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Correlates of arterial stiffness in an ageing population: Role of asymmetric dimethylarginine
Pharmacological Research 60 (2009) 503–507
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Review
Correlates of arterial stiffness in an ageing population: Role of asymmetric dimethylarginine Doan T.M. Ngo a , Aaron L. Sverdlov a , John J. McNeil b , John D. Horowitz a,∗ a Vascular Disease and Therapeutics Research Group, Basil Hetzel Institute, The Queen Elizabeth Hospital, Department of Medicine, The University of Adelaide, 28 Woodville Road, Woodville South, 5011 South Australia, Australia b Monash University, Department of Epidemiology, Melbourne, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 15 May 2009 Received in revised form 16 June 2009 Accepted 16 June 2009 Keywords: Asymmetric dimethylarginine Dimethylarginine dimethylaminohydrolase Augmentation index Arterial stiffness Endothelial function Nitric oxide
a b s t r a c t A number of previous investigators have demonstrated that arterial augmentation index (AIx), a measure of apparent arterial stiffness, reflects in part vascular endothelial function, and that AIx is modulated by nitric oxide (NO) responses. We evaluated AIx in a population of 253 ageing subjects (mean age 63.4 ± 6 (standard deviation, SD) years) and its relationship to (i) plasma levels of asymmetric dimethylarginine (ADMA), a marker and mediator of vascular endothelial dysfunction and (ii) the ratio of ADMA to its non-metabolised enantiomer symmetric dimethylarginine (SDMA), an inverse index of ADMA metabolic clearance. Evaluation was performed by univariate followed by multivariate analyses. On multivariate analyses, both ADMA (ˇ = 0.16, p = 0.01) and ADMA:SDMA (ˇ = 0.21, p < 0.001) ratio were significant direct correlates of AIx. Other significant correlates of AIx on multivariate analysis were: use of angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers (ACEi/ARB) (ˇ = −0.24, p = 0.004), smoking history (ˇ = 0.15, p = 0.007), male gender (ˇ = −0.38, p < 0.001), creatinine clearance (CrCL) (ˇ = −0.25, p < 0.001), and history of hypertension (ˇ = 0.17, p = 0.04). We conclude that (1) endothelial dysfunction engendered by impairment of NO synthesis may represent the basis for increased arterial stiffness in ageing individuals and (2) the fundamental biochemical anomaly may be impairment of ADMA clearance. These pathophysiological factors are likely to be relevant to optimize therapy to ameliorate disorders of arterial compliance in the ageing population. © 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2.
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4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Clinical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Biochemical measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Measurement of aortic augmentation index (AIx) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Clinical univariate correlates of augmentation index (AIx) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Multivariate analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
503 504 504 504 504 504 504 505 505 505 506 506 506 507
1. Introduction
∗ Corresponding author. Tel.: +61 8 8222 6000; fax: +61 8 8222 7201. E-mail address: [email protected] (J.D. Horowitz). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.06.006
Systolic hypertension is an increasingly common condition in ageing individuals, with well-characterised associations with risks of left ventricular (LV) hypertrophy, congestive heart failure, and
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cerebrovascular accidents [1]. The presence of systolic hypertension is in turn the result of increased arterial stiffness, with a resultant acceleration of pulse wave reflection and therefore incremental afterload [2]. Thus, increased arterial stiffness is a cause of cardiovascular morbidity and mortality [3–6]. However, it is now clear that this incremental stiffness is itself variable, with smooth muscle tone, rather than entirely fixed, as a result of vascular remodelling, fibrosis and calcification [7]. The vascular endothelium plays a pivotal role in the regulation of vascular tone via regulating release of vasoactive factors responsible for the maintenance of normal arterial wall composition. Dysfunction of the vascular endothelium, due to decreased nitric oxide (NO) generation or bioavailability, plays an important role in arterial stiffness, and atherosclerosis. Endothelial release of NO attenuates the vasoconstrictor effects of noradrenaline, endothelin, angiotensin II, and serotonin. NO also inhibits vascular smooth