CLINICAL STUDIES
Early endothelial dysfunction in adults at risk from atherosclerosis: different responses to L-arginine
Sara Thorne, MDa,
Michael J. Mullen, MBBSa,
Peter Clarkson, MBBSa,
Ann E. Donalda and
John E. Deanfield, FRCPa
a Vascular Physiology Unit, Great Ormond Street Hospital for Children, London, England, United Kingdom
Manuscript received October 15, 1997;
revised manuscript received March 9, 1998,
accepted April 9, 1998.
Address for correspondence: Dr. John E. Deanfield, Vascular Physiology Unit, Great Ormond Street Hospital for Children, London WC1N 3JE, England, United Kingdom
101723.523{at}compuserve.com
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Abstract
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Objectives. We sought to examine endothelial responses to L-arginine in three groups with isolated risk factors: hypercholesterolemia, smoking and insulin-dependent diabetes mellitus (IDDM).
Background. Endothelial dysfunction occurs early in atherosclerosis, predating clinical disease. We hypothesized that the nature of endothelial injury associated with individual cardiovascular risk factors might be different and that this might affect the response to L-arginine, the substrate for endothelial nitric oxide synthase.
Methods. We studied the effects of intravenous L-arginine on brachial artery flow-mediated dilation (FMD) and glyceryl trinitrate (GTN)–mediated dilation in 36 young subjects (18 to 40 years old) without clinical atherosclerosis: 9 each of normal control subjects, hypercholesterolemic subjects, cigarette smokers and subjects with IDDM.
Results. Baseline FMD was significantly impaired in hypercholesterolemic subjects (mean ± SD 1.7 ± 2.3%), smokers (1.6 ± 1.8%) and diabetic subjects (1.8 ± 1.5%) compared with that in control subjects (6.9 ± 3.3%, p = 0.001). The response to GTN was not significantly different between the subjects with risk factors and control subjects, apart from those with IDDM, in whom it was significantly impaired (p = 0.026). After infusion of L-arginine, there was no change in FMD in control or diabetic subjects. In hypercholesterolemic subjects and smokers, FMD improved from 1.9 ± 1.9% to 4.1 ± 2.1% (p = 0.01) and from 2.0 ± 1.71% to 3.1 ± 2.5% (p = 0.02), respectively.
Conclusions. FMD was impaired in all three risk factor groups; however, they responded differently to L-arginine, FMD being improved in hypercholesterolemic subjects and smokers but unchanged in diabetic subjects. These results indicate differing underlying pathophysiologies that may facilitate the design of treatment strategies for subjects with different risk factors.
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Abbreviations and Acronyms
| | ADMA | = asymmetric dimethyl arginine | | ANOVA | = analysis of variance | | FMD | = flow-mediated dilation | | GTN | = glyceryl trinitrate | | HbA1 | = glycosylated hemoglobin | | HDL | = high density lipoprotein | | IDDM | = insulin-dependent diabetes mellitus | | LDL | = low density lipoprotein | | NO | = nitric oxide |
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Hypercholesterolemia, cigarette smoking and insulin-dependent diabetes mellitus (IDDM) are established risk factors for atherosclerosis and premature cardiovascular disease (1–4) and are known to impair endothelial function, a key factor in early atherogenesis (5–8). Constitutive release of nitric oxide (NO) is a hallmark of healthy endothelium (9). It not only causes vasodilation but also has antiatherogenic effects, inhibiting interactions between platelets, leucocytes and the vessel wall and inhibiting smooth muscle proliferation (10). Loss of this function may therefore be important in the development of early vessel wall damage, leading eventually to atherosclerotic plaque formation (11). The mechanisms by which these three apparently disparate risk factors result in vascular damage remain incompletely understood. Decreased bioavailability of L-arginine may be a common factor, and the administration of L-arginine, the substrate for NO synthase, has been shown to improve endothelial function in hypercholesterolemia (12–15). However, there are conflicting results as to the effects of L-arginine in animal models of diabetes (16,17), and its impact in vivo in either IDDM or smoking has not been reported.
