.01 after Bonferroni correction and adjustment for possible confounding variables). FMD was significantly reduced in smokers compared to controls (6.12 þ 0.6% vs. 8.06 þ 0.9%, p < 0.001). Sex did not significantly affect the differences in NMD and FMD between smokers and controls (p ¼ 0.53 and p ¼ 0.413 for groupegender interaction, respectively). 3. Discussion The novel finding of this study is that, by using low NTG doses, it is possible to demonstrate that smoking is associated with an early impairment of endothelium-independent arterial dilatation in young healthy subjects without any other CVRFs. While an impairment of endothelium-dependent vasodilatation has consistently been found in smokers, controversial results were reported about endothelium-independent vasodilatation [1e8]. Celermajer et al. [1] found a lower NMD in smokers, as compared with controls, but the same group failed to demonstrate a significant independent relation between smoking and NMD in another study [12]. A limitation of most previous studies was that a single high dose of NTG (usually 300e400 mg) was given to assess NMD, which might have made difficult to identify an abnormal response in most cases. A high dose of nitrates, indeed, in most cases might achieve maximal arterial dilatation even when a reduced NMD is present, which might instead be unmasked by a lower dilator effect in response to low NTG doses as compared to healthy controls.
1.4. Statistical analysis Assuming a maximal NMD of 15% (SD, 2.5%) in controls, 8 patients per group were needed to have 80% power to detect as significant (at 0.05 level) a 25% difference in maximal dilatation between groups; 12 subjects per group were enrolled to take multiple comparisons into account. Continuous and discrete variables were compared by unpaired t-test or Fisher exact test, respectively. A generalized linear model with a repeated measure design was applied to compare the NTG doseeresponse curves. Direct comparisons between NMD values for each NTG dose were done by t-test, with results being corrected for multiple (four) comparisons by Bonferroni rule. Results were always adjusted for brachial artery diameter and systolic blood pressure. The influence of sex on the differences between the two groups in FMD and NMD was assessed by including sex as an independent variable in the respective statistical models. A p value <0.05 was considered as statistically significant. The SPSS 20.0 statistical software (SPSS
Table 1 Main clinical characteristics of subjects enrolled in the study and nitrate-mediated results. Smokers (n ¼ 12) Age (years) 24.5 ± 0.9 Sex (M/F) 6/6 BMI (Kg/m2) 22.6 ± 2.3 Cigarettes/day 13.7 ± 2.2 Systolic BP 122.5 ± 7.2 Diastolic BP 79.6 ± 7.8 Heart rate 70.2 ± 9.2 Nitrate-mediated dilatation NTG 10 mg (%) 5.0 ± 1.5 NTG 20 mg (%) 9.5 ± 2.8 NTG 30 mg (%) 12.3 ± 2.8 NTG 40 mg (%) 13.7 ± 1.9
Controls (n ¼ 12)
p
24.5 ± 1.1 6/6 21.9 ± 2.9 e 122.9 ± 7.8 79.8 ± 8.2 74.6 ± 6.5
1.0 1.0 0.47 e 0.89 0.94 0.19
6.0 ± 1.0 11.5 ± 2.9 16.4 ± 3.6 16.3 ± 3.3
>0.05a >0.05a <0.01a >0.05a
BMI ¼ body mass index; BP ¼ blood pressure. NTG ¼ nitroglycerin. a With Bonferroni correction for multiple comparisons.
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G.A. Lanza et al. / Atherosclerosis 240 (2015) 330e332
impaired endothelial NO production. It should be acknowledged that a limitation of our study resides in the small number of subjects enrolled; thus, the ideal dose of NTG to assess NMD needs to be better established in larger studies.
Disclosures None.
References
Fig. 1. Dilatation of the brachial artery following the administration of increasing doses of sublingual nitroglycerin (nitrate-mediated dilatation, NMD). *p < 0.01 vs. controls after Bonferroni correction and adjustment for differences in basal artery diameter, heart rate and systolic blood pressure.
Maruhashi et al. [13] very recently used 75 mg of NTG to assess NMD in 436 subjects, showing that several CVRFs, including smoking, were associated with reduced NMD. Smoking, however, failed to be associated with NMD in multivariable analyses. Our data show that an accurate assessment of NMD can more reliably be obtained using much lower doses of NTG. Specifically, in our study a dose of 30 mg seemed the most appropriate to identify a different response to NTG in young smokers, suggesting that such a dose should probably be preferred when assessing NMD in clinical studies. It should be noticed that the significant dilator effect of such low doses of NTG does not seem to have been observed in previous studies [13,14]. Some methodological differences between these studies and ours can, at least in part, account for these discrepancies. In previous studies NTG was given as sublingual tablets or spray, whereas a saline solution was used in our study, with possible differences in drug availability; methods to assess NMD in several previous studies were potentially more operatordependent, whereas a fully automated system was used in our study; finally, mixed populations of subjects and patients were usually included in previous studies, whereas a very selected group of young subjects, without any other CVRF, was enrolled in this study. The reduced NMD in smokers suggests an impairment of the response of vascular smooth muscle cells to the direct effect of NTG, possibly due to abnormalities in intracellular pathways [15,16], although a reduced release of NO from NTG and/or an increased inactivation of NTG-derived NO [17,18] cannot be excluded. Finally, it is worth noting that the reduced NMD in smokers may complicate the interpretation of the impaired endotheliumdependent vasodilatation. Indeed, a reduced FMD might be caused, at least in part, by a reduced response of smooth muscle cells to a normal endothelial release of NO rather than to an
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