muscle proliferation which could otherwise induce medial thickening and/or myointimal hyperplasia. Furthermore, NO inhibits the interaction of circulating blood elements with the vessel wall. It limits vascular recruitment of platelet aggregation and leukocyte adherence by inhibiting the expression of proinflammatory cytokines, chemokines, and leukocyte adhesion molecules [8]. Nitric oxide is formed in endothelial cells from the amino acid l-arginine by endothelial isoform of NO synthase (eNOS). Asymmetric dimethylarginine (ADMA) is a naturally occurring l-arginine analogue, and hence a competitive inhibitor of eNOS, derived from the proteolysis of proteins containing methylated arginine residues. Symmetric dimethylarginine (SDMA) is the structural isomer of ADMA. Both ADMA and SDMA derive from intranuclear methylation of l-arginine residuals and are released into the cytoplasm after proteolysis. Increased ADMA concentrations have been associated with cardiovascular disease states and risk factors [9]. While, ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH), SDMA is eliminated entirely by renal excretion [10]. Therefore, the ADMA:SDMA ratio represents a “reverse” index of DDAH activity: a high ADMA:SDMA ratio indicates low DDAH activity, and thus selectively impaired ADMA (metabolic) clearance [11,12]. Recently, DDAH has emerged as an important regulator of NO bioavailability and vascular function, affecting tissue and plasma ADMA concentrations. Overexpression of both DDAH isoforms (DDAH1 and DDAH2) in animals reduced ADMA levels and increased NO synthesis, lowers vascular resistance [13,14]. Conversely, defects in DDAH expression and activity could theoretically contribute to arterial stiffness by regulating ADMA kinetics and hence NO synthesis. In this study, we therefore examined the relations of ADMA, SDMA and ADMA:SDMA ratios to aortic augmentation index (AIx), a composite measure of elastic plus muscular artery stiffness and wave reflection [15], in a random ageing Western population with non-selective cardiovascular risk factors. The hypotheses tested were: (i) ADMA concentrations would be correlated with AIx and (ii) ADMA:SDMA ratio, as an index of impaired ADMA clearance, would also be correlated with AIx. 2. Methods 2.1. Subjects Subset members (n = 253) of the general community (aged between 51 and 77 years) participating in the North Western Adelaide Health study were recruited to identify factors associated with presence of aortic valve sclerosis by echocardiography [16]. In the present analysis, we assessed the relationship between ADMA, SDMA, ADMA/SDMA ratios and AIx on all subjects with available data. The study was approved by local ethics com-
mittees, and all subjects gave written informed consent. As a component of evaluation, AIx and ADMA concentrations were evaluated. 2.2. Clinical characteristics All patients’ cardiovascular risk factors such as history of hypertension, hypercholesterolemia, diabetes mellitus, smoking habits, previous angina/ischemic events, were delineated at interview and details are as previously published [15]. All patients underwent transthoracic echocardiography and left ventricular diameters and wall thicknesses were measured from 2D-guided M-mode echocardiography. Height and weight were measured, and body mass index (BMI) was calculated. 2.3. Biochemical measurements Blood was collected from all patients into heparinised tubes, centrifuged at 4 ◦ C at 2700 × g for 20 min, and plasma was stored at −80 ◦ C until assay. Concentrations of ADMA and SDMA in plasma were measured by high-performance liquid chromatography (HPLC) using the derivatisation reagent AccQ-Fluor after solid phase extraction as previously described [17]. Lipid profile, high sensitivity C-reactive protein (hs-CRP), serum creatinine, calcium, and phosphate levels were obtained. Creatinine clearance (CrCl) was calculated according to the Cockcroft–Gault equation, and indexed for body surface area (BSA) using the Dubois and Dubois formula. 