Endothelial dysfunction is the initial step in the. Nitroglycerine-Induced Vasodilation for Assessment of Vascular Function

Transcription

1 Nitroglycerine-Induced Vasodilation for Assessment of Vascular Function A Comparison With Flow-Mediated Vasodilation Tatsuya Maruhashi, Junko Soga, Noritaka Fujimura, Naomi Idei, Shinsuke Mikami, Yumiko Iwamoto, Masato Kajikawa, Takeshi Matsumoto, Takayuki Hidaka, Yasuki Kihara, Kazuaki Chayama, Kensuke Noma, Ayumu Nakashima, Chikara Goto, Yukihito Higashi Objective Nitroglycerine-induced vasodilation has been used as a control test for flow-mediated vasodilation (FMD) to differentiate endothelium-dependent from endothelium-independent response when evaluating endothelial function in humans. Recently, nitroglycerine-induced vasodilation has also been reported to be impaired in patients with atherosclerosis. The purpose of this study was to determine the relationships between nitroglycerine-induced vasodilation and cardiovascular risk factors. Approach and Results We measured nitroglycerine-induced vasodilation and FMD in 436 subjects who underwent health examinations (mean age, 53±19 years; age range, years), including patients with cardiovascular diseases. There was a significant relationship between nitroglycerine-induced vasodilation and FMD (r=0.42; P<0.001). Univariate regression analysis revealed that nitroglycerine-induced vasodilation correlated with age (r= 0.34; P<0.001), systolic blood pressure (r= 0.32; P<0.001), diastolic blood pressure (r= 0.24; P<0.001), heart rate (r= 0.21; P<0.001), glucose (r= 0.23; P<0.001), and smoking pack-year (r= 0.12; P=0.01), as well as Framingham risk score (r= 0.30; P<0.001). Nitroglycerine-induced vasodilation was significantly smaller in patients with cardiovascular disease than in both subjects with and without cardiovascular risk factors (10.5±5.6% versus 13.7±5.4% and 15.3±4.3%; P<0.001, respectively), whereas there was no significant difference in nitroglycerine-induced vasodilation between subjects with and without cardiovascular risk factors. Multivariate analysis revealed that male sex, body mass index, hypertension, diabetes mellitus, baseline brachial artery diameter, and FMD were independent predictors of nitroglycerine-induced vasodilation. Conclusions These findings suggest that nitroglycerine-induced vasodilation may be a marker of the grade of atherosclerosis. FMD should be interpreted as an index of vascular function reflecting both endothelium-dependent vasodilation and endothelium-independent vasodilation in subjects with impaired nitroglycerine-induced vasodilation. (Arterioscler Thromb Vasc Biol. 2013;33: ) Key Words: atherosclerosis cardiovascular risk factors flow-mediated vasodilation nitroglycerine-induced vasodilation Endothelial dysfunction is the initial step in the pathogenesis of atherosclerosis, leading to cardiovascular complications. 1 Measurement of flow-mediated vasodilation (FMD) in the brachial artery, an index of endotheliumdependent vasodilation, has been widely used as a method for assessing endothelial function in humans. 2 9 Although the precise mechanism by which vasodilation occurs during reactive hyperemia in FMD measurement has not been fully elucidated, nitric oxide (NO) has been proposed as a principal mediator of FMD. 10,11 NO, produced as a result of an increase of endothelial NO synthase activity induced by shear stress, diffuses into the tunica media, leading to relaxation of smooth muscle cells and subsequent vasodilation. The assessment of endothelial function by FMD, therefore, presupposes a normal structural condition. Nitroglycerineinduced vasodilation, an index of endothelium-independent vasodilation, assessed by sublingual administration of nitroglycerine, has been used as a control test for FMD measurement to differentiate endothelium-dependent from endothelium-independent vasodilation because both endogenous NO and administered nitroglycerine act on vascular smooth muscle cells. Received on: December 3, 2012; final version accepted on: February 27, From the Department of Cardiovascular Medicine (T. Maruhashi, J.S., N.F., N.I., S.M., Y.I., M.K., T. Matsumoto, T.H., Y.K.), Department of Medicine and Molecular Science (K.C.), Hiroshima University Graduate School of Biomedical Sciences, Hiroshima, Japan; Department of Cardiovascular Regeneration and Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan (K.N., Y.H.); Division