Abstract. Introduction

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1 Clin Physiol Funct Imaging (00) doi: 0./j.-0X x 0 0 Atherosclerosis measured by whole body magnetic resonance angiography and carotid artery ultrasound is related to arterial compliance, but not to endotheliumdependent vasodilation the Prospective Investigation of the Vasculature in Uppsala Senis (PIVUS) study Lars Lind, Jessika Andersson, Tomas Hansen, Lars Johansson, and Håkan Ahlström Departments of Medicine and Radiology, Uppsala University Hospital, Uppsala, Sweden, and AstraZeneca R&D Mölndal, Mölndal, Sweden Crespondence Lars Lind M.D., Department of Medicine, University Hospital, Uppsala, Sweden lars.lind@medsci.uu.se Accepted f publication Received September 00; accepted March 00 Key wds angiography; artery; atherosclerosis; carotid artery; compliance; endothelium; magnetic resonance; ultrasound; vasodilation Introduction Abstract Arterial compliance and endothelium-dependent vasodilation are two maj characteristics of the arteries. These two vascular features are both related to the main cardiovascular risk facts (Creager et al., 0; Linder et al., 0; Celermajer et al., ; Roman et al., ; Johnstone et al., ; Laurent et al., ; Lehmann et al., ), and have both the ability to predict future cardiovascular outcomes in prospective studies (Blacher et al., ; Schachinger et al., 000; London et al., 00; Perticone et al., 00; Lind et al., 00). Atherosclerosis is the maj disease affecting the arteries. It is today recognized as being a generalized disder most likely to Background: Arterial compliance and endothelium-dependent vasodilation are two characteristics of the vessel wall. In the Prospective Investigation of the Vasculature in Uppsala Senis (PIVUS) study, we studied the relationships between arterial compliance and endothelium-dependent vasodilation versus atherosclerosis as measured with two imaging modalities. Methods: In the population-based PIVUS study (0 subjects aged 0), arterial compliance was determined by ultrasound in the carotid artery and the stroke volume to pulse pressure ratio by echocardiography, while endothelium-dependent vasodilation was assessed by the invasive fearm technique with acetylcholine and brachial artery ultrasound. Intima-media thickness was evaluated by ultrasound in the carotid artery (n = ). Stenosis in the carotid, ata, renal, upper and lower leg arteries were determined by magnetic resonance angiography in a random subsample of subjects. Results: After adjustments f gender, Framingham risk sce, obesity, myocardial infarction and stroke, distensibility in the carotid artery and the stroke volume to pulse pressure ratio were both significantly related to a weighted index of stenosis in the five arterial territies evaluated by magnetic resonance angiography (p<0æ0 f both). Distensibility in the carotid artery (P = 0Æ0), but not the stroke volume to pulse pressure ratio (P = 0Æ0), was also significantly related to intima-media thickness. Conclusion: In the elderly population, atherosclerosis is mainly related to arterial compliance, but not to endothelium-dependent vasodilation in peripheral conduit resistance vessels. C P F B affect most arterial territies if present. Endothelium-dependent vasodilation is thought of as an early event in the atherosclerosis development and an impaired endothelium-dependent vasodilation could be detected even befe no maj atherosclerosis could be detected at angiography (Zeiher et al., ). On the contrary, arterial compliance might not be affected during the early stages of atherosclerosis. In large scale epidemiological research, measurements of intima-media thickness in the carotid arteries by ultrasound have been the most commonly used technique to evaluate atheroslerosis. The intima-media thickness has been shown to be related to overt atherosclerotic places and to predict future stroke and myocardial infarction (OÕLeary et al., ). In the Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine Dispatch:..0 Journal: CPF CE: T.M.Prasath Journal Name Manuscript No. Auth Received: No. of pages: PE: Vasugi