2.4. Measurement of aortic augmentation index (AIx) Pulse waveform analysis (PWA) was performed non-invasively with a commercially available SphygmoCor system (AtCor Medical, Sydney, Australia), as previously described [18]. All subjects were asked to lie down in a quiet room for 15 min prior to procedure. Briefly, PWA was computed from the radial artery at the wrist, and recorded by applanation tonometry using a high fidelity micromanometer. Three recordings of 10 sequential waveforms were acquired for each subject; a validated, generalized transfer function was used to generate the corresponding central aortic pressure waveform from which AIx was derived. Only high quality recordings with in-device quality index ≥90% were used. All augmentation indices were corrected for a standard heart rate of 75 bpm. 2.5. Statistical analyses All data are expressed as mean ± standard deviation (SD) unless otherwise stated. Normal distribution was tested for all continuous variables, and skewed data were normalized either by log or square root transformation. Comparisons between groups for non-parametric data were made using the Mann–Whitney test. Correlations between transformed, continuous non-parametric data were made using linear regression. As regards multivariate comparisons, to gain insights into the relations of AIx and methylarginines to the risk factors, we constructed separate backward multiple linear regression models with either ADMA or ADMA:SDMA ratio. In these models, AIx is the outcome variable, and methylarginines and ADMA/SDMA ratio were independently assessed along with other putative correlates of AIx such as: age, gender, plasma SDMA concentrations, plasma calcium × phosphate product (Ca × PO4), history of diabetes mellitus, hypertension, smoking, hypercholesterolemia, previous angina/ischemic events, use of angiotensin-converting enzyme inhibitor (ACEi)/angiotensin-receptor blocker (ARB), statins, calculated CrCL, BMI, hs-CRP, and LV mass index. These variables were
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Table 1 Patient demographics. Age (mean ± SD) (years) Gender (%male) Previous angina/myocardial infarction (%) History of hypercholesterolemia (%) Hypertension (%) Diabetes mellitus (%) Smokers/ex-smokers (%) Family history (%) BMI (kg/m2 ) ACEi/ARB therapy (%) Statin therapy (%)
63.4 ± 6.0 43.5 13.5 58.7 41.8 11.2 14.3 52.2 28.2 ± 5.0 33.1 32.1
Table 2 Univariate correlates of high AIx. Parameter
p value
Older age Female gender No history of previous MI/ischemia Low BMI History of hypercholesterolemia Hypertension No history of diabetes mellitus Smoker/ex-smoker Absence of ACEi/ARB Statin therapy Family history Low CrCL (indexed for BSA) mL/min/1.73 m2 Low left ventricular mass index (g/m2 ) High Ca × PO4 hs-CRP High ADMA Low SDMA High ADMA/SDMA ratio
0.1 <0.001 <0.05 0.09 0.14 0.4 <0.05 <0.05 <0.05 0.8 0.4 <0.001 0.001 0.09 0.9 0.1 0.02 0.002
included either due to statistical significance on univariate analyses or as plausible clinical correlates. All analyses were performed using SPSS 13 software, and a p value of <0.05 was considered to be statistically significant.
3. Results Patients’ baseline and demographic characteristics are presented in Table 1. The clinical characteristics of this cohort were consistent with a Western ageing population with high BMI and multiple cardiovascular risk factors. Approximately 1/3 of subjects were being treated with an ACEi/ARB or statin for hypertension or hypercholesterolemia, respectively.
Fig. 1. Relationship between creatinine clearance (CrCL) indexed for body surface area (BSA) and Aix (R = −0.36, p < 0.001).
3.1. Clinical univariate correlates of augmentation index (AIx) The clinical correlates of augmentation index are summarized as in Table 2. Female gender, absence of a history of ischemia, absence of diabetes, history or current smoking status, and absence of ACEi/ARB use were significantly associated with high AIx. Left ventricular mass index was also inversely associated with AIx. Biochemically, high AIx was correlated with low CrCL (Fig. 1). ADMA concentrations tended to be associated directly with high AIx (R = 0.11, p = 0.1)(Fig. 2A). While high ADMA/SDMA ratios were strongly associated with high AIx (Fig. 2C; R = 0.19, p = 0.002). 3.2. Multivariate analyses Results of multivariate analysis performed utilizing ADMA concentrations as a potential correlate of AIx are summarized in Table 3. ADMA concentrations were directly related to Aix; both SDMA concentrations and CrCL values represented inverse correlates of AIx. Other independently significant correlates of AIx were smoking, hypertension, female gender, and absence of therapy with ACEi/ARB. Multivariate analysis utilizing ADMA:SDMA ratio (Table 4) indicated a strong association between this ratio and AIx, with no change in other independent correlates).
Fig. 2. Correlations between (A) ADMA (R = 0.11, p = 0.1) and (B) ADMA/SDMA (R = 0.0.19, p = 0.002) ratio vs AIx.
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Table 3 Multiple regression analysis: correlates of AIx including ADMA and SDMA in the model.
ACEi/ARB Smoking CrCL Gender (male) Hypertension ADMA SDMA
ˇ Coefficient
p value
−0.23 0.15 −0.27 −0.37 0.18 0.16 −0.26
0.005 0.007 <0.001 <0.001 0.03 0.01 <0.001
Table 4 Multiple regression analysis: correlates of AIx including ADMA:SDMA ratio.