of Regeneration and Medicine, Hiroshima University Hospital, Hiroshima, Japan (K.N., A.N., Y.H.); and Department of General Rehabilitation, Hirohsima International University, Hiroshima, Japan (C.G.). The online-only Data Supplement is available with this article at Correspondence to Yukihito Higashi, MD, PhD, FAHA, Department of Cardiovascular Regeneration and Medicine, Research Institute for Radiation Biology and Medicine, Hiroshima University, Kasumi, Minami-ku, Hiroshima , Japan. yhigashi@hiroshima-u.ac.jp 2013 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 1402 Arterioscler Thromb Vasc Biol June 2013 Recent studies have demonstrated that not only FMD but also nitroglycerine-induced vasodilation is impaired in subjects with cardiovascular risk factors and coronary heart disease Impaired endothelium-independent vasodilation is thought to be associated with structural vascular alterations and alterations in smooth muscle cells as a result of atherosclerosis. Although the relationships between nitroglycerineinduced vasodilation and cardiovascular risk factors, as well as potential determinants of nitroglycerine-induced vasodilation, have been investigated in several studies, differences between nitroglycerine-induced vasodilation and FMD in association with cardiovascular risk factors and the severity of atherosclerosis remain unclear. In addition, although impaired nitroglycerine-induced vasodilation of coronary arteries has been demonstrated to be associated with a significantly higher incidence of cardiovascular events, there is little information on whether nitroglycerine-induced vasodilation of the brachial artery can serve as a marker of the progression of atherosclerosis in a general population. 15 Measurement of nitroglycerine-induced vasodilation of the brachial artery is a simple and noninvasive procedure compared with measurement of FMD or nitroglycerine-induced vasodilation of coronary arteries. We, therefore, measured nitroglycerine-induced vasodilation and FMD of the brachial artery in the same subjects for a direct comparison of these 2 vascular response tests in the relationships with conventional cardiovascular risk factors and severity of atherosclerosis in a general population, including healthy subjects and patients with cardiovascular diseases (CVDs). We also investigated whether measurement of nitroglycerine-induced vasodilation in combination with measurement of FMD or Framingham risk score improves diagnostic accuracy for patients who have cardiovascular risk factors without established CVD or patients with CVD. Materials and Methods Materials and Methods are available in the online-only Supplement. Results Baseline Clinical Characteristics The baseline clinical characteristics are summarized in Table 1. Of the 436 subjects, 307 (70.4%) were men and 129 (29.6%) were women. Two hundred fifty-two (58.6%) had hypertension, 176 (40.8%) had dyslipidemia, 99 (23.0%) had diabetes mellitus, and 270 (63.2%) were smokers. The mean value of nitroglycerine-induced vasodilation was 13.0±5.6% (median, 12.5%; interquartile range, %; range, %) and that of FMD was 4.35±2.81% (median, 4.15%; interquartile range, %; range, 5.0 to 14.7%). There were no significant differences in nitroglycerine-induced vasodilation and FMD between men and women (13.3±5.7% versus 12.3±5.2% and 4.39±2.80% versus 4.26±2.82%; P=0.10 and P=0.65, respectively). Relationships Between Nitroglycerine-Induced Vasodilation and Cardiovascular Risk Factors There was a significant inverse correlation between nitroglycerine-induced vasodilation and Framingham risk score (r= 0.30; P<0.001; Figure 1A). Univariate regression analysis revealed that nitroglycerine-induced vasodilation significantly correlated with age (r= 0.34; P<0.001), systolic blood pressure (r= 0.32; P<0.001), diastolic blood pressure (r= 0.24; P<0.001), heart rate (r= 0.21; P<0.001), glucose (r= 0.23; P<0.001), and smoking pack-year (r= 0.12; P=0.01; Table I in the online-only Data Supplement). In addition, there was a significant correlation between nitroglycerine-induced vasodilation and FMD (r=0.42; P<0.001; Figure IV in the onlineonly Data Supplement). Nitroglycerine-induced vasodilation significantly correlated with FMD both in men (r=0.44; P<0.001) and in women (r=0.37; P<0.001). As for vascular parameters, brachial intima-media thickness and brachial artery diameter significantly correlated with nitroglycerineinduced