2 Atherosclerosis and vascular function, L. Lind et al. 0 0 recent years, also the echogenecity of plaques, reflecting plaque composition, has been shown to have a predictive value (Mathiesen et al., 00). We have just recently shown the echogenecity of the intima-media complex to be closely related to the echogenecity in the plaques (Lind et al., 00) and to be related to maj risk facts (Lind et al., 00), suggesting also this vascular wall characteristic to be of interest. We have recently used whole body magnetic resonance angiography to evaluate the atherosclerosis burden in the in five different arterial territies, the carotid artery, ata, renal arteries, upper leg and lower leg arteries, at the same examination (Hansen et al., 00). Using that approach in the Prospective Investigation of the Vasculature in Uppsala Senis (PIVUS) study, we could confirm the global nature of atherosclerosis. In the present study, we aim to investigate the relationships between arterial compliance and endothelium-dependent vasodilation versus atherosclerosis measured by whole body magnetic resonance angiography and intima-media thickness in the carotid artery by ultrasound in the PIVUS coht (Lind et al., 00) with the hypothesis that both arterial compliance and endothelium-dependent vasodilation are closely related to the degree of atherosclerosis. Material and methods This section has previously been given in detail in different publications (Lind et al., 00, 00; Hansen et al., 00). Total PIVUS sample Subjects All subjects aged 0 were eligible f living in the community of Uppsala, Sweden. The subjects were randomly chosen from the register of community living. 0 subjects participated giving a participation rate of Æ%. Of those, a random of subjects was evaluated with magnetic resonance angiography. The study was approved by the Ethics Committee of the University of Uppsala. All subjects were investigated in the mning after an overnight fast. No medication smoking was allowed after midnight. An arterial cannula was inserted in the brachial artery f blood sampling and later regional infusions of vasodilats. The participants were asked to answer a questionnaire about their medical histy, smoking habits and regular medication. Blood pressure was measured using a calibrated mercury sphygmomanometer in the non-cannulated arm to nearest mmhg after at least min of rest and the average of three recdings was used. Lipid variables and fasting blood glucose were measured by standard labaty techniques. From these data, the Framingham risk sce was calculated (Wilson et al., ). Basic characteristics of the total sample and the magnetic resonance angiography subsample are given in Table. Approximately 0% of the coht repted a histy of conary heart disease, % repted stroke and % diabetes mellitus. Almost half the coht repted any cardiovascular medication (%), with antihypertensive medication being the Subsample with TAS measurement n 0 Females (%) Æ Æ Height (cm) ± Æ ± Æ Weight (kg) ± ± Waist circumference (cm) ± ± BMI (kg m ) ) Æ0 ±Æ Æ ±Æ0 Waist hip ratio 0Æ0 ± 0Æ0 0Æ0 ± 0Æ00 SBP (mmhg) ± ± DBP (mmhg) ± 0 ± Æ LDL-cholesterol (mmol l ) ) Æ ±0Æ Æ ±0Æ HDL-cholesterol (mmol l ) ) Æ ±0Æ Æ ±0Æ Serum triglycerides (mmol l ) ) Æ ±0Æ0 Æ ±0Æ Fasting blood glucose (mmol l ) ) Æ ±Æ Æ ±Æ Current smoking (%) Æ EDV (%) ( 0) (00 0) EIDV (%) ( ) ( ) FMD (%) Æ (0Æ0 Æ) Æ (0Æ0 Æ) CCA distensibility (% per mmhg) 0Æ0 (0Æ0 0Æ) 0Æ0 (0Æ0 0Æ) SV PP ratio (ml mmhg ) ) Æ (0Æ0 Æ) Æ (0Æ Æ) TAS 0Æ (0 0Æ) Means are given ± SD as median and 0th and 0th percentiles in parenthesis. SBP, Systolic blood pressure; DBP, Diastolic blood pressure; BMI, Body mass index; EDV, endothelium-dependent vasodilation (invasive fearm technique); EIDV, endothelium-independent vasodilation (invasive fearm technique); FMD, flow mediated dilatation; CCA, common carotid artery; SV, Stoke volume; PP, pulse pressure; AIx, Augmentation index; TAS, Total atherosclerosis sce. Table Basic characteristics, maj cardiovascular risk facts and measures of endothelium-dependent vasodilation and adipose tissue in the total sample and in the present sample. Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine

3 Atherosclerosis and vascular function, L. Lind et al. 0 0 most prevalent (%). Fifteen percent repted use of statins, while insulin and al antiglycemic drugs were repted in % and % respectively (see reference Lind et al., 00 f details). The invasive fearm technique Fearm blood flow was measured by venous occlusion plethysmography (Elektromedicin, Kullavik, Sweden). After evaluation of resting fearm blood flow, local intra-arterial drug-infusions were given during min f each dose. The infused dosages were and lg min ) f acetylcholine (Clin-Alpha, Switzerland) to evaluate endothelium-dependent vasodilation in fearm resistance vessels and and 0 lg min ) f SNP (Nitropress, Abbot, UK) to