CLINICAL STUDY
Mechanisms responsible for endothelial dysfunction induced by fasting hyperhomocystinemia in normotensive subjects and patients with essential hypertension
Agostino Virdis, MDa,
Lorenzo Ghiadoni, MD, PhDa,
Heloise Cardinal, MDa,
Stefania Favilla, PhDa,
Piero Duranti, BSa,
Renzo Birindelli, BSa,
Armando Magagna, MDa,
Gianpaolo Bernini, MDa,
Guido Salvetti, MDa,
Stefano Taddei, MDa and
Antonio Salvetti, MDa
a Department of Internal Medicine, University of Pisa, Pisa, Italy
Manuscript received August 23, 2000;
revised manuscript received June 1, 2001,
accepted June 20, 2001.
Reprint requests and correspondence: Dr. Agostino Virdis, Department of Internal Medicine, University of Pisa, Via Roma, 56100 Pisa, Italy
avirdis{at}med.unipi.it
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Abstract
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OBJECTIVES
We sought to evaluate whether fasting hyperhomocystinemia reduces endothelial function by oxidative stress in normotensive subjects and hypertensive patients.
BACKGROUND
Subjects with hyperhomocystinemia have endothelial dysfunction.
METHODS
In 23 normotensive subjects and 28 hypertensive patients, classified into normohomocystinemic and hyperhomocystinemic groups according to homocysteine plasma levels (<8.7 and >14.6 µmol/l, respectively), we studied forearm blood flow changes (strain-gauge plethysmography) induced by intrabrachial administration of acetylcholine (0.15 to 15 µg/100 ml tissue per min) or sodium nitroprusside (1 to 4 µg/100 ml per min), an endothelium-dependent and -independent vasodilator, respectively. Acetylcholine was repeated with NG-monomethyl-L-arginine (L-NMMA; 100 µg/100 ml per min), vitamin C (8 mg/100 ml per min) and L-NMMA plus vitamin C.
RESULTS
Normotensive hyperhomocystinemic patients showed a blunted response to acetylcholine and a lower inhibiting effect of L-NMMA on acetylcholine, as compared with normohomocystinemic patients. Although vitamin C was ineffective in normohomocystinemic subjects, it increased the response to acetylcholine and restored the inhibiting effect of L-NMMA on acetylcholine in hyperhomocystinemic patients. Hypertensive hyperhomocystinemic patients showed a reduced response to acetylcholine, as compared with normohomocystinemic subjects. In both subgroups, L-NMMA failed to blunt the response to acetylcholine. The potentiating effect of vitamin C on acetylcholine was greater in hyperhomocystinemic patients than in normohomocystinemic subjects, although it restored the inhibitory effect of L-NMMA on acetylcholine-induced vasodilation to the same extent in both groups. Hyperhomocystinemia did not change the response to sodium nitroprusside.
CONCLUSIONS
In normotensive subjects and hypertensive patients, hyperhomocystinemia impairs endothelium-dependent vasodilation. It could be related to oxidant activity.
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Abbreviations and Acronyms
| | BP | = blood pressure | | FBF | = forearm blood flow | | H-HCY | = hyperhomocystinemia | | L-NMMA | = NG-monomethyl-L-arginine | | NO | = nitric oxide | | SNP | = sodium nitroprusside |
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Homocysteine is a sulfur-containing amino acid formed during dietary methionine metabolism (1). Epidemiologic evidence indicates that mild elevation of the plasma homocysteine level is associated with an increased risk of atherosclerosis (2).
Endothelium plays a primary role in the modulation of vascular tone and structure through production of the relaxing factor nitric oxide (NO), which acts by protecting the vessel wall from development of atherosclerosis and thrombosis (3). A dysfunctioning endothelium, characterized by reduced NO availability induced by oxidative stress, is indicated as an early event in the pathogenesis of atherosclerosis (3). Experimental evidence indicates that hyperhomocystinemia (H-HCY) may induce endothelial injury (4).