vasodilation (r= 0.25 and r= 0.37; P<0.001, respectively). When the same analysis was performed in 257 of the 436 subjects who had no vasoactive medications, similar relationships were observed between nitroglycerine-induced vasodilation and risk factors. Nitroglycerine-induced vasodilation significantly correlated with age (r= 0.31; P<0.001), body mass index (r= 0.22; P<0.001), systolic blood pressure (r= 0.30; P<0.001), diastolic blood pressure (r= 0.26; P<0.001), heart rate (r= 0.14; P=0.03), glucose (r= 0.22; P=0.001), and smoking pack-year (r= 0.19; P=0.003), as well as Framingham risk score (r= 0.38; P<0.001). There was a significant correlation between nitroglycerine-induced vasodilation and FMD (r=0.38; P<0.001). Brachial intimamedia thickness and brachial artery diameter significantly correlated with nitroglycerine-induced vasodilation (r= 0.30 and r= 0.38; P<0.001, respectively). Nitroglycerine-induced vasodilation did not correlate with total cholesterol (r= 0.11; P=0.11), triglycerides (r= 0.13; P=0.06), high-density lipoprotein-cholesterol (r= 0.001; P=0.99), and low-density lipoprotein-cholesterol (r= 0.06; P=0.38). We next categorized subjects into 3 tertiles based on nitroglycerine-induced vasodilation (Table II in the online-only Data Supplement). There were significant decreases in the prevalence of hypertension, dyslipidemia, diabetes mellitus, coronary heart disease, and peripheral artery disease with increase in nitroglycerine-induced vasodilation (Figure 2A and 2B). Multivariate analysis revealed that sex, body mass index, hypertension, diabetes mellitus, brachial artery diameter, and FMD were independent predictors of nitroglycerineinduced vasodilation (Table 2). Relationships Between FMD and Cardiovascular Risk Factors There was a significant inverse correlation between FMD and Framingham risk score (r= 0.34; P<0.001; Figure 1B). Univariate regression analysis revealed that FMD correlated with age (r= 0.38; P<0.001), body mass index (r= 0.13; P=0.006), systolic blood pressure (r= 0.33; P<0.001), diastolic blood pressure (r= 0.35; P<0.001), heart rate (r= 0.11; P=0.02), total cholesterol (r= 0.11; P=0.046), triglycerides (r= 0.12; P=0.02), glucose (r= 0.21; P<0.001), and smoking pack-year (r= 0.21; P<0.001; Table III in the online-only Data Supplement). Brachial intima-media

3 Maruhashi et al Nitroglycerine-Induced Vasodilation 1403 Table 1. Clinical Characteristics of the Subjects Variables Total (n=436) thickness and brachial artery diameter significantly correlated with FMD (r= 0.30 and r= 0.39; P<0.001, respectively). We next categorized subjects into 3 tertiles based on FMD (Table IV in the online-only Data Supplement). There were significant decreases in the prevalence of hypertension, diabetes mellitus, and peripheral artery disease (Figure 2C and 2D). Multivariate analysis revealed that age, hypertension, brachial artery diameter, and nitroglycerine-induced vasodilation were independent predictors of FMD (Table 2). Relationships Between Nitroglycerine-Induced Vasodilation, FMD, and Grade of Atherosclerosis The clinical characteristics in subjects with no cardiovascular risk factors (no-risk group), subjects with at least 1 cardiovascular risk factor, including hypertension, dyslipidemia, diabetes mellitus, and smoking, but without established CVD (at-risk group), and subjects with CVDs (CVD group) are summarized in Table V in the online-only Data Supplement. Both nitroglycerine-induced vasodilation and FMD showed a graded decrease according to the severity of atherosclerosis (Table V in the online-only Data Supplement). Nitroglycerine-induced vasodilation was significantly smaller in the CVD group than in the no-risk group and the at-risk group (no-risk group, 15.3±4.3%; at-risk group, 13.7±5.4%; CVD group, 10.5±5.6%; P<0.001, respectively; Table V in the online-only Data Supplement), but there was no significant difference in nitroglycerine-induced vasodilation between the Men (n=307) Women (n=129) Age, y 53.4± ± ±12.3 <0.001 Body mass index, kg/m ± ± ± Systolic blood pressure, mm Hg 134.2± ± ± Diastolic blood pressure, mm Hg 77.7± ± ± Heart rate, bpm 70.0± ± ±11.9 <0.001 Total cholesterol, mmol/l 4.86± ± ± Triglycerides, mmol/l 1.61± ± ± HDL-cholesterol, mmol/l 1.48± ± ± LDL-cholesterol, mmol/l 2.76± ± ± Glucose, mmol/l 6.42± ± ± Hypertension, n, % 252 (58.6) 164 (54.3) 88 (68.8) Dyslipidemia, n, % 176 (40.8) 107 (35.3) 69 (53.9) <0.001 Diabetes mellitus, n, % 99 (23.0) 59 (19.5) 40 (31.3) Smoking, n, % 270 (63.2) 241 (79.5) 29 (23.4) <0.001 Coronary heart disease, n, % 44 (10.3) 37 (12.3) 7 (5.6) 0.03 Cerebrovascular disease, n, % 21 (5.0) 14 (4.7) 7 (5.6) 0.69 Peripheral artery disease, n, % 53 (12.5) 39 (13.1) 14 (11.2) 0.59 Framingham risk score, % 11.4± ± ± Flow-mediated vasodilation, % 4.35± ± ± Nitroglycerine-induced 13.0± ± ± vasodilation, % Brachial IMT, mm 0.33± ± ±0.06 <0.001 Brachial artery diameter, mm 4.13± ± ±0.58 <0.001 HDL indicates high-density lipoprotein; IMT, intima-media thickness; and LDL, low-density lipoprotein. P Value no-risk group and at-risk group (P=0.13). In contrast, there were significant differences in FMD among the 3 groups (no-risk group, 7.50±2.44%; at-risk group, 4.47±2.75%; CVD group, 3.18±2.32%; P<0.001, respectively; Table V in the online-only Data Supplement). To investigate the difference between nitroglycerine-induced vasodilation and FMD in relation to the grade of atherosclerosis, subjects in the at-risk group were divided into 2 groups: a group of subjects with only 1 risk factor (at-low risk group) and a group of subjects with 2 risk factors (at-high risk group; Table VI in the onlineonly Data Supplement; Figure 3A and 3B). There was no significant difference in nitroglycerine-induced vasodilation between the no-risk group and at-low risk group (P=0.84), whereas FMD was significantly smaller in the at-low risk group than in the no-risk group (P<0.001). Both nitroglycerine-induced vasodilation and FMD were significantly smaller in the at-high risk group than in the at-low risk group (P<0.001, respectively). In the CVD group, nitroglycerineinduced vasodilation was impaired compared with that in the at-high risk group (P=0.006), whereas there was no significant difference in FMD between the at-high risk group and the CVD group (P=0.08). Diagnostic Accuracy of Nitroglycerine- Induced Vasodilation and FMD The receiver operating characteristic (ROC) curves of nitroglycerine-induced vasodilation and FMD for diagnosing

4 1404 Arterioscler Thromb Vasc Biol June 2013 Figure 1. A, Scatter plots show the relationship between nitroglycerine-induced vasodilation and Framingham risk score. B, Scatter plots show the relationship between flow-mediated vasodilation and Framingham risk score. the at-risk group or CVD group are shown in Figure 4. Area under the curve values of ROC curves for nitroglycerineinduced vasodilation and FMD to diagnose the at-risk group were and 0.800, respectively, and that for the combination of nitroglycerine-induced vasodilation and FMD was (Figure 4A). Area under the curve values of ROC curves for nitroglycerine-induced vasodilation and FMD to diagnose the CVD group were and 0.653, respectively, and that for the combination of nitroglycerine-induced vasodilation and FMD was (Figure 4B). Area under curve values of ROC curves for Framingham risk score and the combination of Framingham risk score and nitroglycerineinduced vasodilation to diagnose the CVD group were and 0.755, respectively (Figure V in the online-only Data Supplement). Discussion In the present study, we demonstrated that nitroglycerineinduced vasodilation decreased in relation to cumulative cardiovascular risk factors and significantly correlated with cardiovascular risk factors, as well as FMD. To our knowledge, this is the first report showing an association of nitroglycerine-induced vasodilation with cardiovascular risk factors and prevalence of CVDs and showing the relationship between nitroglycerine-induced vasodilation and FMD in a general population, including healthy subjects and patients with CVD. Endothelial dysfunction is established in the initial step of atherosclerosis and plays an important role in the development of this condition. 1 Recently, measurement of FMD in the brachial artery using high-resolution ultrasound, as an index of endothelium-dependent vasodilation, has been widely used as a method for assessing endothelial function. 2 4,7 9 Nitroglycerine-induced vasodilation, an index of endothelium-independent vasodilation, has been assessed as a control test to ensure that impaired FMD is not a result of underlying vascular smooth muscle dysfunction or alterations in vascular structure but truly a consequence of endothelial dysfunction. 