ACEi/ARB Smoker/ex-smoker CrCL Gender (male) Hypertension ADMA/SDMA ratio
ˇ Coefficient
p value
−0.24 0.15 (0.25 (0.38 (0.17 0.21
0.004 0.007 <0.001 <0.001 0.036 <0.001
4. Discussion Measurements of arterial stiffness have been increasingly used in the clinical assessment of patients as a recognised predictor for cardiovascular (CV) events, including CV mortality [4,19] myocardial infarction [20], and stroke [21]. It has been shown that arterial stiffness increases with age, and is correlated directly with CV risk factors such as hypertension, metabolic syndrome, diabetes, obesity, hypercholesterolemia, and elevated C-reactive protein [22,23]. Increased arterial stiffness has been associated with abnormal activation of vascular pro-fibrotic markers such as matrix metalloproteinases [24], and the renin–angiotensin system [25,26]. In this study, we demonstrated that elevated plasma concentrations of ADMA, a marker and mediator of endothelial dysfunction, are directly correlated with apparent arterial stiffness, as measured by AIx in an ageing population, independent of other CV risk factors. More importantly, the observed direct correlation between AIx and ADMA concentrations is likely to reflect decreased (redox-sensitive) enzymatic clearance via DDAH. ADMA exerts vasoconstrictor responses in normal subjects [27], and indeed has previously been shown to increase arterial stiffness as measured by AIx [28]. However, this finding does not in itself imply that plasma ADMA concentrations are major determinants of AIx values either in normal subjects or in any pathological condition: in this area, the previous literature contains heterogeneous findings. The largest population study on this issue to date, the PREVENCION study, found that ADMA concentrations were not associated with carotid–femoral pulse wave velocity [29]. A study in normal young adults [30] revealed a paradoxical relationship between ADMA and pulse wave velocity which was not significant on multivariate analysis. On the other hand, Weber et al. [31] found a weak correlation between ADMA concentrations and AIx in patients undergoing cardiac catheterization. Finally, in patients undergoing hemodialysis for advanced renal disease, post dialysis falls in ADMA were correlated with decreases in AIx, suggesting a cause-and-effect relationship in these circumstances. We now report for the first time, a positive relationship between ADMA and AIx, independent of other CV risk factors in a cross-sectional ageing population. More remarkedly, our data also suggest that reduced enzymatic clearance of ADMA is a potential mechanism for ADMA accumulation and hence arterial stiffness.
Steady-state ADMA concentrations are predominantly modulated by activity of DDAH [32], but also by rates of ADMA formation, via enzymatic protein catabolism. Several lines of evidence suggest that decreased aortic expression of DDAH is associated with increased ADMA concentrations and decreased NO synthesis [14]; and that the reverse effects are seen with over-expression of DDAH [13,33]. For example, Zoccali et al. [11] found that plasma ADMA concentrations were markedly increased during the recovery phase of acute infection/inflammation with an accompanying rise in ADMA:SDMA ratio, indicating a reduction in DDAH activity. The clearance of SDMA is entirely renal, while that of ADMA is predominantly enzymatic via DDAH-catalysed inactivation. Hence, changes in ADMA:SDMA ratio should correlate inversely with DDAH activity, and have been utilized as a surrogate measure of DDAH activity [11,12]. In the current study, the ADMA:SDMA ratio was strongly correlated with AIx: these findings therefore suggest that impaired DDAH activity may underlie arterial “stiffening” with advanced age. However, how closely ADMA:SDMA ratio relates to DDAH activity remains uncertain, largely due to the impracticability of measuring total body DDAH activity. Despite the difficulties inherent in the process, further studies should address the relationship between intravascular DDAH assays [32,33] and plasma ADMA:SDMA ratio. If impaired DDAH activity indeed represents the “primary” basis for the observed association between AIx and ADMA concentrations, it is possible that this might be triggered by any increase in redox stress [34], or indeed via reduced NO availability itself [35]. Furthermore, the findings in the current study related to an inverse relationship between ACEi/ARB therapy and AIx are consistent with the known effects of these agents, again partially mediated by incremental NO availability as well as reduced redox stress [36,37]. In line with the recent meta-analysis by Kielstein et al. [38], we also observed an inverse relationship between SDMA concentrations and CrCL (data not shown). Correlations between SDMA concentrations and AIx are likely to reflect associated changes in renal function rather than the weak biological effects of SDMA in NO formation [12]. The main caveats of this investigation are that many of the patients were undergoing treatment for hypertension and hypercholesterolemia. This therapy, particularly with ACEi/ARB, may have complicated evaluation of the ADMA vs AIx relationship, both by reducing AIx and by altering ADMA kinetics [39,40]. Furthermore, we did not observe any significant relationship between left ventricular mass index and AIx: again this finding may relate in part to the effects of pharmacotherapy. In conclusion, ADMA and ADMA:SDMA ratio are significant correlates of AIx in this ageing population, presumably reflecting impaired ADMA clearance. Future studies related to therapy of predominantly systolic hypertension in the elderly should therefore, in part, address the hypothesis that amelioration of disorders of arterial compliance is best addressed via optimization of ADMA clearance. From a theoretical point of view, this argues in particular for the advantages of agents such as ACEis, which appear to protect vascular DDAH activity [36,37], as first-line therapy for treatment of disorders of arterial compliance, such as systolic hypertension, in the elderly. Conflict of interest None of the authors have any conflict of interest. Acknowledgements This work was supported in part by research grants from the National Health and Medical Research Council and National Heart Foundation of Australia.