evaluate endothelium-independent vasodilation. Endothelium-dependent vasodilation in fearm resistance vessels was defined as fearm blood flow during infusion of lg min ) of acetylcholine minus resting fearm blood flow divided by resting fearm blood flow. Endothelium-independent vasodilation in fearm resistance vessels was defined as fearm blood flow during infusion of 0 lg min ) of SNP minus resting fearm blood flow divided by resting fearm blood flow. The brachial artery ultrasound technique The brachial artery was assessed by external B-mode ultrasound imaging cm above the elbow (Acuson XP with a 0 MHz linear transducer; Acuson Mountain View, CA, USA). Blood flow increase was induced by inflation of a pneumatic cuff placed around the fearm to a pressure at least mmhg above systolic blood pressure f min. Flow-mediated vasodilation was defined as the maximal brachial artery diameter recded between and 0 s following cuff release minus diameter at rest divided by the diameter at rest. Pulse wave analysis A micromanometer tipped probe (Sphygmoc; Pulse Wave Medical Ltd., Australia) was applied to the surface of the skin overlying the radial artery and the peripheral radial pulse wave was continuously recded. The mean values of around 0 pulse waves were used f analyses. On the Basis of transfer functions, atic systolic and diastolic blood pressure were calculated from the radial recdings with the Sphygmoc software. Carotid artery compliance The diameter of the common carotid artery of the right side cm proximal of the bifurcation was measured at its maximal diameter in systole and the minimal diameter in diastole. The distensibility of the carotid artery was calculated as the change in diameter maximum to minimum in relation to the minimal diameter in diastole divided by the central pulse pressure obtained by pulse wave analysis. Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine Stroke volume to pulse pressure ratio Echocardiography was perfmed (Æ MHz transducer; Acuson XP, CA, USA) and using Teichholz fmula ejection fraction and stroke volume were calculated. The stroke volume to pulse pressure ratio was calculated as stroke volume divided by central pulse pressure (achieved by pulse wave analysis). Carotid artery ultrasound evaluation The carotid artery was assessed by external B-mode ultrasound imaging (Acuson XP with a 0 MHz linear transducer; Acuson Mountain View). The intima-media thickness was evaluated in the far wall in the common carotid artery cm proximal to the bulb. The images were digitized and impted into the AMS (Artery Measurement Software) automated software (Liang et al., 000) f dedicated analysis of intima-media thickness and the grey scale median of the intima-media complex. A maximal 0 mm segment with good image quality was chosen f intima-media thickness-analysis from the carotid artery. The programme automatically identifies the bders of the intima-media thickness of the far wall and the inner diameter of the vessel and calculates intima-media thickness and the diameter from around 00 discrete measurements through the 0 mm long segment. This automated analysis could be manually crected if not found appropriate at visual inspection. The given value f carotid artery intima-media thickness is the mean value from both sides. A region of interest was placed manually around the intimamedia segment that was evaluated f intima-media thickness and the programme calculates the echogenecity in the intimamedia complex from analysis of the individual pixels within the region of interest on a scale from 0 (black) to (white). The blood was used as the reference f black and the adventitia was the reference f white. The GSM-value given is the mean value from both sides. The mean length of the evaluated intima-media segments was Æ0 (SD Æ) mm when subjects with a segment recding less than mm were excluded, leaving subjects with valid recdings. The measurements of intima-media thickness were repeated in random subjects giving a coefficient of variation of carotid artery intima-media thickness of Æ% and Æ% f echogenecity in the intima-media complex. Magnetic resonance angiography Imaging was perfmed on a Æ Tesla MRI system (Gyroscan Intera, Philips Medical Systems, Best, the Netherlands) with a mt m ) gradient system, using the standard quadrature body coil. The whole body was scanned in the supine position using a D RF-spoiled T-weighted gradient echo sequence befe and after injection of ml Gd-DTPA-BMA (OmniscanTM; GE