Moreover, homocysteine increases oxidative stress in vitro (1). Thus, the promotion of endothelial dysfunction through oxidative stress could be one of the potential physiopathologic mechanisms through which H-HCY may lead to atherosclerotic cardiovascular damage. In humans, concordant studies evaluating the effect of long-term fasting H-HCY on endothelial function indicate that subjects with mild H-HCY show impaired flow-mediated endothelium-dependent dilation (5,6).
The mechanism through which fasting H-HCY exerts this adverse effect on endothelial function has not yet been evaluated. Moreover, patients with essential hypertension are characterized by endothelial dysfunction caused by reduced NO availability due to oxidative stress (7,8). At the present time, the possible effect of an interaction between H-HCY and essential hypertension on endothelial function remains to be determined. Therefore, the aim of the present study was to evaluate whether fasting H-HCY impairs endothelium-dependent vasodilation by oxidative stress in the forearm microcirculation of normotensive subjects. In addition, we also evaluated whether H-HCY and essential hypertension exert a synergistic adverse effect on endothelial function.
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Methods
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Patients and homocysteine measurement.
A total of 209 subjects were evaluated in our study. This study group included 90 normotensive subjects (mean age 49.2 ± 13 years; blood pressure [BP] 126.7 ± 9.3/81.7 ± 4.7 mm Hg, homocysteine level 12.5 ± 5.6 µmol/l) and 119 patients with essential hypertension (age 49.4 ± 10 years; BP 151.5 ± 14.6/98.6 ± 5.8 mm Hg, homocysteine level 13.1 ± 8.0 µmol/l). Subjects with a smoking history (>5 cigarettes/day), hypercholesterolemia (total cholesterol >200 mg/dl), diabetes mellitus and cardiac and/or other major diseases were excluded. The protocol was approved by the Ethical Committee of the University of Pisa, and all patients gave written, informed consent to participate in the study.
The subjects were defined as normal according to the absence of a family history of essential hypertension and elevated BP values (Table 1). Patients with essential hypertension were recruited from among the newly diagnosed cases in our outpatient clinic if they reported the presence of positive family history of essential hypertension, whenever supine arterial BP (after 10 min of rest) was consistently >140/90 mm Hg (Table 1). Secondary forms of hypertension were excluded by routine diagnostic procedures. Any pharmacologic treatment was discontinued for at least two weeks before performing the study.
In each individual venous blood was sampled after an overnight fast. For homocysteine, vitamin B12 and folate measurements, blood was put into tubes containing EDTA, placed on ice and immediately centrifugated. Samples were stored at –70°C until analysis. Serum homocysteine was measured by high-performance liquid chromatography (9), and vitamin B12 and folate by radioimmunoassay (ICN System).
According to previous evidence (5,6), H-HCY was defined as a homocysteine concentration corresponding to the 75th percentile of fasting homocysteine from the entire group of individuals recruited. In our study group of 209 individuals, the upper quartile of fasting homocysteine concentrations corresponded to  14.6 µmol/l, with no differences between normotensive subjects and hypertensive patients. From this group, 48 individuals withheld their consent to participate in the vascular study, and in 29 individuals, the experimental procedure was not performed because of technical problems. After these exclusions, a final group of 132 individuals (61 normotensive subjects and 71 hypertensive patients) was investigated. Finally, we included 12 normotensive subjects and 16 hypertensive patients with homocysteine levels in the upper quartile (14.6 µmol/l), and 11 normotensive subjects and 12 hypertensive patients with homocysteine levels in the lower quartile ( 8.7 µmol/l) (Table 1).
Experimental procedure.
Vascular reactivity was assessed by the perfused forearm technique. Briefly, the brachial artery was cannulated for drug infusion at systemically ineffective rates, as well as intra-arterial BP and heart rate monitoring. Forearm blood flow (FBF) was measured in experimental and the contralateral forearm by strain-gauge venous plethysmography. Circulation to the hand was excluded 1 min before FBF measurement by inflating a pediatric cuff around the wrist at suprasystolic BP. Details on the method have already been published (7).
Experimental design.
