From the Cardiovascular Disease Unit, Department of Experimental and Clinical Medicine G. Salvatore, University of Catanzaro Magna Graecia, Catanzaro, Italy.
Address correspondence and reprint requests to Francesco Perticone, MD, Dipartimento di Medicina Sperimentale e Clinica G. Salvatore, Policlinico Mater DominiVia T. Campanella, 88100 Catanzaro, Italy. E-mail: perticone{at}unicz.it .
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ABSTRACT |
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INTRODUCTION |
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The normal endothelium plays a key role in the regulation of vascular tone and in preventing the progression of atherosclerosis through the production and release of both contracting and relaxing factors (5). Nitric oxide (NO) represents the major endogenous relaxing factor (6,7,8,9), and its production is stimulated by physical stimuli (e.g., shear stress) (9) and by several agonists (e.g., acetylcholine [ACh], bradykinin, substance P, and serotonin) (8). The activation of guanylate cyclase and the subsequent accumulation of cGMP are the main mechanisms of NO-induced vasodilation. In contrast, sodium nitroprusside (SNP) is an endothelium-independent vasodilator capable of inducing vasodilation by providing an inorganic source of NO (10). Major risk factors for atherosclerotic vascular diseases (e.g., hypertension, smoking, diabetes, and hypercholesterolemia) have been associated with endothelial dysfunction due to increased oxidative stress (11,12,13,14,15,16). Recent reports have also indicated that insulin contributes to the maintenance of vascular tone through a selective physiological action in vasodilating skeletal muscle vasculature. Specifically, the insulin-mediated vasodilation has been attributed to endothelial NO release (17,18). Nevertheless, endothelial dysfunction has also been reported in obese insulin-resistant subjects, but the underlying mechanisms have not been clarified (19).
Given this information, we evaluated the relationship between body weight and endothelium-dependent vasodilation, and we investigated whether body fat distribution affects ACh-mediated vasodilation. Moreover, we evaluated whether oxygen free radicals could be responsible for the impairment in the L-arginineNO pathway of obese subjects by studying the effects of a vitamin C infusion, a potent antioxidant compound. Finally, using indomethacin as an experimental tool, we also evaluated the possibility that oxygen free radicals could originate from cyclooxygenase activity.
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RESEARCH DESIGN AND METHODS |
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Anthropometric measurements. A trained examiner (F.S.) collected measurements of height, weight, and circumference according to a standardized protocol. BMI was calculated as kilograms per square meter, and the waist was measured at its smallest point with the abdomen relaxed. The hip circumference was measured at its largest point: the tape was held at the top of the patient's hipbone and then wrapped carefully around the torso.
Central fat distribution was defined on the basis of the sex-specific 85th
percentile of the waist-to-hip ratio (WHR) values as indicated by the Italian
Consensus Conference on Obesity
(20). According to these
guidelines, the cutoff value of central obesity was considered 0.81 for
women and
0.92 for men.
IR evaluation. An oral glucose tolerance test was performed to exclude diabetes in undiagnosed patients. Subjects were seated for the test between 8:00 and 9:00 A.M. after fasting overnight for at least 12 h. Fasting glucose and insulin values were averaged from the values obtained 15 and 5 min before administration of a 75-g glucose solution.
IR was estimated using the homeostasis model assessment (HOMA) from the fasting glucose and insulin concentrations (21). The HOMA-IR is commonly used in clinical studies, and it was recently used in a population-based study (22,23).
Measurements of forearm blood flow. All studies were performed at 9:00 A.M. after subjects had fasted overnight, with the subjects lying supine in a quiet air-conditioned room (22-24°C). We used the study protocol previously described by Panza et al. (24) and subsequently used by our group (25,26).
The forearm blood flow (FBF) was measured as the slope of the change in the forearm volume (27). The mean of at least three measurements was calculated at each time point. Forearm vascular resistance (VR), expressed in arbitrary units, was calculated by dividing mean blood pressure (BP) by FBF. To avoid underestimation of FBF measurements, the forearm circumference in all subjects was required to be <28 cm.
Vascular function
Endothelium-dependent and endothelium-independent
vasodilation. All participants rested 30 min after artery
cannulation to obtain a stable baseline before data collection; FBF and VR
were repeated every 5 min until stable. Endothelium-dependent and
endothelium-independent vasodilation were assessed by the dose-response curve
to intra-arterial infusions at increasing doses of ACh (7.5, 15, and 30 µg
· ml-1 · min-1, each for 5 min) and SNP
(0.8, 1.6, and 3.2 µg · ml-1 · min-1,
each for 5 min), respectively. The drug infusion rate, adjusted for the
forearm volume of each subject, was 1 ml/min.
