Age and sex differences in the relationship between inherited and lifestyle risk factors and subclinical carotid atherosclerosis: the Tromsø study

Atherosclerosis 154 (2001) 437 448 www.elsevier.com/locate/atherosclerosis Age and sex differences in the relationship between inherited and lifestyle risk factors and subclinical carotid atherosclerosis: the Tromsø study Eva Stensland-Bugge *, Kaare H. Bønaa, Oddmund Joakimsen Institute of Community Medicine, Uni ersity of Tromso, N-9037 Tromsø, Norway Received 22 November 1999; received in revised form 21 March 2000; accepted 24 March 2000 Abstract Background: Ultrasound measurement of carotid artery intima-media thickness (IMT) is regarded as a valid index of atherosclerosis. Age and sex differences in the distribution of, and risk factors for, IMT have not been investigated thoroughly. Methods: In 1994 1995 a total of 6408 men and women aged 25 84 years living in the municipality of Tromsø, Norway, underwent ultrasound examination of carotid artery IMT and measurements of cardiovascular risk factors. Results: Age, systolic blood pressure, total cholesterol, HDL cholesterol, body mass index, and smoking were independent predictors of IMT in both sexes. Fibrinogen levels and physical activity were associated with IMT in men only, whereas triglyceride levels were associated with IMT independently of HDL cholesterol in women only. A family history of cardiovascular disease (CVD) was an independent predictor of IMT in both sexes, also when controlling for traditional CVD risk factors. The magnitude of the association between most risk factors and IMT did not differ depending on age, but the effects of physical activity and triglycerides were more pronounced at higher age. Conclusion: These data suggest that there are significant age and sex differences in the distribution and the determinants of subclinical atherosclerosis. 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Carotid arteries; Ultrasonics; Epidemiology; Atherosclerosis; Risk factors 1. Introduction The development of high-resolution ultrasound techniques for visualisation and measurement of the intimamedia layer of the carotid artery has made it possible to quantify intima-media thickness (IMT) with an acceptable degree of precision and accuracy [1]. Several studies have demonstrated that an increased IMT is associated with unfavourable cardiovascular risk factor levels, [2 5] prevalent cardiovascular disease [6] and atherosclerosis in other parts of the arterial system [7], indicating that carotid artery IMT may be regarded as a valid index of generalized atherosclerosis. Epidemiological studies of cardiovascular disease risk factors have usually emphasised clinical end-points such * Corresponding author. Tel.: +47-77-644816; Fax: +47-77- 644831. E-mail address: eva.stensland-bugge@ism.uit.no (E. Stensland- Bugge). as myocardial infarction and sudden death as dependent variables. These clinical end-points represent the interaction of different patophysiological mechanisms, such as the development of atherosclerosis, the acute occlusion usually caused by thrombosis, and the susceptibility to myocardial ischemia, all of which probably have different risk factors. Ultrasound now makes it possible to conduct population-based studies focusing specifically on the determinants of subclinical atherosclerosis. Furthermore, because ultrasound measures the dependent variable on a continuous scale, the power to quantify the effect of risk factors, as well as interaction among risk factors, is increased in ultrasound studies as compared with studies in which the end point is defined only by the presence or absence of clinical disease. Present knowledge about the epidemiology of IMT is limited since previous studies were restricted to subjects of a narrow age range, [2,3] included men only [4], used combined data from different studies [5], or had other 0021-9150/01/$ - see front matter 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0021-9150(00)00486-X

438 E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 limitations in study design. In particular, little population-based information is available on possible age and sex differences in inherited and lifestyle risk factors for subclinical atherosclerosis. We therefore performed ultrasonography of the right carotid artery on 6408 men and women aged 25 84 years who participated in a population health survey in the municipality of Tromsø, Norway. 