Endothelium-dependent vasodilation was estimated by a dose-response curve to intra-arterial acetylcholine (Farmigea S.p.A., Pisa, Italy; cumulative increase in infusion rates by 0.15, 0.45, 1.5, 4.5 and 15 µg/100 ml forearm tissue per min, 5 min each dose), whereas endothelium-independent vasodilation was estimated by intra-arterial sodium nitroprusside (SNP; Malesci, Milan, Italy; cumulative increase by 1, 2 and 4 µg/100 ml per min, 5 min each dose).
To evaluate the NO availability, acetylcholine was repeated under intra-arterial infusion of NG-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor (Clinalfa AG, Läufelfingen, Switzerland; 100 µg/100 ml per min) (10). Because L-NMMA modifies blood flow, the effect of acetylcholine in the presence of the NO clamp, which allows assessment of endothelial agonists in the presence of NO synthase blockade without changes in blood flow (11), was also evaluated. Thus, after 10 min of L-NMMA infusion, SNP was co-infused to neutralize the L-NMMA–induced vasoconstriction and restore baseline FBF. Then, SNP and L-NMMA were continued throughout the acetylcholine infusion (NO clamp system). In preliminary cases, a dose-ranging analysis was performed for SNP to identify the rate at which this compound could reverse the constrictor effect of L-NMMA (0.4 and 0.3 µg/100 ml tissue per min for 5 min in normotensive subjects and hypertensive patients, respectively). Moreover, in an adjunct group of hypertensive patients, prolonged infusion of a constant dose of SNP indicated that steady-state FBF was obtained after 2 min and remained at the restored baseline level up to 25 min of SNP infusion, hence demonstrating the stability of the NO clamp (data not shown).
To assess oxidative stress production, acetylcholine was again repeated under intra-arterial administration of vitamin C (Bracco, Milan, Italy; 8 mg/100 ml per min), an antioxidant (12) that was previously validated at this infusion rate in our experimental conditions (7). Finally, to evaluate whether oxidative stress could determine a decrease in NO availability, another dose-response curve to acetylcholine was repeated under simultaneous infusion of L-NMMA and vitamin C. Vitamin C was started 10 min before acetylcholine and continued throughout. After 10 min of L-NMMA before the infusion, the NO clamp technique was repeated.
Experimental data have shown that NO can act as a negative feedback modulator of NO synthase (13). Therefore, to assess the possibility that SNP co-infusion could change the inhibiting effect of L-NMMA in an adjunct group of young healthy normohomocystinemic subjects (n = 6), we produced a dose-response curve to acetylcholine during saline and during L-NMMA in the absence and presence of SNP co-infusion. Finally, to assess whether exogenous NO by SNP, per se, could affect the response to acetylcholine, independently of NO synthase blockade, we repeated a dose-response curve to acetylcholine under the NO clamp technique by replacing L-NMMA with the endothelium-independent vasoconstrictor noradrenaline (150 ng/100 ml per min).
A 30-min washout period was allowed between each dose-response curve, whereas a 60-min period was allowed when L-NMMA was infused.
Data analysis.
Because BP did not significantly change during the study, data were analyzed in terms of changes in FBF, and FBF increments were taken as evidence of local vasodilation. The FBF analyses were performed by an observer (A.V.) who had no knowledge of the homocysteine plasma values. Differences between two mean values were compared by use of the paired or unpaired Student t test, as appropriate. Dose-response curves to acetylcholine and SNP were analyzed by means of analysis of variance and analysis of co-variance, as appropriate, for repeated measures, and the Scheffé test was applied for multiple comparisons. The determinants of vasodilation to acetylcholine and SNP were assessed within the whole study group of normotensive subjects and patients with essential hypertension by means of univariate and multivariate linear regression analyses of homocysteine, folate, vitamin B12, cholesterol and glucose levels, age, gender and BP. The results are expressed as the mean value ± SEM.
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Results
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The clinical characteristics were similar between normohomocystinemic and hyperhomocystinemic individuals, within both the normotensive and hypertensive groups (Table 1).
H-HCY and endothelium-dependent and -independent vasodilation.