We therefore studied the effects of intravenous L-arginine on endothelial-dependent dilation in the conduit arteries of young subjects with hypercholesterolemia or IDDM or who smoked and compared the responses with those in control subjects without risk factors. The findings showed a heterogeneity of response, with improvement in flow-mediated dilation (FMD) in hypercholesterolemic subjects and smokers but no effect in subjects with IDDM, suggesting that the nature of early vascular injury differs between these three risk factor groups. This finding may need to be taken into account when designing therapeutic strategies aimed at the reversal or prevention of early arterial damage.
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Methods
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Subjects.
Twenty-seven subjects were selected for study if they were  40 years old and had no clinical vascular disease but had one of three major cardiovascular risk factors: hypercholesterolemia, cigarette smoking or IDDM. None had hypertension or a family history of premature vascular disease or were taking vasoactive or lipid-lowering medication. Hypercholesterolemic subjects (total plasma cholesterol  5.7 mmol/liter) were selected from a database. Current cigarette smokers were studied if they had smoked 2 pack-years (1 pack-year = 20 cigarettes/day for 1 year). Subjects with IDDM for >2 years had all presented with ketoacidosis at time of diagnosis and were recruited from diabetic clinics. None had clinical evidence of retinopathy or microalbuminuria (urinary albumin/creatinine ratios <0.01). Findings were compared with those of nine age- and gender-matched volunteers recruited from the hospital staff in whom the above risk factors had been excluded. All subjects gave written informed consent, and ethical approval was granted by the local ethics committee.
Study protocol.
Vascular responses in the brachial artery were studied in each subject on two separate days (3 to 10 days apart, with no therapeutic interventions between) during which either L-arginine (Martindale Pharmaceuticals UK) (0.1 g/kg body weight in 100 ml of 0.9% saline) or 100 ml 0.9% saline was administered intravenously over 20 min. The order of administration was randomized in double-blind fashion, and the same operator (A.E.D.) performed all ultrasound scans. Baseline blood samples were taken at rest, and L-arginine levels were also measured immediately and 20 min after infusions. Total plasma cholesterol was measured using the cholesterol C-system high performance CHOD-PAP method, and plasma triglycerides were measured using the GPO-PAP high performance enzymatic colorimetric test (both Boehringer-Mannheim GmbH, Diagnostica). High density lipoprotein (HDL) cholesterol was measured after precipitation of apoprotein B-containing lipoproteins, and low density lipoprotein (LDL) cholesterol was calculated according to the Friedwald formula (18). Plasma L-arginine was measured by ion exchange separation and reaction with ninhydrin (Pharmacia Alpha Plus amino acid analyzer). Glycosylated hemoglobin (HbA1) (Rapid EP system, Helena Laboratories) and urinary microalbumin were measured in the diabetic group. Noninvasive high resolution brachial artery ultrasound scans were performed before and after each infusion, using a modification of a technique that we have described previously (19). Arterial endothelial and smooth muscle function were assessed by examining brachial artery responses to endothelial dependent (flow mediated) (20) and independent (glyceryl trinitrate [GTN]–mediated) stimuli. A baseline scan was performed after 15 min of supine rest. A pneumatic tourniquet was inflated around the forearm for 4.5 min to a pressure of 300 mm Hg and then rapidly deflated, leading to reactive hyperemia in the hand and increased brachial artery blood flow. Brachial artery diameter and wall motion were recorded during the 55 to 65 s after cuff deflation using a high resolution (diameter evolution <3µm) A-mode echo tracking device (AMA, The Netherlands) and a 7-MHz linear array transducer with a stereotactic clamp and a standard Acuson 128XP10 system (Acuson). B- and M-mode images were obtained, and the exact position of each processed radio frequency peak corresponding to the vessel wall lumen interface, tagged using electronic trackers, was determined by an interpolation technique. Calculation of end-diastolic brachial artery diameter was performed automatically. At each stage of the experiment, continuous wall motion was recorded for 5 s, allowing the mean diameter of 4 to 10 cardiac cycles to be taken. Volumetric blood flow was calculated using pulsed-wave Doppler with a 1.5-mm sampling gate at an angle of 70° to the longitudinal axis, in the center of the artery, both at rest and at maximal flow during the first 15 s after tourniquet release, and images and velocity measurements were obtained simultaneously. Reactive hyperemia was expressed as the percent increase in blood flow. Because Doppler measurements were taken from the center of the vessel, calculations of flow may be overestimated; but because reactive hyperemia was calculated as a ratio of baseline and post–cuff deflation flows, relative values are accurate. After L-arginine or saline infusion, further baseline and reactive hyperemia measurements were made. After a 10-min recovery period, brachial artery response to GTN was measured.