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Review
Correlates of arterial stiffness in an ageing population: Role of asymmetric dimethylarginine Doan T.M. Ngo a , Aaron L. Sverdlov a , John J. McNeil b , John D. Horowitz a,∗ a Vascular Disease and Therapeutics Research Group, Basil Hetzel Institute, The Queen Elizabeth Hospital, Department of Medicine, The University of Adelaide, 28 Woodville Road, Woodville South, 5011 South Australia, Australia b Monash University, Department of Epidemiology, Melbourne, Victoria, Australia
a r t i c l e
i n f o
Article history: Received 15 May 2009 Received in revised form 16 June 2009 Accepted 16 June 2009 Keywords: Asymmetric dimethylarginine Dimethylarginine dimethylaminohydrolase Augmentation index Arterial stiffness Endothelial function Nitric oxide
a b s t r a c t A number of previous investigators have demonstrated that arterial augmentation index (AIx), a measure of apparent arterial stiffness, reflects in part vascular endothelial function, and that AIx is modulated by nitric oxide (NO) responses. We evaluated AIx in a population of 253 ageing subjects (mean age 63.4 ± 6 (standard deviation, SD) years) and its relationship to (i) plasma levels of asymmetric dimethylarginine (ADMA), a marker and mediator of vascular endothelial dysfunction and (ii) the ratio of ADMA to its non-metabolised enantiomer symmetric dimethylarginine (SDMA), an inverse index of ADMA metabolic clearance. Evaluation was performed by univariate followed by multivariate analyses. On multivariate analyses, both ADMA (ˇ = 0.16, p = 0.01) and ADMA:SDMA (ˇ = 0.21, p < 0.001) ratio were significant direct correlates of AIx. Other significant correlates of AIx on multivariate analysis were: use of angiotensin-converting enzyme inhibitors/angiotensin-receptor blockers (ACEi/ARB) (ˇ = −0.24, p = 0.004), smoking history (ˇ = 0.15, p = 0.007), male gender (ˇ = −0.38, p < 0.001), creatinine clearance (CrCL) (ˇ = −0.25, p < 0.001), and history of hypertension (ˇ = 0.17, p = 0.04). We conclude that (1) endothelial dysfunction engendered by impairment of NO synthesis may represent the basis for increased arterial stiffness in ageing individuals and (2) the fundamental biochemical anomaly may be impairment of ADMA clearance. These pathophysiological factors are likely to be relevant to optimize therapy to ameliorate disorders of arterial compliance in the ageing population. © 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Clinical characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Biochemical measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Measurement of aortic augmentation index (AIx) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Statistical analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Clinical univariate correlates of augmentation index (AIx) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Multivariate analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
503 504 504 504 504 504 504 505 505 505 506 506 506 507
1. Introduction
∗ Corresponding author. Tel.: +61 8 8222 6000; fax: +61 8 8222 7201. E-mail address: [email protected] (J.D. Horowitz). 1043-6618/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2009.06.006
Systolic hypertension is an increasingly common condition in ageing individuals, with well-characterised associations with risks of left ventricular (LV) hypertrophy, congestive heart failure, and
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cerebrovascular accidents [1]. The presence of systolic hypertension is in turn the result of increased arterial stiffness, with a resultant acceleration of pulse wave reflection and therefore incremental afterload [2]. Thus, increased arterial stiffness is a cause of cardiovascular morbidity and mortality [3–6]. However, it is now clear that this incremental stiffness is itself variable, with smooth muscle tone, rather than entirely fixed, as a result of vascular remodelling, fibrosis and calcification [7]. The vascular endothelium plays a pivotal role in the regulation of vascular tone via regulating release of vasoactive factors responsible for the maintenance of normal arterial wall composition. Dysfunction of the vascular endothelium, due to decreased nitric oxide (NO) generation or bioavailability, plays an important role in arterial stiffness, and atherosclerosis. Endothelial release of NO attenuates the vasoconstrictor effects of noradrenaline, endothelin, angiotensin II, and serotonin. NO also inhibits vascular smooth muscle proliferation which could otherwise induce medial thickening and/or myointimal hyperplasia. Furthermore, NO inhibits the