Vasodilation to acetylcholine was significantly (p < 0.01) blunted in patients with essential hypertension (FBF from 3.4 ± 0.4 to a maximum of 13.7 ± 4.3 ml/100 ml forearm tissue per min with the highest dose—an increase of 303%), as compared with normotensive control subjects (FBF from 2.8 ± 0.3 to 15.3 ± 3.9 ml/100 ml per min—an increase of 446%), whereas the response to SNP was similar in both groups (FBF from 3.4 ± 0.4 to 17.7 ± 2.6 [420%] and from 2.8 ± 0.3 to 15.9 ± 3.3 ml/100 ml per min [467%] in hypertensive patients and normotensive subjects, respectively; p = NS). Hyperhomocystinemic normotensive individuals showed a blunted response to acetylcholine, as compared with normohomocystinemic subjects (Table 2, Fig. 1). Similarly, hyperhomocystinemic patients with essential hypertension showed a further significantly blunted response to acetylcholine, as compared with both hypertensive patients with normal homocysteine levels (Fig. 1), and hyperhomocystinemic normotensive individuals (Table 2). In contrast, the response to SNP was not different between normohomocystinemic and hyperhomocystinemic individuals, in either the normotensive or hypertensive group (Table 2, Fig. 1). Considering the entire group of 61 normotensive subjects and 71 essential hypertensive patients who took part in the vascular study, on univariate analysis, according to a linear regression, the homocysteine plasma level was inversely correlated with vasodilation to acetylcholine, expressed as the percent increase at the highest infusion rate, both in normotensive subjects (r = –0.73, p < 0.00001) and hypertensive patients (r = –0.57, p < 0.001) (Fig. 2). In contrast, no correlation was found between the homocysteine level and the response to SNP (normotensive subjects: r = –0.03, p = NS; hypertensive patients: r = –0.07, p = NS). In both normotensive subjects and hypertensive patients, no relationship was found between the vascular response to acetylcholine and folate (r = 0.2 and r = –0.03, respectively; p = NS), vitamin B12 (r = 0.2 and r = –0.04, p = NS), total cholesterol (r = 0.3 and r = –0.08, p = NS), high density lipoprotein cholesterol (r = 0.8 and r = 0.16, p = NS), low density lipoprotein cholesterol (r = 0.2 and r = –0.05, p = NS), glucose (r = 0.04 and r = 0.02, p = NS), age (r = 0.2 and r = 0.1, p = NS), systolic BP (r = 0.07 and r = –0.07, p = NS) and diastolic BP (r = 0.19 and r = –0.14, p = NS). On multivariate analysis, the maximal response to acetylcholine was again inversely correlated with the homocysteine level, in both normotensive subjects (R2 = 0.69, p < 0.0001) and patients with essential hypertension (R2 = 0.51, p < 0.01), independent of the effects of other variables. However, when the data from normotensive subjects were considered together with data from patients with essential hypertension on multivariate analysis (R2 = 0.55), we found that homocysteine (p < 0.0001) and diastolic BP (p < 0.05) were the significant determinants of the response to acetylcholine.
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Table 2 Maximal Absolute (ml/100 ml tissue per min) and Percent Forearm Blood Flow Modifications Above Baseline During Acetylcholine, SNP, L-NMMA and Vitamin C Infusion
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Figure 1 Acetylcholine- and sodium nitroprusside (SNP)-induced increases in forearm blood flow (FBF) in normotensive subjects (top) and patients with essential hypertension (bottom), classified into those with normohomocystinemia (N-HCY; open circles) and hyperhomocystinemia (H-HCY; solid circles).
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Figure 2 Scatterplot of relationship between homocysteine (x axis) and maximal forearm blood flow response to acetylcholine (Ach; y axis) in normotensive subjects (top) and patients with essential hypertension (bottom). VD = vasodilation.
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H-HCY and responses to L-NMMA and vitamin C.
In normotensive subjects with hyperhomocystinemia, the inhibiting effect exerted by L-NMMA on acetylcholine (–27.6%), although still present, was lower than that observed in normohomocystinemic subjects (–52.7%), as compared with acetylcholine alone (Table 2, Fig. 3).