4 Atherosclerosis and vascular function, L. Lind et al. 0 0 Healthcare, Oslo, Nway) at a rate of 0Æ mls ). The acquired slice thickness was mm with a resolution of Æ Æ mm. Imaging did not include the conary arteries. The arterial tree was categized into five territies: (i) the carotids including internal carotid artery and common carotid artery, (ii) the ata including both the thacic and abdominal part, (iii) the renal arteries, (iv) the pelvic upper limbs including common iliac artery, external iliac artery, common femal artery, superficial femal artery and popliteal artery, (v) the lower legs including tibio-peroneal trunk, anteri tibial artery, peroneal artery and posteri tibial artery. To obtain a comparable graded number reflecting the atherosclerosis in each territy, an atherosclerotic sce was calculated f each territy. A nmal vessel segment received null points, less than % stenosis was given one point and % reduction me of the vessel diameter including occlusions was given two points. The points f the vessel segments in a territy were summarized. That sum was than divided with the maximum sum that would be achieved if all included segments had a me than % stenosis occlusion. A global total atherosclerosis sce was defined as the sum of the five territies (Hansen et al., 00). Aneurysms and vessel segments that could not be evaluated were excluded from the calculations. Statistics Non-nmally distributed variables were transfmed to achieve a nmal distribution. Relationships between pairs of variables were evaluated by PearsonÕs SpearmansÕ crelation coefficient. Multiple regression analysis was applied to relate several independent variables to a dependent variable. Two-tailed significance values were given with p<0æ0 regarded as significant. The statistical programme package StatView (SAS Inc., NC, USA) was used. Results Endothelial function and arterial compliance versus carotid artery ultrasound Table Relationships between endothelium-dependent vasodilation (invasive fearm technique, EDV), endothelium-independent vasodilation (invasive fearm technique, EIDV), flow mediated dilatation (FMD), distensibility of the common carotid artery (CCA) and stroke volume to pulse pressure ratio (SV PP) and intima-media thickness (IMT) and the grey scale median of the intima-media complex (IM- GSM) of the common carotid artery evaluated by ultrasound given as crelation coefficients and P-values in parenthesis if significant. IMT IM-GSM EDV )0Æ0 )0Æ0 FMD )0Æ0 0Æ0 EIDV )0Æ0 (0Æ0) 0Æ0 CCA distensibility )0Æ (0Æ000) 0Æ0 SV PP ratio )0Æ0 (0Æ00) )0Æ0 Table Multiple regression analysis with distensibility of the common carotid artery (CCA) and stroke volume to pulse pressure ratio (SV PP) as dependent variables in two different models with intimamedia thickness (IMT) of the common carotid artery, gender, Framingham risk sce, BMI, myocardial infarction and stroke as independent variables. CCA distensibility Regression coefficient P-value SV PP ratio Regression coefficient P-value IMT )0Æ 0Æ0 )0Æ 0Æ0 Gender )0Æ 0Æ000 )0Æ 0Æ000 Framingham sce )0Æ0 0Æ000 )0Æ0 0Æ000 BMI )0Æ00 0Æ 0Æ0 0Æ000 Myocardial infarction )0Æ0 0Æ )0Æ0 0Æ0 Stroke 0Æ00 0Æ 0Æ0 0Æ In univariate analysis, intima-media thickness was inversely related to endothelium-independent vasodilation in fearm resistance vessels, carotid artery distensibility and the stroke volume to pulse pressure ratio, but not significantly related to endothelium-dependent vasodilation in fearm resistance vessels flow-mediated vasodilation (Table ). Echogenecity in the intima-media complex was not significantly related to any of the variables reflecting endothelial function arterial compliance. In multivariate analysis with distensibility of the carotid artery, stroke volume to pulse pressure ratio and endotheliumindependent vasodilation in fearm resistance vessels as dependent variables in three different models with intimamedia thickness, gender, Framingham risk sce, BMI, myocardial infarction and stroke as independent variables in all models, carotid artery distensibility, but not stroke volume to pulse pressure ratio (P = 0Æ0) endothelium-independent vasodilation in fearm resistance vessels (P = 0Æ), were significantly related to total atherosclerotic sce (see Table f details). Endothelial function and arterial compliance versus total atherosclerotic sce at magnetic resonance angiography Total atherosclerotic sce was significantly related in univariate analysis to carotid artery