Effects of vitamin C on endothelium-dependent and endothelium-independent vasodilation. To evaluate whether oxygen free radicals can selectively impair endothelium-dependent or endothelium-independent vasodilation in obese subjects, both ACh and SNP were infused under controlled conditions (saline infusion) and in the presence of intrabrachial vitamin C (24 mg/min), which was administered 5 min before the agonists and continued throughout. This vitamin C concentration has been shown to both protect human plasma from free radicalmediated lipid peroxidation (11) and improve impaired ACh-induced vasodilation in smokers (12) and hypercholesterolemic (11,13), hypertensive (14), and diabetic patients (15).
Effects of cyclooxygenase inhibition on ACh-stimulated vasodilation. We have evaluated the effects of cyclooxygenase activity (a source of oxygen free radicals) on endothelium-dependent vasodilation. A dose-response curve to intrabrachial ACh administration was performed during the coinfusion of indomethacin (a cyclooxygenase inhibitor) at a constant dose of 500 µg/min starting 10 min before ACh administration and continuing throughout.
Drugs. ACh (Sigma, Milan, Italy), vitamin C (Bracco, Milan, Italy), and indomethacin (Liometacen, Chiesi Farmaceutici SpA, Parma, Italy) were obtained from commercially available sources and diluted freshly to the desired concentration by the addition of saline. SNP (Malesci, Florence, Italy) was diluted in a 5% glucose solution immediately before each infusion and protected from light with aluminum foil.
Statistical analysis. Analysis of variance (ANOVA) was performed for clinical and biological data, and the differences between means were compared using unpaired Student's t tests. The responses to ACh and SNP were compared by ANOVA for repeated measurements, and when the analysis was significant, the Tukey's test was applied. Simple linear regression analysis was performed to assess the relationship between the peak increase in FBF in response to ACh infusion and variables such as indexes of obesity (i.e., BMI, waist circumference, and WHR), fasting insulin, HOMA-IR, and other factors reported to impair endothelium-dependent vasodilation (i.e., age, cholesterol, and systolic and diastolic BP). Subsequently, variables that achieved statistical significance were entered into a stepwise multiple regression model to assess the magnitude of their individual effect on the peak FBF response to intra-arterial infusions of ACh. In this analysis, we included only fasting insulin because HOMA-IR is a function of both fasting insulin and glucose. Thus, we considered fasting insulin the fittest variable to avoid a possible colinearity. Significant differences were assumed to be present at P < 0.05. All group data are reported as means ± SD.
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RESULTS |
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Endothelium-dependent and endothelium-independent vasodilation
ACh study. Basal FBF was 3.8 ± 0.6, 3.6 ±
0.7, and 3.6 ± 0.8 ml · 100 ml-1 of tissue ·
min-1 in groups A, B, and C, respectively (P = 0.673 by
ANOVA). Similarly, no significant (P = 0.268 by ANOVA) differences in
VR among the groups were observed: 26.5 ± 4.7 U for group A, 29.1
± 5.6 U for group B, and 29.2 ± 6.0 U for group C.
In response to the intra-arterial infusion of ACh, FBF significantly increased (P < 0.0001) in a dose-dependent fashion in all groups. However, obese subjects (group C) had a significantly lower responsiveness to ACh (i.e., FBF from 3.6 ± 0.8 to a maximum of 6.5 ± 1.8 ml · 100 ml-1 tissue ·min-1) than subjects included in both group A (3.8 ± 0.6 to a maximum of 19.8 ± 2.8 ml · 100 ml-1 tissue · min-1) and group B (3.6 ± 0.7 to a maximum of 10.8 ± 2.7 ml · 100 ml-1 tissue · min-1) (Fig. 1). VR significantly decreased (P < 0.0001) in a dose-dependent manner in all groups, and changes in VR were significantly more pronounced in group A than in groups B and C. Similarly, the changes induced by ACh in FBF and VR were significantly different between groups A and B (Fig. 1). ACh and vitamin C coinfusion. The blunted vasodilation in response to ACh that was documented in obese subjects significantly increased during coinfusion of vitamin C (Fig. 1). However, the oxygen free radical scavenger did not significantly change the response to ACh in the subjects included in group A (FBF from 3.7 ± 0.5 to 19.1 ± 3.1 ml · 100 ml-1 tissue · min-1 with the highest dose), but in the subjects of both group B (3.6 ± 0.6 to 14.8 ± 2.5 ml · 100 ml-1 tissue · min-1 with the highest dose; P < 0.0001 vs. ACh during saline) and group C (3.5 ± 0.6 to 12.3 ± 2.4 ml · 100 ml-1 tissue · min-1 with the highest dose; P < 0.001 vs. ACh during saline), the intrabrachial administration of vitamin C significantly improved the vasodilating effect of the muscarinic agonist. In addition, the comparison among the groups was statistically significant (P < 0.0001 by ANOVA).