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Figure 3 Acetylcholine-induced increase in forearm blood flow (FBF) during saline (open circles) or NG-monomethyl-L-arginine (L-NMMA) (solid circles), in the absence (control) and presence of vitamin C in normohomocystinemic (N-HCY) and hyperhomocystinemic (H-HCY) normotensive subjects.
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In contrast, in both subgroups of patients with essential hypertension, L-NMMA infusion failed to affect vasodilation to acetylcholine (2% in normohomocystinemic patients vs. 2% in hyperhomocystinemic patients), as compared with acetylcholine alone (Table 2, Fig. 4). In normotensive subjects with normohomocystinemia, vitamin C did not modify vasodilation to acetylcholine (1%), as compared with acetylcholine alone. In contrast, the antioxidant significantly increased the response to acetylcholine in the hyperhomocystinemic subgroup (45%), as compared with acetylcholine alone, and the maximal vasodilation to acetylcholine was no longer different between the two subgroups (Table 2, Fig. 3). Finally, when the effect of L-NMMA was retested under vitamin C administration, the NO synthase inhibitor did not modify its inhibitory effect on acetylcholine in normohomocystinemic subjects (–2%), as compared with acetylcholine with L-NMMA alone, although it increased the inhibitory effect on acetylcholine in hyperhomocystinemic subjects (–44%), as compared with acetylcholine with vitamin C alone. Therefore, with vitamin C, the inhibiting effect of L-NMMA on acetylcholine was no longer different between the two groups (Table 2, Fig. 3). In patients with essential hypertension, the potentiating effect of vitamin C on acetylcholine-induced vasodilation was significantly greater in hyperhomocystinemic patients than in normohomocystinemic patients (97% and 24%, respectively), as compared with acetylcholine alone) (Table 2, Fig. 4). Therefore, after vitamin C infusion, the maximal vasodilation to acetylcholine was no longer different between hyperhomocystinemic and normohomocystinemic patients. Finally, under antioxidant infusion, the inhibiting effect of L-NMMA on vasodilation to acetylcholine was restored to the same extent in both hyperhomocystinemic and normohomocystinemic hypertensive patients (Table 2, Fig. 4).
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Figure 4 Acetylcholine-induced increase in forearm blood flow (FBF) during saline (open circles) or NG-monomethyl-L-arginine (L-NMMA) (solid circles), in the absence (control) and presence of vitamin C in normohomocystinemic (N-HCY) and hyperhomocystinemic (H-HCY) patients with essential hypertension.
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A highly significant direct correlation between homocystinemia and the potentiating effect of vitamin C on acetylcholine was found in normotensive subjects (r = 0.83, p < 0.001) and patients with essential hypertension (r = 0.82, p < 0.001).
In all patients, contralateral FBF did not significantly change throughout the study (data not shown).
Effect of SNP on the vascular response to acetylcholine.
In this adjunct group of healthy subjects, the vasodilation to acetylcholine (FBF from 2.7 ± 0.4 to 18.1 ± 3.9 ml/100 ml per min) was significantly (p < 0.0001) blunted by L-NMMA (FBF from 1.7 ± 0.3 to 6.1 ± 1.9 ml/100 ml per min). During NO clamping, L-NMMA, again, blunted the vasodilation to acetylcholine (FBF at baseline: 2.8 ± 0.3; FBF with L-NMMA: 1.7 ± 0.3; FBF with L-NMMA plus SNP: 2.8 ± 0.3; FBF with L-NMMA plus SNP plus acetylcholine: 10.1 ± 2.5 ml/100 ml per min). The percent inhibition of L-NMMA on acetylcholine was similar in the absence and presence of SNP co-infusion (54.6% and 54.3%, respectively). Finally, the vasodilation to acetylcholine with noradrenaline and SNP (FBF at baseline: 2.7 ± 0.3; FBF with noradrenaline: 1.8 ± 0.4; FBF with noradrenaline plus SNP: 2.7 ± 0.3; FBF with noradrenaline plus SNP plus acetylcholine: 17.9 ± 3.5 ml/100 ml per min) was similar to that observed with saline infusion.