distensibility and endothelium-independent vasodilation in fearm resistance vessels, while a tendency was also seen f the stroke volume to pulse pressure ratio (P = 0Æ0). However, no relationships between endothelium-dependent vasodilation in fearm resistance vessels, flowmediated vasodilation and total atherosclerotic sce were found (see Table ). When the vascular variables being significantly related to total atherosclerotic sce in the univariate analysis were evaluated regarding their relationships with the AS in the five different vascular territies, it was mainly the carotid artery, ata and the upper leg arteries that were related to the distensibility of the Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine

5 Atherosclerosis and vascular function, L. Lind et al. 0 0 Table Relationships between change in reflectance index at pulse wave analysis (RI), endothelium-dependent vasodilation (invasive fearm technique, EDV), endothelium-independent vasodilation (invasive fearm technique, EIDV), flow mediated dilatation (FMD), distensibility of the common carotid artery (CCA) and stroke volume to pulse pressure ratio (SV PP) and total atherosclerosis sce (TAS) measured by MR angiography given as crelation coefficient and P-value in parenthesis. In the five columns at the left, the P-value f relationship in the five different territies is given f indices of vascular function being related to TAS. carotid artery, stroke volume to pulse pressure ratio and endothelium-independent vasodilation in fearm resistance vessels. Atherosclerosis in the renal artery the lower leg arteries were not significantly related to any of the vascular measurements (see Table f details). In multivariate analysis with distensibility of the carotid artery, stroke volume to pulse pressure ratio and endotheliumindependent vasodilation in fearm resistance vessels as dependent variables in three different models with total atherosclerosis sce, gender, Framingham risk sce, BMI, myocardial infarction and stroke as independent variables in all models, carotid artery distensibility and stroke volume to pulse pressure but not endothelium-independent vasodilation in fearm resistance vessels (P = 0Æ), were significantly related to total atherosclerotic sce (see Table f details). Discussion TAS Carotid Ata Renal Upper leg Lower leg EDV )0Æ0 FMD )0Æ0 EIDV )0Æ (0Æ0) (0Æ0) (0Æ) (0Æ0) (0Æ) (0Æ) CCA distensibility )0Æ (0Æ000) (0Æ00) (0Æ0) (0Æ) (0Æ0) (0Æ) SV PP ratio )0Æ (0Æ0) (0Æ) (0Æ0) (0Æ) (0Æ0) (0Æ) The present study showed that indices of arterial compliance (carotid artery distensibility and the stroke volume to pulse pressure ratio), but not endothelium-dependent vasodilation in peripheral resistance arteries a conduit artery, were related to atherosclerotic stenosis evaluated by magnetic resonance angiography after adjustment f the classical cardiovascular risk facts. A similar picture emerged when intima-media thickness of the carotid artery was used as a measure of atherosclerosis. Table Multiple regression analysis with distensibility of the common carotid artery (CCA) and stroke volume to pulse pressure ratio (SV PP) as dependent variables in two different models with total atherosclerosis sce (TAS), gender, Framingham risk sce, BMI, myocardial infarction and stroke as independent variables. CCA distensibility Regression coefficient P-value SV PP ratio Regression coefficient P-value TAS )0Æ 0Æ0 )0Æ 0Æ0 Gender )0Æ 0Æ00 )0Æ 0Æ000 Framingham sce )0Æ0 0Æ000 )0Æ0 0Æ000 BMI 0Æ00 0Æ 0Æ0 0Æ00 Myocardial infarction 0Æ 0Æ )0Æ 0Æ0 Stroke )0Æ0 0Æ 0Æ0 0Æ Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine Endothelium-dependent vasodilation In the present study, endothelium-dependent vasodilation was not significantly related to the degree of stenosis to intimamedia thickness. This might at the first sight seem contradicty since we have previously found all three techniques to be related with Framingham risk sce, an established marker of conary risk (Lind et al., 00). However, this unexpected finding could be explained by different mechanisms. First, atherosclerosis is a TAS TAS ln EDV ln FMD Figure Relationships between total atherosclerosis sce (TAS) evaluated by MR angiography and endothelium-dependent vasodilation evaluated with the invasive fearm technique (ln EDV, relative increase in FBF at the highest dose Ach, top panel, ln-transfmed, r = )0Æ0, P = 0Æ) and the brachial artery ultrasound technique (ln FMD, lower panel, ln-transfmed, r = )0Æ0, P = 0Æ).