16,17 However, it has been shown that nitroglycerine-induced vasodilation per se is impaired in adults with cardiovascular risk factors, or with coronary atherosclerosis and in children at risk for atherosclerosis Consistent with these previous observations, our results demonstrated that nitroglycerine-induced vasodilation significantly correlated with cardiovascular risk factors, including age, systolic blood pressure, diastolic blood pressure, glucose level, smoking status, and the prevalence of hypertension, dyslipidemia, diabetes mellitus, coronary heart disease, and peripheral artery disease. In addition, nitroglycerine-induced vasodilation inversely correlated with Framingham risk score, which is a risk calculator and an index of cumulative cardiovascular risk commonly used for assessing the probability of heart attack or death from heart disease within 10 years. These findings suggest that nitroglycerine-induced vasodilation can be used as a marker of atherosclerosis and may be helpful for estimation of the extent of atherosclerosis and for risk stratification of patients with cardiovascular risk factors. The prevalence of smoking was not associated with the blunted nitroglycerine-induced vasodilation. This might be because of the definition of smoking as subjects who had ever smoked, which did not take into account smoking amount. Actually, smoking pack-year was inversely correlated with nitroglycerine-induced vasodilation and significantly decreased with increase in nitroglycerine-induced vasodilation. Although we have not investigated the mechanism underlying the attenuation of nitroglycerine-induced vasodilation in patients with cardiovascular risk factors and CVD, involvement of increases in superoxide production in smooth muscle cells has been demonstrated. 18 Superoxide inhibits not only NO production in the endothelium but also intravascular signaling processes in smooth muscle cells by inhibiting the activity of soluble guanylyl cyclase and cgmp-dependent kinase. 19 In addition to these alterations of smooth muscle cell function, vascular structural change in intima-media layers may be involved in impaired vascular response to nitroglycerine. Interestingly, in the present study, nitroglycerine-induced vasodilation was significantly correlated with intima-media thickness of the brachial artery. An increase in connective tissue matrix in thickened intima-media layers may contribute to limitation of relaxation, and proliferation of vascular smooth muscle cells may cause a relative decrease in NO derived from nitroglycerine, leading to impaired dilator response to nitroglycerine. 20 Although the precise mechanisms for acute detection of shear stress and subsequent signal transduction to modulate vasomotor tone are not fully understood, endothelium-derived NO is considered to be a principle mediator of FMD. 10,11 Shear stress mediated by reactive hyperemia stimulates endothelial cells, resulting in activation of endothelial

5 Maruhashi et al Nitroglycerine-Induced Vasodilation 1405 Figure 2. A, Bar graphs show the prevalence of cardiovascular risk factors in subjects categorized according to tertiles of nitroglycerineinduced vasodilation. B, Bar graphs show the prevalence of cardiovascular disease (CVD) in subjects categorized according to tertiles of nitroglycerine-induced vasodilation. C, Bar graphs show the prevalence of cardiovascular risk factors in subjects categorized according to tertiles of flow-mediated vasodilation. D, Bar graphs show the prevalence of CVD in subjects categorized according to tertiles of flowmediated vasodilation. NO synthase and subsequent generation of NO, which diffuses into adjacent smooth muscle cells and interacts with soluble guanylyl cyclase, leading to smooth muscle relaxation Table 2. Variables Multivariate Analysis of the Relationships Between Vascular Functions and Variables Nitroglycerine-Induced Vasodilation and dilation of the brachial artery. Therefore, assessment of FMD is based on the premise that endothelium-independent vasodilation is not altered. However, in the present study, Flow-Mediated Vasodilation Parameter Estimate SE P Value Parameter Estimate SE P Value Age, y <0.001 Male Body mass index, kg/m 2 Hypertension Dyslipidemia Diabetes mellitus < Smoking Brachial artery < <0.001 diameter, mm Nitroglycerine-induced <0.001 vasodilation, % Flow-mediated <0.001 vasodilation, % Age hypertension The adjusted r 2 of the model for both nitroglycerine-induced vasodilation and flow-mediated vasodilation was 0.33.