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Discussion
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The present study was designed to evaluate whether fasting H-HCY impairs endothelium-dependent vasodilation by producing oxidative stress in the forearm microcirculation of normotensive subjects. In addition, it was also evaluated whether H-HCY and essential hypertension exert a synergistic adverse effect on endothelial function and, if so, whether this effect could be mediated by oxidative stress. In normotensive hyperhomocystinemic subjects, the vasodilation to acetylcholine was significantly reduced, as compared with that in normohomocystinemic individuals, an alteration specific for the endothelium, because the vascular response to SNP was similar between the two subgroups. In addition, when the whole group was analyzed, the response to acetylcholine, but not to SNP, showed a significant inverse correlation with plasma homocysteine concentrations. The relationship between homocysteine and endothelium-dependent vasodilation appears to be linear, demonstrating a continuous effect without a threshold value. Therefore, these data extend to the forearm resistance vessels, with previous studies demonstrating that fasting H-HCY is associated with impaired endothelium-dependent vasodilation in the conduit vessels of normotensive subjects (5,6).
Mechanisms responsible for H-HCY–induced endothelial dysfunction.
We employed L-NMMA and vitamin C to assess NO availability and oxidative stress, respectively. The effect of L-NMMA on the response to acetylcholine was assessed by the NO clamp technique, which allows restoration of basal arterial flow with SNP infusion. Control experiments demonstrate that SNP co-infusion has no direct effect on vasodilation to acetylcholine. In hyperhomocystinemic normotensive subjects, the diminished response to acetylcholine seems to be mediated by a reduction in NO availability, because in this subgroup, the inhibiting effect of L-NMMA on acetylcholine-induced vasodilation was significantly blunted, as compared with that in normohomocystinemic subjects.
The finding that acetylcholine still exerts a vasodilating effect in normohomocystinemic subjects receiving L-NMMA, is attributable to the fact that this L-arginine analogue is a competitive antagonist for NO synthase, and the dose used is unable to completely abolish NO production. However, we cannot exclude the alternative possibility that mediators different from NO, such as the hyperpolarizing factor or prostacyclin, account for the vasodilation to acetylcholine under NO synthase inhibition (3).
With regard to oxidative stress, in hyperhomocystinemic subjects, vitamin C administration reversed the homocysteine-induced endothelial dysfunction, because the antioxidant improved both vasodilation to acetylcholine and the inhibiting effect of L-NMMA on acetylcholine. It is worth noting that under vitamin C administration, the degree of vasodilation to acetylcholine and the inhibition exerted by L-NMMA were no longer different from the effects observed in normohomocystinemic subjects. Finally, in the overall group of normotensive subjects, homocystinemia showed a direct correlation with the potentiating effect of vitamin C on acetylcholine. Taken together, these results seem to indicate that in normotensive subjects, H-HCY impairs endothelium-dependent vasodilation by reducing NO availability. This alteration could be caused by increased oxidative stress in H-HCY. Such a hypothesis is in agreement with in vitro studies showing that homocysteine produces oxidative stress, represented, above all, by superoxide anions, which are known to inactivate NO (14). Although not planned in the present study, we recognize that it would be interesting to evaluate the effect of another L-arginine derivative, L-NAME. Given its property of inhibiting both NO and superoxide production by NO synthase, L-NAME should be able to provide information on the source of oxidative stress and, likewise, on NO availability.
H-HCY–induced endothelial dysfunction and oxidative stress.
In humans, the relationship between oxidative stress and H-HCY was previously examined, with conf1icting results. Chambers et al. (15) and Kanani et al. (16) found that, in healthy subjects, oral administration of vitamin C prevented the decrease in flow-mediated vasodilation after methionine loading. In contrast, Hanratty et al. (17) and Chao et al. (18) reported no change in oxidative status (as assessed by thiobarbituric acid–reactive substances and phosphatidylcholine hydroperoxide, respectively) after methionine loading. In addition, as far as fasting hyperhomocystinemia is concerned, Blom et al. (19) reported that lipid peroxidation (as assessed by thiobarbituric acid–reactive substances) was not increased in homocystinuric patients. A possible explanation for this discrepancy could be related to the different tools employed to evaluate the production of oxidative stress (vitamin C vs. plasma markers of lipid peroxidation). In fact, the antioxidant effect of vitamin C not only scavenges superoxide anions or inhibits low density lipoprotein oxidation, but also spares intracellular glutathione, which, together with vitamin C, is the primary regulator of the intracellular redox state (20), whereas the thiobarbituric acid–reactive substances are only systemic markers of oxidative stress (21). It is important to point out, however, that in our experimental conditions we can only speculate that vitamin C acts on endothelial function as an antioxidant, because we cannot exclude a different mechanism, such as stimulation of NO production (22).