Statistics.
Descriptive data are expressed as mean value ± SD, and statistical significance was inferred at a p value <0.05. Baseline FMD (Fig. 1) was calculated as the mean of the pre–L-arginine and pre–saline infusion FMD values, measured on the two separate days. Comparisons within individual groups were made by paired t tests. To compare responses between groups a two-way repeated measures analysis of variance (ANOVA) was used, and appropriate adjustments were made for multiple comparisons. For the whole group, the relation between the dependent variable FMD, change in FMD after L-arginine or placebo and response to GTN and the independent variables age; gender; vessel size; presence or absence of diabetes; triglycerides; and total, HDL and LDL cholesterol was explored using simple univariate analysis, and then the same variables were entered into a stepwise multivariate linear regression analysis. Within the diabetic and smoking groups, similar analyses were performed, introducing duration of diabetes and HbA1 or total number of pack-years as independent variables.
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Figure 1 FMD at baseline in the four subject groups. FMD (mean of presaline and pre–L-arginine levels) was significantly lower in the hypercholesterolemic, smoking and diabetic groups than in the control group. Bold lines represent mean values.
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Results
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Subject characteristics (Table 1).
There were no significant differences in age, gender or baseline heart rate and blood pressure between the groups or in lipid profile between the control subjects, smokers and diabetic subjects. Total plasma and LDL cholesterol were higher (p < 0.0001), and HDL cholesterol lower (p = 0.02) in the hypercholesterolemic group than in the control group. The mean number of pack-years smoked by the smoker group was 10 ± 6.6 (range 2 to 20). In the diabetic group, mean age at diagnosis was 15.1 ± 8.9 years (range 5 to 32), and mean duration of diabetes was 14.9 ± 7.4 years (range 3 to 29). Mean glycosylated hemoglobin levels were 10.8 ± 2.5% (range 8.1% to 14.4%). Venous blood glucose in all subjects was between 3 and 10 mmol/liter at the time of each scan. Plasma L-arginine rose 25-fold from a mean of 77 ± 43.7 µmol/liter (range 47 to 212) to 1,992 ± 1,052.6 (range 504 to 4,017) µmol/liter. There were no differences between the groups in preinfusion and postinfusion plasma L-arginine concentrations. No subject had side effects after the L-arginine or placebo infusions; two complained of shortlived headache after GTN.
Preinfusion vascular study.
There were no significant differences within any of the groups between pre–saline and pre–L-arginine infusion FMD (Fig. 2), and mean rest vessel size, blood flow and percent increase in blood flow during reactive hyperemia did not differ significantly between the four groups (data not shown), indicating both the reproducibility of the technique and the stable physiologic status of the subjects. Baseline FMD was significantly reduced in hypercholesterolemic, smoking and diabetic subjects compared with that in control subjects (p = 0.001) (Fig. 1).
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Figure 2 FMD responses to L-arginine and saline. There were no significant changes in FMD after saline infusion in any subject group. After L-arginine infusion, FMD improved significantly in the hypercholesterolemic subjects (b) (p = 0.01) and smokers (c) (p = 0.02) but was unchanged in the control (a) and diabetic groups (d). In hypercholesterolemic subjects and smokers, the percent change in FMD was significantly greater after L-arginine than saline infusion (p = 0.03 and p = 0.02, respectively); there were no differences in percent change in FMD in either the control or diabetic groups. There were no significant differences between presaline and pre–L-arginine FMD values (p values not shown). Bold lines represent mean value ± SD.
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Postinfusion vascular studies (Fig. 2 and 3).