6 1406 Arterioscler Thromb Vasc Biol June 2013 Figure 3. A, Bar graphs show nitroglycerine-induced vasodilation in the no risk group, at-low risk group, at-high risk group, and cardiovascular disease (CVD) group. B, Bar graphs show flow-mediated vasodilation in the no-risk group, at-low risk group, at-high risk group, and CVD group. nitroglycerine-induced vasodilation, an index of endotheliumindependent vasodilation, was decreased in subjects with cardiovascular risk factors and CVD. Although it is not possible to prove causality of the association between nitroglycerineinduced vasodilation and FMD in this cross-sectional study, measurement of nitroglycerine-induced vasodilation should be performed for appropriate interpretation of FMD, given that FMD caused by reactive hyperemia occurs as a result of vascular smooth muscle relaxation regardless of the mechanism. In subjects without cardiovascular risk factors (no-risk group), both nitroglycerine-induced vasodilation and FMD were maintained. In subjects with cardiovascular risk factor (at-risk group), FMD was impaired compared with that in the no-risk group, but there was no significant difference in nitroglycerine-induced vasodilation between the no-risk group and the at-risk group. In addition, area under the curve value of the ROC curve for FMD to diagnose the at-risk group was higher than that for nitroglycerine-induced vasodilation. Figure 4. Receiver operating characteristic curves of nitroglycerine-induced vasodilation (nitroglycerine-induced vasodilation), flow-mediated vasodilation (FMD), and their combination (FMD+nitroglycerine-induced vasodilation) for patients without clinical evidence of cardiovascular diseases (CVD; A) and patients with CVD (B). These findings suggest that FMD is an appropriate marker for detecting asymptomatic subjects at an early stage of atherosclerosis. When subjects in the at-risk group were divided into 2 groups, that is, subjects with 1 risk factor (at-low risk group) and subjects with 2 risk factors (at-high risk group), the difference in the relationship of severity of atherosclerosis with nitroglycerine-induced vasodilation and FMD was more pronounced. In the at-low risk group, FMD was impaired compared with that in the no-risk group, whereas nitroglycerine-induced vasodilation was not altered, indicating that endothelial dysfunction is an early event that precedes the impairment of endothelium-independent vasodilation. Considering that the decrease in nitroglycerine-induced vasodilation is attributed, at least in part, to vascular structural change, this result is consistent with results of previous studies showing that endothelial dysfunction precedes the development of morphological vascular changes. 21,22 However, both FMD and nitroglycerine-induced vasodilation were significantly smaller in the at-high risk group and in subjects with

7 Maruhashi et al Nitroglycerine-Induced Vasodilation 1407 CVD (CVD group) than in the no-risk group and the at-low risk group. Therefore, in these subjects, it may be theoretically impossible to discern whether impaired FMD is a result of endothelial dysfunction or concurrent abnormalities of endothelial function and vascular smooth muscle function. Thus, in subjects with decrease in nitroglycerine-induced vasodilation, FMD should be interpreted as an index of vascular function reflecting both endothelium-dependent and endotheliumindependent vasodilation. As for the diagnostic accuracy for patients with CVD, area under the curve value of the ROC curve for nitroglycerine-induced vasodilation to diagnose the CVD group was almost equal to that for FMD, and the addition of nitroglycerine-induced vasodilation to FMD did not improve the prediction of CVD group. Moreover, the addition of nitroglycerine-induced vasodilation to Framingham risk score did not improve the prediction of CVD group. Taken together, nitroglycerine-induced vasodilation added very little predictive information on top of Framingham risk score for detecting patients with clinically evident atherosclerotic disease. In previous studies, there was no significant difference in nitroglycerine-induced vasodilation between healthy control subjects and subjects with cardiovascular risk factors or CVD. 2,3,23 25 Although we do not know the reasons for the discrepancy in the results of our study and previous studies, some possibilities are postulated: (1) relatively low-risk subjects, such as subjects with only 1 risk factor, were enrolled in previous studies, and therefore the results showing no significant difference in nitroglycerine-induced vasodilation between healthy control subjects and individuals at increased risk for atherosclerosis may be, at least in part, because of the subject selection, and (2) Raitakari et al 13 raised the possibility that a high dose of nitroglycerine elicits maximal artery dilatation in patients with coronary artery disease, leading to escaped detection of impaired nitroglycerine-induced vasodilation, suggesting that the difference in dose of administered nitroglycerine ( versus 75 µg) led to the misinterpretation that nitroglycerine-induced vasodilation remains unaltered in subjects with