Essential hypertension and H-HCY.
The present results indicate a blunted response to acetylcholine, but not to SNP, in hypertensive patients as compared with normotensive subjects, thereby confirming (7,8) the presence of endothelial dysfunction in essential hypertension. Moreover, hyperhomocystinemic hypertensive patients showed a further reduced vasodilation to acetylcholine, but not to SNP, as compared with normohomocystinemic hypertensive patients and hyperhomocystinemic normotensive subjects. Moreover, when the whole group was analyzed, acetylcholine, but not SNP, was inversely correlated with plasma homocysteine. It is worth noting that in the hypertensive group, on multivariate analysis, BP was not independently correlated with the maximal response to acetylcholine, thereby confirming a lack of interaction between these two variables (23). Only when the entire group was considered (normotensive subjects plus hypertensive patients) did diastolic BP come into play as a significant determinant of the response to acetylcholine. This finding was expected, given the inclusion of two groups characterized by a larger range of BP values and responses to acetylcholine.
Taken together, these data demonstrate a synergistic adverse effect between essential hypertension and H-HCY on endothelial function—a finding that is in agreement with the documented additive effect of the simultaneous presence of different cardiovascular risk factors (24–26) on endothelial dysfunction.
However, the relationship between H-HCY and impaired endothelium-dependent vasodilation in hypertensive patients is more complex. In patients with essential hypertension, vasodilation to acetylcholine is resistant to L-NMMA, independent of the dose of the NO synthase antagonist used (27), because such patients are characterized by the presence of an oxidative stress-induced reduction in NO availability (7). Therefore, mediator(s) other than NO (possibly a hyperpolarizing factor) account for the vascular relaxing responses to endothelial agonists (8). Thus, the profound reduction in endothelium-dependent vasodilation to acetylcholine observed in hypertensive patients with H-HCY cannot be explained by a reduction in NO availability. In contrast, increased production of oxidative stress seems to be responsible for the endothelial dysfunction in hyperhomocystinemic hypertensive patients, because in this subgroup, the potentiating effect exerted by vitamin C was significantly greater than that in patients with normal homocysteine levels. It should be noted that with vitamin C administration, the response to acetylcholine was no longer different between hyperhomocystinemic and normohomocystinemic hypertensive patients, and the inhibiting effect of L-NMMA on acetylcholine was restored in both subgroups. Moreover, in the whole group of patients with essential hypertension, a highly significant direct correlation was found between homocystinemia and the potentiating effect of vitamin C on acetylcholine.
Overall, these results seem to indicate that in patients with essential hypertension, H-HCY causes a further reduction in endothelial function by exacerbating the production of oxidative stress. Such augmented production of oxygen free radicals seems to act by impairing the alternative endothelial pathways that act as compensatory mechanisms for the reduced NO availability that is characteristic of essential hypertension. Administration of an antioxidant such as vitamin C can correct oxidative stress-induced endothelial dysfunction in hypertensive patients with either normal or increased homocysteine plasma levels by restoring NO availability.
Conclusions.
This study indicates that in the forearm microcirculation of normotensive subjects, H-HCY impairs endothelial function by producing oxidative stress that reduces NO availability. Moreover, in patients with essential hypertension, H-HCY can lead to a further reduction in endothelial function by exacerbating the production of oxidative stress and, consequently, by impairing NO-independent endothelial responses. This profound impact of H-HCY on endothelial function in both healthy subjects and patients with essential hypertension could be one of the possible mechanisms through which H-HCY can lead to an increased risk of cardiovascular disease.
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