After L-arginine or saline infusions, there were no changes in heart rate, blood pressure, rest vessel size, rest blood flow or reactive hyperemia, indicating that the infusions had no effect on distal vascular tone. FMD improved significantly after L-arginine infusion in the hypercholesterolemic and smoking groups, from 1.9 ± 1.9% to 4.1 ± 2.1% (p = 0.03) and from 2.0 ± 1.71% to 3.1 ± 2.5% (p = 0.02), respectively. However, there was no significant change in the control (6.9 ± 3.3% to 6.7 ± 3.3%, p = 0.22) and diabetic (1.8 ± 1.5% to 2.2 ± 2.1%, p = 0.28) groups. On two-way repeated measures ANOVA, there were significant differences in the response to L-arginine in the hypercholesterolemic (p = 0.001) and smoking (p = 0.02) groups but not in the diabetic group (p = 0.9) compared to that in the control group. There was no change in FMD or any significant difference between the groups in response to saline infusion. There were no significant differences in brachial artery dilation in response to GTN between the control (23.1 ± 7.3%) and hypercholesterolemia or smoking groups (18.7 ± 6.9%, p = 0.09 and 20.1 ± 9.9%, p = 0.46, respectively). However, in the diabetic group dilation in response to GTN was significantly reduced compared with that in the control group (13.3 ± 9.6%, p = 0.026). There was no significant difference in response to GTN after L-arginine compared with that after saline infusion in any of the groups.
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Figure 3 GTN-induced dilation in the four subject groups. In the hypercholesterolemic subjects and smokers, GTN-induced dilation was not significantly different than that in control subjects but was significantly reduced in diabetic subjects (p = 0.026). Bold lines represent mean values.
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Analysis of results revealed no "order effect" dependent on whether the saline or L-arginine infusions were given first. There were no significant relations between the three dependent variables FMD, change in FMD after placebo and response to GTN and age, vessel size, lipid profile or presence or absence of diabetes for the whole group, or with HbA1, duration and age at diagnosis of diabetes, total pack-years smoked within groups. On univariate but not multivariate analysis, change in FMD in response to L-arginine correlated significantly and directly with total plasma cholesterol and triglyceride levels (r = 0.33, p = 0.049 and r = 0.36, p = 0.032 respectively).
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Discussion
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The present study shows that young subjects with different cardiovascular risk factors and impaired endothelial-dependent relaxation have differing responses to L-arginine, with FMD improving in hypercholesterolemic subjects and smokers but not in subjects with IDDM, a new finding continuing our previous work. Thus, even at this early preclinical stage of atherosclerosis, the effects of L-arginine are determined by the differing nature or extent of vascular damage associated with cardiovascular risk factors, a finding that should influence the development of strategies to reverse or prevent early arterial damage.
FMD and the L-arginine/NO pathway.
There is increasing evidence that the endothelial L-arginine/NO pathway has a key antiatherogenic role (10). The same cardiovascular risk factors that are causally implicated in the development of clinical atherosclerotic disease cause impairment of endothelium-dependent dilation. This impairment has been demonstrated in the coronary and peripheral microvascular circulations (5,21–23) and in large conduit arteries using a high resolution ultrasound technique (19,24–27). FMD measurements are reproducible (28), blocked by NG-monomethyl-L-arginine (LNMMA) (29) and correlate with those demonstrated invasively in the coronary vasculature (30). Impairment of dilation in response to flow represents a failure of endothelial-dependent homeostasis but provides no information on the nature of the vascular injury responsible. The reported beneficial effects of L-arginine administration in vivo remain controversial. The purposes of our present study were thus twofold: 1) to describe the effects of L-arginine in conduit arteries of young subjects; and 2) to use the response to L-arginine to further characterize the vascular abnormalities associated with three different risk factor groups. We therefore identified young subjects who had a single powerful cardiovascular risk factor and who were otherwise matched for age, gender and other physical characteristics known to affect shear stress–mediated endothelial function. The young age of the study group avoided the confounding influence of increasing age on FMD (31). The small size of the study group may have also contributed to the lack of a relation between FMD and age or vessel size (19). We previously showed (15) improvement in FMD after dietary supplementation with L-arginine in young hypercholesterolemic subjects, but the effects of this approach in cigarette smokers and in patients with IDDM have yet to be determined. The findings of this short-term study, which resulted in transiently high circulating levels of L-arginine, cannot be extrapolated directly to predict benefits that might be expected from long-term therapy with lower L-arginine concentrations. We did not set out to determine the relative improvements that could be achieved and thus only compared responses in the risk factor groups with those found in the risk factor–free control group. Separate studies would be required to determine whether the degree of response differs between risk factor groups and whether similar improvements can be achieved with long-term oral therapy.
Hypercholesterolemia.