cardiovascular risk factors. There are several limitations in the present study. First, this study was performed without withholding medications. One hundred seventy-nine (41.1%) of the subjects received vasoactive medications, 87 (20.0%) received lipid-lowering medication, and 49 (11.2%) received hypoglycemic drugs. Because the study subjects were recruited from health-screening examinations, it would have been inappropriate to withhold medications. Although we cannot deny the possibility that some medications, especially vasoactive medications, affect FMD or nitroglycerine-induced vasodilation, Gokce et al 26 reported that administration of vasoactive medication not including nitrates does not significantly affect the value of FMD and nitroglycerine-induced vasodilation. In addition, subjects who received nitrates, which might confound the results of nitroglycerine-induced vasodilation measurement, were excluded from this study. Second, we did not investigate mitochondrial aldehyde dehydrogenase 2 (ALDH2) genotype of the subjects. ALDH2 is regarded as an important enzyme for nitroglycerine bioactivation. An ALDH2 variant, ALDH2*504Lys, is present only in East Asian populations, including Japanese with frequencies as high as 40%. 27 This mutation causes a great reduction in activity of ALDH2 in heterozygotes and abolishes activity in homozygotes. We cannot deny the possibility that subjects with the ALDH2 variant were included in this study and that their responses to nitroglycerine were influenced by reduced bioactivation of nitroglycerine. However, Sakata et al 28 demonstrated that nitroglycerine-induced vasodilation did not differ among ALDH2 genotypes, suggesting that ALDH2 genotypes might have little influence on the results of this study. Third, we did not investigate the relationships between duration of cardiovascular risk factors and vascular response. Although the precise reason why nitroglycerine-induced vasodilation significantly correlated with cardiovascular risk factors and there was no significant difference in nitroglycerine-induced vasodilation between the no risk group and at-low risk group is unknown, duration of cardiovascular risk factors is possibly involved. Impaired nitroglycerine response may be associated with alteration of vascular structure. No significant difference in nitroglycerine-induced vasodilation between the no risk group and at-low risk group might be associated with a short duration of cardiovascular risk factors because of the relatively young age of subjects in the at-low risk group. Fourth, the dose of nitroglycerine used was based on subjects tolerability. We did not choose a dose in the midrange of the dose response curve of each patient. In conclusion, nitroglycerine-induced vasodilation is associated with cardiovascular risk factors and may be a marker of atherosclerosis. FMD declines with increase in the severity of the disease because it incorporates the deterioration of nitroglycerine-induced vasodilation. As a predictive marker of CVDs, nitroglycerine-induced vasodilation provided very little additive information on top of Framingham risk score. Further studies are needed to determine whether nitroglycerine-induced vasodilation can be used as a surrogate marker of future cardiovascular events. Acknowledgments We thank Megumi Wakisaka, Miki Kumiji, Kiichiro Kawano, and Satoko Michiyama for their excellent secretarial assistance. Sources of Funding This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan ( and ) and a grant-in-aid of Japanese Arteriosclerosis Prevention Fund. None. Disclosures References 1. Ross R. Atherosclerosis an inflammatory disease. N Engl J Med. 1999;340: Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet. 1992;340: Celermajer DS, Sorensen KE, Bull C, Robinson J, Deanfield JE. Endothelium-dependent dilation in the systemic arteries of asymptomatic subjects relates to coronary risk factors and their interaction. J Am Coll Cardiol. 1994;24:

8 1408 Arterioscler Thromb Vasc Biol June Benjamin EJ, Larson MG, Keyes MJ, Mitchell GF, Vasan RS, Keaney JF Jr, Lehman BT, Fan S, Osypiuk E, Vita JA. Clinical correlates and heritability of flow-mediated dilation in the community: the Framingham Heart Study. Circulation. 2004;109: Yeboah J, Crouse JR, Hsu FC, Burke GL, Herrington DM. Brachial flowmediated dilation predicts incident cardiovascular events in older adults: the Cardiovascular Health Study. Circulation. 2007;115: Yeboah J, Folsom AR, Burke GL, Johnson C, Polak JF, Post W, Lima JA, Crouse JR, Herrington DM. Predictive value of brachial flow-mediated dilation for incident cardiovascular events in a population-based study: the multi-ethnic study of atherosclerosis. Circulation. 2009;120: Idei N, Nishioka K, Soga J, Hidaka T, Hata T, Fujii Y, Fujimura N, Maruhashi T, Mikami S, Teragawa H, Kihara Y, Noma K, Chayama K, Higashi Y. Vascular function and circulating progenitor cells in thromboangiitis obliterans (Buerger s disease) and atherosclerosis obliterans. Hypertension. 