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ACh and indomethacin coinfusion. ACh-stimulated vasodilation increased significantly during indomethacin coinfusion (Fig. 2). When the activity of the cyclooxygenase inhibitor was compared among the subjects of group A, ACh-dependent vasodilation was not significantly increased by indomethacin (FBF from 3.7 ± 0.6 to 19.3 ± 2.9 ml · 100 ml-1 tissue · min-1 with the highest dose; NS vs. ACh during saline). On the contrary, ACh-induced vasodilation was significantly increased by indomethacin in the subjects of group B (3.5 ± 0.5 to 15.2 ± 2.3 ml · 100 ml-1 tissue · min-1 with the highest dose; P < 0.0001 vs. ACh during saline) and group C (3.6 ± 0.5 to 12.5 ± 2.1 ml · 100 ml-1 tissue · min-1 with the highest dose; P < 0.0001 vs. ACh during saline). The enhancement of ACh-induced vasodilation exerted by indomethacin in obese subjects (groups B and C) was similar to the potentiation produced by coinfusion of vitamin C in the same subjects.
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SNP study. Figure 1 shows the percent increase in FBF above baseline in response to intra-arterial infusions of SNP. The vasodilating responses to the endothelium-independent vasodilator SNP were similar in all groups.
SNP and vitamin C coinfusion. The vitamin C coinfusion did not significantly change either FBF or VR during SNP infusions (Fig. 1). The results also remained unchanged when data were analyzed according to WHR. Thus, all subjects displayed normal endothelium-independent vasodilation.
Correlational analyses. Figure 3 shows the inverse relationship between the peak increase in FBF after ACh infusion and BMI (r = -0.676, P < 0.0001), which accounts for 45.7% of the variation in FBF response in the whole population. Subsequently, we tested the effect of fat distribution on endothelium-dependent vasodilation by using both waist circumference (r = -0.581, P < 0.0001) and WHR (r = -0.631, P < 0.0001) (Fig. 4) as criteria for central obesity. The last relationship accounts for 39.8% of the variation, and it is more evident in men (r = -0.696, P < 0.0001) than in women (r = -0.538, P < 0.0001). Therefore, our data suggest that central fat distribution may also affect endothelium-dependent vasodilation. When we investigated the effect of insulin sensitivity on the peak increase in FBF after ACh infusion, we found an inverse linear relationship between HOMA-IR (Table 2) and fasting insulin (graphically reported in Fig. 5). Taking into account that both BMI and WHR affect insulin sensitivity, these data suggest that IR indeed mediates the negative effects of BMI and WHR on endothelium-dependent vasodilation. Finally, we performed a simple linear regression analysis between the peak increase in FBF during ACh infusions and both waist circumference and IR for the subject population as a whole and subdivided by sex. This analysis (Table 2) demonstrated that increasing degrees of obesity, waist circumference, WHR, fasting insulin, and IR are associated with different degrees of endothelial dysfunction.
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Furthermore, because impaired ACh-stimulated vasodilation has been
associated with aging (27),
hypercholesterolemia
(22,24),
and essential hypertension
(25), we also investigated the
relationship between the peak increase in FBF and age, cholesterolemia, and BP
values. This analysis did not show any significant correlation with the
maximal FBF response to ACh. In addition, in
Table 3 we report the
relationship between insulin sensitivity, indexes of obesity/body fat
distribution, and % changes in the FBF response to ACh caused by the
coinfusion of vitamin C and indomethacin. The analysis demonstrates that
greater improvement in FBF was observed in the subjects with the highest
degree of obesity or IR whereas less improvement was seen in the leaner
subjects, confirming that obesity and/or IR are the main causes of the
endothelial dysfunction.
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Finally, in a stepwise multivariate regression analysis (Table 4), the independent determinants of the peak increase in FBF were fasting insulin, WHR, and BMI in the total subject population and in the men; on the contrary, only BMI was significantly related to FBF in the women.
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DISCUSSION |
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Another finding of our study is that, like BMI, central fat distribution also affects the peak FBF response to intrabrachial infusion of ACh. This inverse relationship between FBF and both BMI and WHR is affected by sex, as shown in Figs. 3 and 4; in particular, BMI predicts 48.3 and 57.9% and WHR predicts 48.4 and 28.9% of the maximum response to ACh in men and women, respectively.