2. Methods 2.1. Study design and subjects The Tromsø Study was started in 1974 and is a single center prospective follow-up study of inhabitants in the municipality of Tromsø, Norway. The main focus is on cardiovascular diseases. The study was approved by the regional board of research ethics, and each subject gave informed consent. The fourth survey of the Tromsø population was carried out in 1994 1995. The survey was conducted by the University of Tromsø in co-operation with the National Health Screening Service and comprised two screening visits with an interval of 4 12 weeks. All inhabitants born before 1970 were invited to the first visit, and all subjects born in 1920 1939 and 5 10% random samples in the other 5-year birth cohorts were invited to the second visit. A total of 6889 subjects, 79% of the eligible population, attended both visits. Of these, 319 male subjects born in 1940 1949 were specially invited to participate because they took part in a family intervention study on high-risk males in a previous survey. These subjects were excluded from the analyses in the present study. A protocol similar to that used in the previous surveys in this population and the Norwegian county studies was followed [8]. The examination included standardized measurements of height, weight, blood pressure, nonfasting serum lipids, and blood cell counts. Two questionnaires covered previous and present diseases and symptoms, use of antihypertensive drugs, life style factors, and family history of stroke and myocardial infarction. The questionnaires were checked for logical inconsistencies at the examination. The second visit comprised ultrasonographic examination of the carotid artery, sitting and standing blood pressures, and blood sampling. 2.2. Ultrasonographic scanning and measurement of risk factors IMT measurements of the right carotid artery were obtained with the use of a high-resolution ultrasound Acuson 128 XP/ 10c ART-upgraded scanner (Mountain View, CA,) equipped with a linear transducer with 7 MHz in B-mode. Three frozen images of IMT from each of three locations of the carotid artery: the near and far walls of the common carotid artery (CCA) and the far wall of the bifurcation, were stored on high-resolution videotapes. The loss of parallel configuration of the near and far walls of the CCA served as a reference point for the start of the carotid bifurcation. The ultrasonic images were analyzed off-line with a computerized technique for automated ultrasonic image analysis. Measurements of IMT were performed in 10-mm segments, and mean IMT from the three pre-selected images was calculated for each location. Plaques (defined as focal widening of the IMT relative to adjacent segments) were included in measurements of intima-media thickness. The scanning and reading procedure and the reproducibility of the ultrasound measurements have been published previously [1]. Height and weight were measured with the subjects in light clothing without shoes: Body mass index was calculated as weight in kg divided by the square of height in meters. Blood pressure was recorded by a specially trained technician using an automatic device (Dinamap Vital Signs Monitor 1846, Critikon) before the ultrasound examination and blood sampling. After the participant had been seated for 2 min, three recordings were made at 2-min intervals. The lowest value of the second and third measurement is used in the report. Nonfasting serum total cholesterol and triglycerides were analyzed by enzymatic colorimetric methods with commercial kits (CHOD-PAP for cholesterol and GPO- PAP for triglycerides; Boehringer-Mannheim). Serum HDL cholesterol was measured after the precipitation of lower density lipoprotein with heparin and manganese chloride. Serum fibrinogen was measured using the PT-Fibrinogen reagent (Instrumentation Laboratory, Italy). The analyses were done at the Department of Clinical Chemistry, University Hospital of Tromsø. For blood pressure, total cholesterol, HDL cholesterol, and triglycerides, we used the mean of values obtained at the first and second visit. Information about current cigarette smoking, physical activity, and a history of cardiovascular disease (CVD) was obtained from a self-administered questionnaire. Participants were asked whether a brother, sister, mother, or father had suffered a myocardial infarction (MI) before the age of 60 or a stroke, and 6008 subjects responded to these questions. Categories of physical activity (defined as activity during recreational hours which makes subjects perspire or become breathless) were graded from 1 to 4 according to which of the following categories would best describe the participant s usual level of weekly physical activity: 1, none; 2, less than 1 h weekly; 3, 1 2 h weekly and 4, 3 h or more weekly. We dichotomised physical activity into not active (category 1) or active (categories 2,3, and 4).