2011;57: Soga J, Noma K, Hata T, Hidaka T, Fujii Y, Idei N, Fujimura N, Mikami S, Maruhashi T, Kihara Y, Chayama K, Kato H, Liao JK, Higashi Y; ROCK Study Group. Rho-associated kinase activity, endothelial function, and cardiovascular risk factors. Arterioscler Thromb Vasc Biol. 2011;31: Maruhashi T, Soga J, Fujimura N, et al. Hyperbilirubinemia, augmentation of endothelial function, and decrease in oxidative stress in Gilbert syndrome. Circulation. 2012;126: Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in the vasodilator response to increased flow in vivo. Hypertension. 1986;8: Joannides R, Haefeli WE, Linder L, Richard V, Bakkali EH, Thuillez C, Lüscher TF. Nitric oxide is responsible for flow-dependent dilatation of human peripheral conduit arteries in vivo. Circulation. 1995;91: Adams MR, Robinson J, McCredie R, Seale JP, Sorensen KE, Deanfield JE, Celermajer DS. Smooth muscle dysfunction occurs independently of impaired endothelium-dependent dilation in adults at risk of atherosclerosis. J Am Coll Cardiol. 1998;32: Raitakari OT, Seale JP, Celermajer DS. Impaired vascular responses to nitroglycerin in subjects with coronary atherosclerosis. Am J Cardiol. 2001;87:217 9, A Järvisalo MJ, Lehtimäki T, Raitakari OT. Determinants of arterial nitratemediated dilatation in children: role of oxidized low-density lipoprotein, endothelial function, and carotid intima-media thickness. Circulation. 2004;109: Schächinger V, Britten MB, Zeiher AM. Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation. 2000;101: Corretti MC, Anderson TJ, Benjamin EJ, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Herman M, Herrington D, Vallance P, Vita J, Vogel R; International Brachial Artery Reactivity Task Significance Force. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery: a report of the International Brachial Artery Reactivity Task Force. J Am Coll Cardiol. 2002;39: Inoue T, Matsuoka H, Higashi Y, Ueda S, Sata M, Shimada KE, Ishibashi Y, Node K; Vascular Failure Workshop Group. Flow-mediated vasodilation as a diagnostic modality for vascular failure. Hypertens Res. 2008;31: Münzel T, Afanas ev IB, Kleschyov AL, Harrison DG. Detection of superoxide in vascular tissue. Arterioscler Thromb Vasc Biol. 2002;22: Münzel T, Feil R, Mülsch A, Lohmann SM, Hofmann F, Walter U. Physiology and pathophysiology of vascular signaling controlled by guanosine 3,5 -cyclic monophosphate-dependent protein kinase [corrected]. Circulation. 2003;108: Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362: Juonala M, Viikari JS, Laitinen T, Marniemi J, Helenius H, Rönnemaa T, Raitakari OT. Interrelations between brachial endothelial function and carotid intima-media thickness in young adults: the cardiovascular risk in young Finns study. Circulation. 2004;110: Halcox JP, Donald AE, Ellins E, Witte DR, Shipley MJ, Brunner EJ, Marmot MG, Deanfield JE. Endothelial function predicts progression of carotid intima-media thickness. Circulation. 2009;119: Imamura M, Biro S, Kihara T, Yoshifuku S, Takasaki K, Otsuji Y, Minagoe S, Toyama Y, Tei C. Repeated thermal therapy improves impaired vascular endothelial function in patients with coronary risk factors. J Am Coll Cardiol. 2001;38: Singh TP, Groehn H, Kazmers A. Vascular function and carotid intimalmedial thickness in children with insulin-dependent diabetes mellitus. J Am Coll Cardiol. 2003;41: Kato M, Hisatome I, Tomikura Y, Kotani K, Kinugawa T, Ogino K, Ishida K, Igawa O, Shigemasa C, Somers VK. Status of endothelial dependent vasodilation in patients with hyperuricemia. Am J Cardiol. 2005;96: Gokce N, Holbrook M, Hunter LM, Palmisano J, Vigalok E, Keaney JF Jr, Vita JA. Acute effects of vasoactive drug treatment on brachial artery reactivity. J Am Coll Cardiol. 2002;40: Li H, Borinskaya S, Yoshimura K, et al. Refined geographic distribution of the oriental ALDH2*504Lys (nee 487Lys) variant. Ann Hum Genet. 2009;73: Sakata S, Yoshihara T, Arima H, Shiraishi F, Oniki H, Takahashi-Yanaga F, Matsumura K, Sasaguri T. Differential effects of organic nitrates on arterial diameter among healthy Japanese participants with different mitochondrial aldehyde dehydrogenase 2 genotypes: randomised crossover trial. BMJ Open. 2011;1:e Nitroglycerine-induced vasodilation of the brachial artery, assessed by sublingual administration of nitroglycerine, has been used as a control test for flow-mediated vasodilation (FMD) measurement to differentiate endothelium-dependent from endothelium-independent response. We measured nitroglycerine-induced vasodilation and FMD in the same subjects for a direct comparison of these 2 vascular response tests in 436 subjects. Both nitroglycerine-induced vasodilation and FMD correlated with cardiovascular risk factors, as well as Framingham risk score. In the low-risk patients, FMD was already impaired, whereas nitroglycerine-induced vasodilation was not altered. However, both FMD and nitroglycerine-induced vasodilation were impaired in the high-risk patients and patients with cardiovascular disease. Thus, in these patients, FMD should be interpreted as an index of vascular function reflecting both endothelium-dependent vasodilation and endothelium-independent vasodilation. As a predictive marker of cardiovascular diseases, nitroglycerine-induced vasodilation provided very little additive information on top of Framingham risk score.