6 Atherosclerosis and vascular function, L. Lind et al. 0 0 TAS TAS ln CCA distensibility ln SV/PP Figure Relationships between total atherosclerosis sce (TAS) evaluated by MR angiography and three indices of arterial compliance; distensibility in the common carotid artery (CCA distensibility, ln-transfmed, r = )0Æ, P =0Æ000) and the stroke volume to pulse pressure ratio (SV PP, ln-transfmed, r = )0Æ, P = 0Æ0). disder mainly affecting conduit arteries. The invasive fearm model with acetylcholine infusion is thought to mainly evaluate endothelium-dependent vasodilation in fearm resistance vessels, a type of vessels not generally affected by atherosclerosis. Secondly, flow-mediated vasodilation is measured in a conduit artery, but as recently shown in two separate studies (Witte et al., 00; Lind, 00), flow-mediated vasodilation is affected by the reduced arterial compliance in the elderly making this technique of less value in this age-group. Third, both flow-mediated vasodilation and endothelium-dependent vasodilation in fearm resistance vessels are measured in the upper extremity, not clinically affected by atherosclerosis to a maj extent, although it recently has been shown that intimamedia thickness in the carotid and brachial arteries are crelated (Agewall et al., 00). A number of studies have shown flow-mediated vasodilation (Hashimoto et al., ; Haraki et al., 00; Olsen et al., 00; Pedos et al., 00; Jarvisalo et al., 00; Juonala et al., 00; Suzuki et al., 00; Yan et al., 00) to be inversely related to the thickness of the intima-media complex of the carotid arteries, mainly in different diseased populations. One study has also repted a relationship between endothelium-dependent vasodilation in fearm resistance vessels and intima-media thickness in hypertensives (Ghiadoni et al., ). However, it should be remembered that intima-media thickness is not only determined by atherosclerosis and that intima-media thickness is not equivalent to the degree of stenosis evaluated by magnetic resonance angiography in the present study. There are however no studies in the past relating the degree of stenosis in the maj conduit arteries to endothelium-dependent vasodilation. Arterial compliance Arterial compliance was consistently related to atherosclerotic stenosis. In this case, two different markers of arterial compliance, carotid artery distensibility and the stroke volume to pulse pressure ratio were related to total atherosclerotic sce. We have previously shown these two markers of arterial compliance to be independently related to conary risk in this coht (Lind et al., 00), but they were still both related to total atherosclerotic sce after adjustment f conary risk facts. Arterial compliance is possibly me affected by structural changes in the vascular wall than endothelium-dependent vasodilation and therefe a relationship with atherosclerosis is me likely to be disclosed. Both carotid artery distensibility and the stroke volume to pulse pressure ratio have previously been linked to intima-media thickness of the carotid arteries (Riley et al., ; Liang et al., ; de Simone et al., 00; Jourdan et al., 00). This was also seen in the present study in the univariate analysis, but following multiple adjustments only carotid artery distensibility was related to intima-media thickness. Location of atherosclerotic stenosis In the present study, we differed between five arterial territies regarding the extent of atherosclerotic stenosis at the magnetic resonance angiography. The degree of atherosclerosis in the carotid artery, ata and the upper leg arteries contributed most to the variation in carotid artery distensibility and the stroke volume to pulse pressure ratio, while atherosclerosis in the renal arteries and lower leg did not. Although atherosclerosis is a generalized disder and we previously have shown that occurrence of atherosclerosis in these five arterial territies are inter-related (Hansen et al., 00), there are regional differences and the carotid artery, ata and the upper leg arteries are in fact the vascular segments in which carotid artery distensibility and the stroke volume to pulse pressure ratio are evaluated. If endothelium-dependent vasodilation and arterial compliance would be evaluated in the renal lower leg arteries another picture might have emerged. Reproducibility We have previously shown the reproducibility (coefficient of variation, CV) at repeated measurements to be % f baseline brachial artery diameter and % f flow-mediated vasodilation (Lind et al., 000) and the cresponding CVs f Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine

7 Atherosclerosis and vascular function, L. Lind et al. 0 0 endothelium-dependent vasodilation in fearm resistance vessels and endothelium-independent vasodilation in fearm resistance vessels to be 0% (Lind et al., ). F the compliance measures carotid artery distensibility and stroke volume to pulse pressure ratio the CVs were % and % respectively. When measurements of intima-media thickness were repeated in random subjects a coefficient of variation of carotid artery intima-media thickness of Æ% and Æ% f echogenecity in the intima-media complex were obtained (Lind et al., 00) and repeated readings in the magnetic resonance angiography images yielded a kappa-value of 0Æ f intraobserver variability and 0Æ f inter-observer variability (Hansen et al., 00). Thus, the reliability f the atherosclerotic measures was generally good. The lack of relationship between flow-mediated vasodilation and the atherosclerotic measures might be due to a po reproducibility of flow-mediated vasodilation, but almost 000 individuals with values f both flow-mediated vasodilation and intima-media thickness would nevertheless have disclosed any existing relationship. The most consistent finding was seen f carotid artery distensibility versus the atherosclerotic measures despite the fact that the CV f carotid artery distensibility was rather high (%), further suppting the assumption that the sample size is sufficient to disclose pathophysiological associations. Relationships between vascular measures We have previously shown that no relationship exists between endothelial function in conduit arteries (flow-mediated vasodilation) and resistance vessels (endothelium-dependent vasodilation in fearm resistance vessels) in the PIVUS study (Lind et al., 00), as well as in younger subjects (Lind et al., 00). On the contrary, a fairly close relationship exists between the two compliance measures used in the present study (Lind et al., 00). We have also recently shown that no close associations exists between the two endothelial function measures and the two arterial compliance methods used in the present study (Lind et al., 00). Validation of the measurements In terms of validation, we have previously shown that both of the two endothelial function measures (Lind et al., 00) and both of the two arterial compliance methods (Lind et al., 00) used in the present study are related to conary risk as estimated by the Framingham risk sce. Since we do not have enough follow-up time yet in the PIVUS study to relate the vascular measures to future cardiovascular events, we have to await these data in the present coht, but both of the two endothelial function measures (Schachinger et al., 000; Perticone et al., 00) and both of the two arterial compliance methods (Blacher et al., ; Lind et al., 00) have been shown to predict CV events in other cohts. This is also true f the atherosclerosis measures intima-media thickness and echogenecity in the intima-media complex (Wohlin et al., 00). No data yet exists on the value of total atherosclerotic sce to predict CV events, but we have recently shown total atherosclerotic sce to be related to conary risk (Hansen et al., 00). Limitations The present sample is limited to Caucasians aged 0. Caution should therefe be made to draw conclusions to other ethnic and age groups. The PIVUS study had a moderate participation rate. However, an analysis of non-participants showed the present sample to be fairly representative of the total population regarding most cardiovascular disders and drug intake (Lind et al., 00). Endothelium-independent vasodilation in fearm resistance vessels was only assessed by one of the methods f practical and ethical reasons not to prolong the investigation procedure. We have previously shown that endothelium-independent vasodilation in fearm resistance vessels evaluated by SNP infusion in the brachial artery and nitroglycerine provoked change in brachial artery diameter are closely related (Lind et al., 000), so additional measurements of endothelium-dependent vasodilation in fearm resistance vessels would probably not add substantial infmation to the study. Another limitation is that atic pulse wave velocity, the most commonly used index of arterial compliance, was not evaluated in the present coht. 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Laurent S, Caviezel B, Beck L, Girerd X, Billaud E, Boutouyrie P, Hoeks A, Safar M. Carotid artery distensibility and distending pressure in hypertensive humans. Hypertension (); :. Lehmann ED, Hopkins KD, Rawesh A, Joseph RC, Kongola K, Coppack SW, Gosling RG. Relation between number of cardiovascular risk facts events and noninvasive Doppler ultrasound assessments of atic compliance. Hypertension (); :. Liang YL, Teede H, Kotsopoulos D, Shiel L, Cameron JD, Dart AM, McGrath BP. Non-invasive measurements of arterial structure and function: repeatability, interrelationships and trial sample size. Clin Sci (Lond). (); :. Liang Q., Wendelhag I., Wikstrand J., Gustavsson T. A Multiscale Dynamic Programming Procedure f Boundary Detection in Ultrasonic Artery Images. IEEE Transactions on Medical Imaging, (000); :. Lind L. Arterial compliance influences the measurement of flow-mediated vasodilation, but not acetylcholine-mediated fearm blood flow. The Prospective Investigation of the Vasculature in Uppsala Senis (PIVUS) study. Atherosclerosis (00); 0:. Lind L. Arterial compliance and endothelium-dependent vasodilation are independently related to conary risk in the elderly: the Prospective Investigation of the Vasculature in Uppsala Senis (PIVUS) study. Clin Physiol Funct Imaging (00);???:???????? (in press). Lind L, Sarabi M, Millgård J. Methodological aspects of the evaluation of endotelium-dependent vasodilatation in the human fearm. Clin Physiol (); :. Lind L, Hall J, Larsson A, Annuk M, Fellström