The differing responses to L-arginine may either reflect differences in the pathogenesis of atherosclerosis between the three risk factor groups or differences in the way each risk factor responds to L-arginine. Impaired endothelial dependent dilation in hypercholesterolemia has been the most extensively researched. Our findings are consistent with previous studies showing that L-arginine improves endothelial function in animals (13) and humans (12,14,15), but the mechanisms of action of L-arginine are unclear. L-Arginine is unlikely to act merely by increasing NO substrate availability (32), and NOS may interact with intracellular L-glutamine (33) and the endogenous competitive inhibitor asymmetric dimethyl arginine (ADMA) (34). However, NO production may be preserved or even increased in hypercholesterolemia (35,36), so that impaired vascular responses may be due to accelerated NO breakdown (37). Our study suggests that L-arginine is acting neither as a direct vasodilator nor to increase basal NO activity. We found that the magnitude of response to L-arginine for the entire study group was directly related to total plasma cholesterol and triglyceride levels, suggesting that the detrimental endothelial effects of hyperlipidemia might be particularly amenable to reversal by L-arginine.
Smoking.
The importance of smoking as a primary risk factor for coronary disease is likely to increase over the next four decades, unless the rise in smoking among young people can be reversed (38). Cigarette smoking increases oxidative stress because of low circulating levels of antioxidants and increased levels of oxygen-derived free radicals and lipid peroxides that degrade NO (39,40), enhance monocyte adhesion (41) and increase the susceptibility of LDL to oxidation. Oxidized LDL exerts numerous cytotoxic and atherogenic effects (42,43) as well as interfering with the L-arginine/NO pathway by decreasing both L-arginine uptake and NO synthase expression (44). The observation that antioxidant vitamins can both reduce smoking-induced lipid peroxidation and endothelial dysfunction suggests a way forward in planning therapeutic strategies (45,46). The present study purposefully examined separate risk factors and showed that each can cause damage in isolation. However, experimental (23,47) and epidemiologic (48) evidence indicates that smoking and cholesterol levels may be importantly linked. Apart from encouraging smokers to quit, a combination of L-arginine, antioxidant and cholesterol-lowering therapy might therefore act synergistically to maximize NO bioavailability.
Diabetes.
Of the three risk factors studied, IDDM appears to be the worst, with no improvement in FMD after L-arginine infusion. This finding is in accord with its known adverse impact on early arterial injury and cardiovascular mortality (3). Although reduced FMD and impaired response to GTN have been reported previously, both by us and other workers (27,49), there is not yet a consensus on these findings (50). These effects complicate the interpretation of the nature of vascular injury and may reflect reduced smooth muscle responses to local NO or abnormalities in the vessel wall matrix itself. An additional element to vascular damage is therefore likely to be present in IDDM, and hyperglycemia-related oxidant stress may be an important factor. Autooxidation of glucose, stimulation of the polyol pathway and the formation of advanced glycosylation end products all increase oxidant stress in diabetes, and the effect is compounded by the reduced levels of antioxidants that occur in diabetes (6,51,52). There is also evidence of a failure to regulate intracellular arginine levels in diabetes (53). It is therefore not surprising that the diabetic group showed no improvement in vascular reactivity with L-arginine. Our previous work has shown that plasma LDL and FMD are related in IDDM (27). An aggressive approach to other additional risk factors may thus be beneficial in IDDM, although the incremental benefit that L-arginine may have in addition to lipid-lowering and antioxidant therapy is unknown (54). Further studies are needed to assess both their roles and those of competitive inhibitors such as ADMA in the differing perturbations of NO synthase in the different risk factor groups.
Conclusions.
We showed that the endothelial dysfunction characteristic of young people with different cardiovascular risk factors may differ in its pathophysiology, and this difference is presumably responsible for the distinct vascular responses observed in response to the short-term administration of L-arginine in the diabetic cohort. Interventions to alter the natural history of atherogenesis are likely to be most effective early in young people, when intuitively arterial damage is more likely to be reversible. The information from the present study will help to design therapeutic strategies appropriate to different cardiovascular risk factors, and to optimize endothelial protection, multifactorial approaches are likely to be needed.
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Footnotes
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Dr. Mullen is supported by a Bristol Myers Squibb Cardiovascular Fellowship; Dr. Clarkson and Dr. Thorne by the British Heart Foundation, London; Ms. Donald by a grant from CORDA, London, England, United Kingdom; Bristol-Myers Squibb, Hounslow, Middlesex, England, United Kingdom.
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