interaction of circulating blood elements with the vessel wall. It limits vascular recruitment of platelet aggregation and leukocyte adherence by inhibiting the expression of proinflammatory cytokines, chemokines, and leukocyte adhesion molecules [8]. Nitric oxide is formed in endothelial cells from the amino acid l-arginine by endothelial isoform of NO synthase (eNOS). Asymmetric dimethylarginine (ADMA) is a naturally occurring l-arginine analogue, and hence a competitive inhibitor of eNOS, derived from the proteolysis of proteins containing methylated arginine residues. Symmetric dimethylarginine (SDMA) is the structural isomer of ADMA. Both ADMA and SDMA derive from intranuclear methylation of l-arginine residuals and are released into the cytoplasm after proteolysis. Increased ADMA concentrations have been associated with cardiovascular disease states and risk factors [9]. While, ADMA is enzymatically degraded by dimethylarginine dimethylaminohydrolase (DDAH), SDMA is eliminated entirely by renal excretion [10]. Therefore, the ADMA:SDMA ratio represents a “reverse” index of DDAH activity: a high ADMA:SDMA ratio indicates low DDAH activity, and thus selectively impaired ADMA (metabolic) clearance [11,12]. Recently, DDAH has emerged as an important regulator of NO bioavailability and vascular function, affecting tissue and plasma ADMA concentrations. Overexpression of both DDAH isoforms (DDAH1 and DDAH2) in animals reduced ADMA levels and increased NO synthesis, lowers vascular resistance [13,14]. Conversely, defects in DDAH expression and activity could theoretically contribute to arterial stiffness by regulating ADMA kinetics and hence NO synthesis. In this study, we therefore examined the relations of ADMA, SDMA and ADMA:SDMA ratios to aortic augmentation index (AIx), a composite measure of elastic plus muscular artery stiffness and wave reflection [15], in a random ageing Western population with non-selective cardiovascular risk factors. The hypotheses tested were: (i) ADMA concentrations would be correlated with AIx and (ii) ADMA:SDMA ratio, as an index of impaired ADMA clearance, would also be correlated with AIx. 2. Methods 2.1. Subjects Subset members (n = 253) of the general community (aged between 51 and 77 years) participating in the North Western Adelaide Health study were recruited to identify factors associated with presence of aortic valve sclerosis by echocardiography [16]. In the present analysis, we assessed the relationship between ADMA, SDMA, ADMA/SDMA ratios and AIx on all subjects with available data. The study was approved by local ethics com-
mittees, and all subjects gave written informed consent. As a component of evaluation, AIx and ADMA concentrations were evaluated. 2.2. Clinical characteristics All patients’ cardiovascular risk factors such as history of hypertension, hypercholesterolemia, diabetes mellitus, smoking habits, previous angina/ischemic events, were delineated at interview and details are as previously published [15]. All patients underwent transthoracic echocardiography and left ventricular diameters and wall thicknesses were measured from 2D-guided M-mode echocardiography. Height and weight were measured, and body mass index (BMI) was calculated. 2.3. Biochemical measurements Blood was collected from all patients into heparinised tubes, centrifuged at 4 ◦ C at 2700 × g for 20 min, and plasma was stored at −80 ◦ C until assay. Concentrations of ADMA and SDMA in plasma were measured by high-performance liquid chromatography (HPLC) using the derivatisation reagent AccQ-Fluor after solid phase extraction as previously described [17]. Lipid profile, high sensitivity C-reactive protein (hs-CRP), serum creatinine, calcium, and phosphate levels were obtained. Creatinine clearance (CrCl) was calculated according to the Cockcroft–Gault equation, and indexed for body surface area (BSA) using the Dubois and Dubois formula. 2.4. Measurement of aortic augmentation index (AIx) Pulse waveform analysis (PWA) was performed non-invasively with a commercially available SphygmoCor system (AtCor Medical, Sydney, Australia), as previously described [18]. All subjects were asked to lie down in a quiet room for 15 min prior to procedure. Briefly, PWA was computed from the radial artery at the wrist, and recorded by applanation tonometry using a high fidelity micromanometer. Three recordings of 10 sequential waveforms were acquired for each subject; a validated, generalized transfer function was used to generate the corresponding central aortic pressure waveform from which AIx was derived. Only high quality recordings with in-device quality index ≥90% were used. All augmentation indices were corrected for a standard heart rate of 75 bpm. 2.5. Statistical analyses All data are expressed as mean ± standard deviation (SD) unless otherwise stated. Normal distribution was tested for all continuous variables, and skewed data were normalized either by log or square root transformation. Comparisons between groups for non-parametric data were made using the Mann–Whitney test. Correlations between transformed, continuous non-parametric data were made using linear regression. As regards multivariate comparisons, to gain insights into the relations of AIx and methylarginines to the risk factors, we constructed separate backward multiple linear regression models with either ADMA or ADMA:SDMA ratio. In these models, AIx is the outcome variable, and methylarginines and ADMA/SDMA ratio were independently assessed along with other putative correlates of AIx such as: age, gender, plasma SDMA concentrations, plasma calcium × phosphate product (Ca × PO4), history of diabetes mellitus, hypertension, smoking, hypercholesterolemia, previous angina/ischemic events, use of angiotensin-converting enzyme inhibitor (ACEi)/angiotensin-receptor blocker (ARB), statins, calculated CrCL, BMI, hs-CRP, and LV mass index. These variables were
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Table 1 Patient demographics. Age (mean ± SD) (years) Gender (%male) Previous angina/myocardial infarction (%) History of hypercholesterolemia (%) Hypertension (%) Diabetes mellitus (%) Smokers/ex-smokers (%) Family history (%) BMI (kg/m2 ) ACEi/ARB therapy (%) Statin therapy (%)
63.4 ± 6.0 43.5 13.5 58.7 41.8 11.2 14.3 52.2 28.2 ± 5.0 33.1 32.1
Table 2 Univariate correlates of high AIx. Parameter
p value
Older age Female gender No history of previous MI/ischemia Low BMI History of hypercholesterolemia Hypertension No history of diabetes mellitus Smoker/ex-smoker Absence of ACEi/ARB Statin therapy Family history Low CrCL (indexed for BSA) mL/min/1.73 m2 Low left ventricular mass index (g/m2 ) High Ca × PO4 hs-CRP High ADMA Low SDMA High ADMA/SDMA ratio
0.1 <0.001 <0.05 0.09 0.14 0.4 <0.05 <0.05 <0.05 0.8 0.4 <0.001 0.001 0.09 0.9 0.1 0.02 0.002
included either due to statistical significance on univariate analyses or as plausible clinical correlates. All analyses were performed using SPSS 13 software, and a p value of <0.05 was considered to be statistically significant.
3. Results Patients’ baseline and demographic characteristics are presented in Table 1. The clinical characteristics of this cohort were consistent with a Western ageing population with high BMI and multiple cardiovascular risk factors. Approximately 1/3 of subjects were being treated with an ACEi/ARB or statin for hypertension or hypercholesterolemia, respectively.
Fig. 1. Relationship between creatinine clearance (CrCL) indexed for body surface area (BSA) and Aix (R = −0.36, p < 0.001).
3.1. Clinical univariate correlates of augmentation index (AIx) The clinical correlates of augmentation index are summarized as in Table 2. Female gender, absence of a history of ischemia, absence of diabetes, history or current smoking status, and absence of ACEi/ARB use were significantly associated with high AIx. Left ventricular mass index was also inversely associated with AIx. Biochemically, high AIx was correlated with low CrCL (Fig. 1). ADMA concentrations tended to be associated directly with high AIx (R = 0.11, p = 0.1)(Fig. 2A). While high ADMA/SDMA ratios were strongly associated with high AIx (Fig. 2C; R = 0.19, p = 0.002). 3.2. Multivariate analyses Results of multivariate analysis performed utilizing ADMA concentrations as a potential correlate of AIx are summarized in Table 3. ADMA concentrations were directly related to Aix; both SDMA concentrations and CrCL values represented inverse correlates of AIx. Other independently significant correlates of AIx were smoking, hypertension, female gender, and absence of therapy with ACEi/ARB. Multivariate analysis utilizing ADMA:SDMA ratio (Table 4) indicated a strong association between this ratio and AIx, with no change in other independent correlates).
Fig. 2. Correlations between (A) ADMA (R = 0.11, p = 0.1) and (B) ADMA/SDMA (R = 0.0.19, p = 0.002) ratio vs AIx.
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Table 3 Multiple regression analysis: correlates of AIx including ADMA and SDMA in the model.
ACEi/ARB Smoking CrCL Gender (male) Hypertension ADMA SDMA
ˇ Coefficient
p value
−0.23 0.15 −0.27 −0.37 0.18 0.16 −0.26
0.005 0.007 <0.001 <0.001 0.03 0.01 <0.001
Table 4 Multiple regression analysis: correlates of AIx including ADMA:SDMA ratio.