E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 439 2.3. Statistical analysis Analyses were done separately for men and women, and sex-specific results are presented unless otherwise specified. The distribution of IMT is presented by 5- year age-specific percentile-distributions of mean IMT in three locations of the carotid artery (Fig. 1). The average of the mean IMT in the three locations (i.e. the near and far walls of the CCA and the far wall of the bifurcation) was calculated and is used as the dependent variable in the analyses unless otherwise specified. All subjects included in the analyses had IMT values from the common carotid artery (n, 6357), and 95% of the subjects also had IMT values from the carotid bifurcation (n, 6015). Values of triglycerides were log transformed. To assess univariate relationships between lifestyle risk factors and IMT (Table 2) we used a general linear model (the GLM procedure in the SAS statistical software [9]) with IMT as the dependent variable and risk factors as predictor variables, as described by Howard et al. [5]. The study population was in these analyses divided in five age-strata ( 40, 40 49, 50 59, 60 69, and 70+ years old) within each sex. To assess the effect of a continuous risk factor on IMT, a single Table 1 Characteristics of the study population: the Tromsø study a Men Women n=2984 n=3373 Age (years) Agegroups n (mean age) 60.9 (9.8) 60.8 (10.3) 40 years 176 (32.9) 216 (33.0) 40 49 years 120 (45.4) 177 (44.8) 50 59 years 852 (56.4) 923 (56.4) 60 69 years 1290 (64.3) 1435 (64.7) 70 years 546 (72.2) 622 (72.4) Systolic blood pressure (mmhg) 140.3 (18.9) 139.5 (22.4) Diastolic blood pressure 81.0 (11.0) 78.0 (11.7) (mmhg) Serum total cholesterol (mmol/1) 6.47 (1.15) 6.91 (1.31) Serum HDL cholesterol (mmol/1) Serum triglycerides (mmol/1) 1.40 (0.38) 1.68 (0.94) 1.67 (0.42) 1.52 (0.85) Current smokers (%) 33.8 31.1 Number of cigarettes daily 13 (7) 10 (5) among smokers Body mass index (kg/m 2 ) 25.9 (3.3) 26.0 (4.5) Plasma fibrinogen (g/1) 3.35 (0.90) 3.44 (0.81) Participating in physical activity (%) 44.4 26.5 Mean intima-media thickness (mm) Far wall common carotid 0.81 (0.21) 0.74 (0.17) artery Near wall common carotid artery Far wall carotid bifurcation 0.85 (0.19) 1.08 (0.36) 0.80 (0.18) 0.97 (0.30) Average of the three above 0.91 (0.20) 0.83 (0.17) locations Maximum intima-media thickness (mm) Far wall common carotid 1.01 (0.28) 0.92 (0.23) artery Near wall common carotid artery Far wall carotid bifurcation 1.06 (0.24) 1.49 (0.61) 0.99 (0.23) 1.34 (0.52) Average of the three above locations Mean common carotid lumen 1.18 (0.30) 6.77 (0.92) 1.08 (0.25) 6.17 (0.73) diameter (mm) Pre alence of cardio ascular disease (%) Treated hypertension 14.3 13.6 Myocardial infarction 9.7 3.2 Angina pectoris 11.7 7.7 Diabetes mellitus 3.4 3.2 Stroke 3.3 2.4 a Values are means (S.D.), number of subjects (mean age) and percentages; HDL, high-density lipoprotein. Fig. 1. Percentile distribution of mean IMT in the far wall of the common carotid artery (top panel), the near wall of the common carotid artery (middle panel) and the far wall of the carotid bifurcation (bottom panel) related to age. Men are shown in solid lines, women in dashed lines. Percentiles shown are from top to bottom the 95th, 50th and 5th percentile. The Tromsø Study. regression model was fit for each risk factor with parameters to estimate the regression relationship between the risk factor and IMT within each age sexstratum. This model required a slope and an intercept for each of the ten strata. A similar model was fit to dichotomous variables. The slope parameter associated with a continuous risk factor corresponds to the differ-

440 E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 Table 2 Estimated magnitude of risk factor effects on intima-media thickness (mm), and probability values for test of differences in effect of risk factors across age strata: the Tromsø study a Risk factor S.D. Effect of risk factor P-value for sex P-value for trend in effect of risk factors across Age sex difference age strata c interaction Men Women Estimate P-value Estimate P-value Men Women Men and women Systolic blood pressure 20.8 0.033 0.007 0.0001 0.038 0.006 0.0001 0.61 0.07 0.95 0.20 0.23 (mmhg) Diastolic blood pressure 11.5 0.009 0.005 0.097 0.015 0.005 0.001 0.35 0.76 0.70 0.62 0.78 (mmhg) Total cholesterol (mmol/1) 1.26 0.020 0.005 0.0001 0.017 0.005 0.0003 0.63 0.34 0.77 0.38 0.53 HDL cholesterol (mmol/1) 0.42 0.015 0.006 0.013 0.010 0.004 0.017 0.50 0.83 0.12 0.30 0.83 Triglycerides (mmol/l) b 0.49 0.015 0.004 0.0002 0.018 0.004 0.0001 0.52 0.13 0.037 0.01 0.97 Smoking (no/yes) 0.020 0.009 0.02 0.000 0.008 0.97 0.095 0.99 0.32 0.51 0.84 Body mass index (kg/m 2 ) 3.97 0.028 0.005 0.0001 0.016 0.004 0.0001 0.097 0.92 0.79 0.82 0.69 Fibrinogen (g/l) 0.85 0.022 0.005 0.0001 0.008 0.004 0.058 0.029 0.36 0.38 0.21 0.53 Physical activity (no/yes) 0.018 0.009 0.046 0.002 0.008 0.78 0.19 0.041 0.28 0.025 0.96 a HDL, high-density lipoprotein; Values are estimated effect+se for 1-S.D. change for continuous risk factors, and for differences between having and not having dichotomous risk factors. b Values of triglycerides were log transformed in the analyses. c The participants were grouped in five age-strata as shown in Table 1.