B, Lithell H. Evaluation of endothelium-dependent vasodilation in the human peripheral circulation. Clin Physiol (000); 0:. Lind L, Hall J, Johansson K. Evaluation of four different methods to measure endothelium-dependent vasodilation in the human peripheral circulation. Clin Sci (00); 0:. Lind L, Andren B, Sundstrom J. The stroke volume pulse pressure ratio predicts conary heart disease mtality in a population of elderly men. J Hypertens (00); : 0. Lind L, Fs N, Hall J, Marttala K, Stenbg A. A comparison of three different methods to evaluate endothelium-dependent vasodilation in the elderly: the Prospective Investigation of the Vasculature in Uppsala Senis (PIVUS) study. Arterioscler Thromb Vasc Biol (00); :. Lind L, Fs N, Hall J, Marttala K, Stenbg A. A comparison of three different methods to determine arterial compliance in the elderly: the Prospective Investigation of the Vasculature in Uppsala Senis (PIVUS) study. J Hypertens (00); : 0 0. Lind L, Andersson J, Ronn M, Gustavsson T. The echogenecity of the intima-media complex in the common carotid artery is closely related to the echogenecity in plaques. Atherosclerosis (00);???:???????? (in press). Lind L, Andersson J, Rönn M, Gustavsson T, Holdfelt P, Hulthe J, Elmgren A, Zilmer K, Zilmer M. Brachial artery intima-media thickness and echogenecity in relation to lipids and markers of oxidative stress in elderly subjects the prospective investigation of the vasculature in Uppsala senis (PIVUS) study. Lipids (00); :. Linder L, Kiowski W, Buhler FR, Lüscher TF. Indirect evidence f the release of endothelium-derived relaxing fact in the human fearm circulation in vivo: Blunted response in essential hypertension. Circulation (0); :. London GM, Blacher J, Pannier B, Guerin AP, Marchais SJ, Safar ME. Arterial wave reflections and survival in end-stage renal failure. Hypertension (00); :. Mathiesen EB, Bonaa KH, Joakimsen O. Echolucent plaques are associated with high risk of ischemic cerebrovascular events in carotid stenosis: the tromso study. Circulation (00); 0:. OÕLeary DH, Polak JF, Kronmal RA, Manolio TA, Burke GL, Wolfson SK Jr. Carotid-artery intima and media thickness as a risk fact f myocardial infarction and stroke in older adults. Cardiovascular Health Study Collabative Research Group. N Engl J Med (); :. Olsen MH, Wachtell K, Aalkjaer C, Dige-Petersen H, Rokkedal J, Ibsen H. Vasodilaty capacity and vascular structure in long-standing hypertension: a LIFE substudy. Losartan Intervention F Endpoint-Reduction in Hypertension. Am J Hypertens (00); :. Perticone F, Ceravolo R, Pujia A, Ventura G, Iacopino S, Scozzafava A, Ferraro A, Chello M, Mastroberto P, Verdecchia P, Schillaci G. Prognostic significance of endothelial dysfunction in hypertensive patients. Circulation (00); 0:. Pedos P, Golob M, Jensterle M. Interrelationship between peripheral arterial occlusive disease, carotid atherosclerosis and flow mediated dilation of the brachial artery. Int Angiol (00); :. Riley WA, Evans GW, Sharrett AR, Burke GL, Barnes RW. Variation of common carotid artery elasticity with intimal-medial thickness: the ARIC Study. Atherosclerosis Risk in Communities. Ultrasound Med Biol (); :. Roman MJ, Pini R, Pickering TG, Devereux RB. Non-invasive measurements of arterial compliance in hypertensive compared with nmotensive adults. J Hypertens (); 0: S S. Schachinger V, Britten MB, Zeiher AM. Prognostic impact of conary vasodilat dysfunction on adverse long-term outcome of conary heart disease. Circulation (000); 0: 0. de Simone G, McClelland R, Gottdiener JS, Celentano A, Kronmal RA, Gardin JM. Relation of hemodynamics and risk facts to ventricular vascular interactions in the elderly: the Cardiovascular Health Study. J Hypertens (00); : 0. Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine

9 Atherosclerosis and vascular function, L. Lind et al. 0 0 Ó 00 The Auths Journal compilation Ó 00 Scandinavian Society of Clinical Physiology and Nuclear Medicine Suzuki M, Takamisawa I, Suzuki K, Hiuge A, Hio T, Yoshimasa Y, Harano Y. Close association of endothelial dysfunction with insulin resistance and carotid wall thickening in hypertension. Am J Hypertens (00); :. Wilson PW, DÕAgostino RB, Levy D, Belanger AM, Silbershatz H, Kannel WB. Prediction of conary heart disease using risk fact categies. Circulation (); :. Witte DR, van der Graaf Y, Grobbee DE, Bots ML, SMART Study Group. Measurement of flow-mediated dilatation of the brachial artery is affected by local elastic vessel wall properties in high-risk patients. Atherosclerosis (00); :. Wohlin M, Sundström J, Andrén B, Larsson A, Lind L. An echolucent carotid artery intima-media complex is a new and independent predict of mtality in an elderly male coht. Atherosclerosis (00);???:???????? (in press). Yan RT, Anderson TJ, Charbonneau F, Title L, Verma S, Lonn E. Relationship between carotid artery intima-media thickness and brachial artery flow-mediated dilation in middle-aged healthy men. J Am Coll Cardiol (00); : 0. Zeiher AM, Drexler H, Wollschläger H, Just H. Modulation of conary vasomot tone in humans. Progressive endothelial dysfunction with different early stages of conary atherosclerosis. Circulation (); :.

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