We also demonstrated that indexes of insulin sensitivity, which are linearly related to BMI and WHR, predict endothelial dysfunction in obese subjects. In fact, our data demonstrate that fasting insulin predicts 51.8 and 50.9% of the peak increase in FBF induced by ACh infusion in men and women, respectively. In addition, subsequent multiple regression analysis confirmed these data, as reported in Table 4. These findings are in agreement with the observation that the physiological vasodilating action of insulin is present in insulin-sensitive but not in insulin-resistant patients (30,31). In the same way, recent reports have shown that endothelial NO may mediate insulin-stimulated vasodilation in skeletal muscle (17,18), suggesting a direct physiological link between endothelial function and insulin sensitivity. Therefore, subjects who are relatively insulin-resistant appear to have a corresponding decrease in NO production. With respect to hypertension (14,24,26), hypercholesterolemia (11,13), diabetes (15), and aging (16)well-known conditions associated with endothelial dysfunctionit is necessary to point out that all our subjects were normotensive, normoglycemic, and had cholesterol levels that did not exceed 200 mg/dl. Finally, all our subjects were relatively young, and we were unable to find a significant relationship between age and the peak increase in FBF (r = 0.016, P = 0.892). On the basis of this information, it is possible to affirm that indexes of insulin sensitivity, which are linearly related to indexes of obesity, largely explain the endothelial dysfunction in obese subjects.
However, our data do not completely clarify the association between endothelial dysfunction and both obesity and IR. In fact, the depressed ACh-stimulated vasodilation demonstrated in this study can be explained only in part by oxidative stress because the vitamin C coinfusion improved but did not completely normalize the endothelial response to ACh. Therefore, additional factors may be involved in the development of endothelial dysfunction in obese subjects. It is important to emphasize that, whereas insulin at low physiological levels increases endothelium-dependent vasodilation in normal subjects, much higher insulin levels, such as those detected in insulin-resistant obese and diabetic patients, fail to enhance endothelium-dependent vasodilation (19). This vasoactive action of insulin may be explained by its ability to act at the level of endothelial cells that modulate the production and release of NO. Nevertheless, this study fails to demonstrate the mechanisms by which the vasodilating effect of insulin is blunted in obese and diabetic patients, which suggests that the endothelium is probably resistant to insulin's modulating effect on NO production and release. On the other hand, there is evidence that sympathetic activity is increased in insulin-resistant states as a consequence of decreased activity of the inhibitory brain-stem center (32). The increase in vaso-constrictor substances could impair endothelium-dependent vasodilation and, thus, could reduce the FBF increase response to ACh. In the same way, Cardillo et al. (33) recently reported that in skeletal muscle, insulin stimulates both endothelin and NO activities. Therefore, on the basis of this evidence, it is possible to hypothesize that an imbalance between the release of ET-1 and NO in response to insulin may be involved in the endothelial dysfunction of obesity and other insulin-resistant states.
In conclusion, we have demonstrated that 1) obesity/IR, independently of other risk factors, is associated with reduced ACh-stimulated vasodilation and 2) the endothelial dysfunction is due, at least in part, to increased oxidative stress, which can be improved by the administration of intrabrachial vitamin C or indomethacin. Considering the key role of normal endothelium in antiproliferative and antiatherosclerotic processes, we suggest that endothelial dysfunction in human obesity may confer an increased risk of macrovascular diseases in obese insulin-resistant subjects. Finally, it is important to emphasize that this risk is higher in men than in women and that both fasting insulin and WHR, which were analyzed in multivariate stepwise logistic regression, appear as independent predictors of endothelial dysfunction. Thus, our analysis demonstrates that insulin sensitivity and central fat distribution are two interrelated predictors of endothelial dysfunction.
Clinical implications. The increased cardiovascular morbidity and mortality observed in human obesity may be caused by a quick progression of the atherosclerotic process in the presence of some risk factors for atherosclerosis. In this way, the endothelium has an important influence on vascular physiology and appears to be central in mediating damage to the vessel wall when patients are exposed to conventional risk factors for atherosclerosis. Thus, it is reasonable to hypothesize that caloric restrictions (by diet and/or pharmacological treatment) and metabolic and cardiovascular adaptations to weight loss might be useful in improving endothelial dysfunction and reducing cardiovascular risk. Nevertheless, further studies are required to determine the mechanistic explanation for the observed relationship between obesity/IR and depressed endothelium-dependent vasodilation.
Finally, we remark that we used intra-arterial vitamin C infusion to evaluate only the endothelial function because at present there is no firm evidence that ascorbic acid supplementation decreases cardiovascular mortality.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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ACh, acetylcholine; ANOVA, analysis of variance, BP, blood pressure; FBF, forearm blood flow; HOMA, homeostasis model assessment; IR, insulin resistance; NO, nitric oxide; SNP, sodium nitroprusside; VR, vascular resistance; WHR, waist-to-hip ratio.
Received for publication February 15, 2000 and accepted in revised form September 18, 2000
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REFERENCES |
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