E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 441 Table 3 Age-adjusted mean intima-media thickness in men and women without or with a family history of stroke and/or myocardial infarction: the Tromsø study Sex/age No family history of stroke or myocardial infarction Family history of myocardial Family history of stroke Family history of stroke and infarction myocardial infarction n IMT SE (mm) n IMT SE (mm) n IMT SE (mm) n IMT SE (mm) p-value a Men 25 59 641 0.802 0.006 192 0.806 0.010 202 0.815 0.010 81 0.812 0.016 0.70 60 84 887 0.955 0.006 281 0.975 0.011 447 0.980 0.009* 138 1.010 0.016** 0.004 All men 1528 0.896 0.004 473 0.909 0.008 649 0.916 0.007* 219 0.933 0.012** 0.006 Women 25 59 671 0.730 0.005 217 0.760 0.008** 242 0.740 0.008 111 0.751 0.012 0.01 60 84 862 0.876 0.005 322 0.885 0.009 493 0.898 0.007* 221 0.928 0.011*** 0.0001 All women 1533 0.820 0.004 539 0.835 0.006* 735 0.835 0.005* 332 0.858 0.008*** 0.0001 a P-value for overall F-test (analysis of variance with age as covariate). * P 0.05; ** P 0.01; *** P 0.001 compared to no family history of stroke or myocardial infarction.

442 E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 Table 4 Determinants of mean intima-media thickness (mm) by multiple linear regression in men and women: the Tromsø study a Men Women P-value P-value Age (years) 0.087 0.004 0.0001 0.071 0.003 0.0001 Systolic blood pressure (mmhg) 0.033 0.004 0.0001 0.031 0.003 0.0001 Total cholesterol (mmol/1) 0.013 0.004 0.0003 0.009 0.003 0.0036 HDL cholesterol (mmol/1) 0.012 0.004 0.003 0.008 0.003 0.0085 Triglycerides (mmol/1) b 0.004 0.004 0.35 0.008 0.004 0.020 Smoking (no/yes) 0.025 0.007 0.0001 0.014 0.006 0.015 Body mass index (kg/m 2 ) 0.020 0.004 0.0001 0.006 0.003 0.017 Fibrinogen (g/l) 0.013 0.003 0.0001 0.003 0.003 0.29 Physical activity (no/yes) 0.018 0.006 0.003 0.007 0.006 0.21 Family history of MI (no/yes) 0.008 0.007 0.25 0.013 0.005 0.024 Family history of stroke (no/yes) 0.014 0.007 0.035 0.017 0.005 0.002 Adj. R 2 0.31 0.36 a Values are regression coefficients+se for a 1-S.D. change in continuous variables and for presence/absence of dichotomous variables; HDL, high-density lipoprotein; MI, myocardial infarction. b Values of triglycerides were log transformed in the analyses. ence in IMT associated with a 1 SD change in the risk factor, and the slope parameter associated with a dichotomous factor corresponds to the difference in IMT between those with and those without the risk factor. The SD scale was used to ease comparability between continuous variables, but does not extend to the dichotomous variables. We first assessed the age-adjusted effect of risk factors in men and women separately, and tested whether the effects differed between men and women. Then a trend test was applied in which a linear relationship between age-strata and the magnitude of the estimated impact of the risk factor was assumed. This trend test was performed in men and women separately and then in a pooled analysis of both sexes. An ageby-sex interaction test was applied to see whether there was a sex difference in the effect of the risk factor across age. Multiple linear regression analysis was used to assess the independent relationship between risk factors and IMT (Table 4). To test for interaction with sex, a pooled regression analysis was run including the following interaction terms: sex age, sex body mass index, sex physical activity, and sex fibrinogen. To investigate interaction with age, a pooled regression analysis including the interaction terms age triglycerides and age physical activity was run. Adjustment for time since last meal did not change coefficients notably. We did not adjust for multiple comparisons [10]. Age-adjusted (Table 3, Table 5, Fig. 2 and Fig. 5), age-and sexadjusted (Fig. 4), and multivariate-adjusted (Fig. 4) mean values of IMT within strata were calculated with ANCOVA. Two-sided P-values less than 0.05 were considered as statistically significant. The SAS software package [9] was used. 3. Results Of the 6889 survey participants, a total of 6727 subjects were examined with B-mode ultrasound, and 6408 were eligible for the present study. Among these subjects, high quality images of IMT of the CCA and the bifurcation were obtained in 6357 (99.2%) and 6015 subjects (93.9%), respectively. All subjects with missing images from the CCA also had missing images from the bifurcation (n=51). Seven percent of women and 4% of men had missing values of IMT from the bifurcation (P 0.001 for difference between sexes). Subjects