ACEi/ARB Smoker/ex-smoker CrCL Gender (male) Hypertension ADMA/SDMA ratio
ˇ Coefficient
p value
−0.24 0.15 (0.25 (0.38 (0.17 0.21
0.004 0.007 <0.001 <0.001 0.036 <0.001
4. Discussion Measurements of arterial stiffness have been increasingly used in the clinical assessment of patients as a recognised predictor for cardiovascular (CV) events, including CV mortality [4,19] myocardial infarction [20], and stroke [21]. It has been shown that arterial stiffness increases with age, and is correlated directly with CV risk factors such as hypertension, metabolic syndrome, diabetes, obesity, hypercholesterolemia, and elevated C-reactive protein [22,23]. Increased arterial stiffness has been associated with abnormal activation of vascular pro-fibrotic markers such as matrix metalloproteinases [24], and the renin–angiotensin system [25,26]. In this study, we demonstrated that elevated plasma concentrations of ADMA, a marker and mediator of endothelial dysfunction, are directly correlated with apparent arterial stiffness, as measured by AIx in an ageing population, independent of other CV risk factors. More importantly, the observed direct correlation between AIx and ADMA concentrations is likely to reflect decreased (redox-sensitive) enzymatic clearance via DDAH. ADMA exerts vasoconstrictor responses in normal subjects [27], and indeed has previously been shown to increase arterial stiffness as measured by AIx [28]. However, this finding does not in itself imply that plasma ADMA concentrations are major determinants of AIx values either in normal subjects or in any pathological condition: in this area, the previous literature contains heterogeneous findings. The largest population study on this issue to date, the PREVENCION study, found that ADMA concentrations were not associated with carotid–femoral pulse wave velocity [29]. A study in normal young adults [30] revealed a paradoxical relationship between ADMA and pulse wave velocity which was not significant on multivariate analysis. On the other hand, Weber et al. [31] found a weak correlation between ADMA concentrations and AIx in patients undergoing cardiac catheterization. Finally, in patients undergoing hemodialysis for advanced renal disease, post dialysis falls in ADMA were correlated with decreases in AIx, suggesting a cause-and-effect relationship in these circumstances. We now report for the first time, a positive relationship between ADMA and AIx, independent of other CV risk factors in a cross-sectional ageing population. More remarkedly, our data also suggest that reduced enzymatic clearance of ADMA is a potential mechanism for ADMA accumulation and hence arterial stiffness.
Steady-state ADMA concentrations are predominantly modulated by activity of DDAH [32], but also by rates of ADMA formation, via enzymatic protein catabolism. Several lines of evidence suggest that decreased aortic expression of DDAH is associated with increased ADMA concentrations and decreased NO synthesis [14]; and that the reverse effects are seen with over-expression of DDAH [13,33]. For example, Zoccali et al. [11] found that plasma ADMA concentrations were markedly increased during the recovery phase of acute infection/inflammation with an accompanying rise in ADMA:SDMA ratio, indicating a reduction in DDAH activity. The clearance of SDMA is entirely renal, while that of ADMA is predominantly enzymatic via DDAH-catalysed inactivation. Hence, changes in ADMA:SDMA ratio should correlate inversely with DDAH activity, and have been utilized as a surrogate measure of DDAH activity [11,12]. In the current study, the ADMA:SDMA ratio was strongly correlated with AIx: these findings therefore suggest that impaired DDAH activity may underlie arterial “stiffening” with advanced age. However, how closely ADMA:SDMA ratio relates to DDAH activity remains uncertain, largely due to the impracticability of measuring total body DDAH activity. Despite the difficulties inherent in the process, further studies should address the relationship between intravascular DDAH assays [32,33] and plasma ADMA:SDMA ratio. If impaired DDAH activity indeed represents the “primary” basis for the observed association between AIx and ADMA concentrations, it is possible that this might be triggered by any increase in redox stress [34], or indeed via reduced NO availability itself [35]. Furthermore, the findings in the current study related to an inverse relationship between ACEi/ARB therapy and AIx are consistent with the known effects of these agents, again partially mediated by incremental NO availability as well as reduced redox stress [36,37]. In line with the recent meta-analysis by Kielstein et al. [38], we also observed an inverse relationship between SDMA concentrations and CrCL (data not shown). Correlations between SDMA concentrations and AIx are likely to reflect associated changes in renal function rather than the weak biological effects of SDMA in NO formation [12]. The main caveats of this investigation are that many of the patients were undergoing treatment for hypertension and hypercholesterolemia. This therapy, particularly with ACEi/ARB, may have complicated evaluation of the ADMA vs AIx relationship, both by reducing AIx and by altering ADMA kinetics [39,40]. Furthermore, we did not observe any significant relationship between left ventricular mass index and AIx: again this finding may relate in part to the effects of pharmacotherapy. In conclusion, ADMA and ADMA:SDMA ratio are significant correlates of AIx in this ageing population, presumably reflecting impaired ADMA clearance. Future studies related to therapy of predominantly systolic hypertension in the elderly should therefore, in part, address the hypothesis that amelioration of disorders of arterial compliance is best addressed via optimization of ADMA clearance. From a theoretical point of view, this argues in particular for the advantages of agents such as ACEis, which appear to protect vascular DDAH activity [36,37], as first-line therapy for treatment of disorders of arterial compliance, such as systolic hypertension, in the elderly. Conflict of interest None of the authors have any conflict of interest. Acknowledgements This work was supported in part by research grants from the National Health and Medical Research Council and National Heart Foundation of Australia.
D.T.M. Ngo et al. / Pharmacological Research 60 (2009) 503–507
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