in whom images were obtained from the CCA, but not from the bifurcation (n=342), had higher levels of body mass index than subjects where images were obtained both from the CCA and the bifurcation (n= 6015) (28.1 vs. 25.8 kg/m 2, respectively; P 0.0001), but did not differ in age, systolic blood pressure, or total cholesterol. Mean levels of total cholesterol, HDL cholesterol, and fibrinogen were higher in women than in men (all P 0.001), whereas men had higher triglyceride levels (P 0.001) than women (Table 1). Thirty-four percent of men and 31% of women were smokers. More men than women reported a history of myocardial infarction (P 0.001), angina pectoris (P 0.001) or stroke (P= 0.03), whereas the prevalence of treated hypertension and diabetes mellitus was similar in men and women. 3.1. Distribution of intima-media thickness IMT was smallest in the far wall of the CCA, and greatest in the far wall of the bifurcation (Table 1, Fig. 1). In both sexes there was a graded and almost linear increase in the median IMT from the age of 25 years

and throughout the whole age-range (Fig. 1). Men had a thicker mean intima-media layer than women in all three locations (P 0.0001 for all locations) (Table 1, Fig. 1). This sex difference was small in younger agegroups and increased with age as the average progression per year of mean IMT was greater in men than in women:, 0.010 mm versus 0.009 mm, respectively (P 0.01). The range of IMT values also increased with age in both sexes, but the increase was greater in men than women (Fig. 1). The increase in range of IMT values started at an earlier age in the bifurcation than in the CCA. For both sexes the distribution of IMT showed a positive skewness (Fig. 1). This skewness increased with age, was more pronounced in men than in women, and more pronounced in the bifurcation than in the CCA. Mean and maximum IMT values within each location were highly correlated (r 0.9 in all three locations). The correlation between the mean IMT values in the three different locations ranged from r=0.38 to r= 0.50. E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 443 Table 5 Comparison of carotid artery intima-media thickness (mm) among healthy subjects and subjects with cardiovascular disease: the Tromsø study a N Intima-media thickness mean b max c Men Healthy d 2127 0.882 0.004 1.139 0.005 Myocardial infarction 289 0.926 0.010*** 1.226 0.014*** Angina pectoris 347 0.948 0.009*** 1.261 0.013*** Diabetes mellitus 102 0.974 0.016*** 1.264 0.024*** Treated hypertension 425 0.941 0.008*** 1.232 0.012*** Stroke 97 0.936 0.016*** 1.217 0.024*** Women Healthy d 2633 0.812 0.003 1.045 0.004 Myocardial infarction 108 0.861 0.014*** 1.157 0.020*** Angina pectoris 260 0.857 0.009*** 1.131 0.013*** Diabetes mellitus 107 0.855 0.014** 1.095 0.020* Treated hypertension 458 0.870 0.007*** 1.135 0.010*** Stroke 80 0.847 0.016* 1.142 0.023*** a Values are age-adjusted mean IMT SE. b Mean IMT, average of mean IMT in the near and far walls of the CCA and the far wall of the bifurcation. c Max IMT, average of max IMT in the near and far walls of the CCA and the far wall of the bifurcation. d No history of myocardial infarction, angina pectoris, diabetes mellitus, treated hypertension or stroke. * P 0.05. ** P 0.01. *** P 0.001 compared with healthy subjects. Fig. 2. Age-adjusted values of mean IMT ( SE) by systolic blood pressure (top panel), total cholesterol (middle panel) and HDL cholesterol (bottom panel). Men are shown in solid lines, women in dashed lines. The Tromsø Study. 3.2. Effects of cardio ascular risk factors on IMT in men and women Table 2 shows the effects of risk factors on IMT. Systolic blood pressure, total cholesterol, HDL cholesterol, triglycerides, and body mass index were significantly associated with IMT in both men and women. Systolic blood pressure had a greater effect on IMT than other risk factors. In both sexes there was a linear increase in IMT by increasing levels of continuous risk factors, illustrated by systolic blood pressure (P 0.0001 for linear trend) and serum cholesterol (P 0.0001 for linear trend), and a decrease in IMT by increasing levels of HDL cholesterol (P 0.0001 for linear trend) without evidence of a cut-off point below or plateau above which no association was seen (Fig. 2). In the univariate analyses current smoking was not associated with IMT in women, whereas an association was seen in men (Table 2). Increasing levels of fibrinogen was associated with elevated IMT in men only (Table 2). Physical activity was associated with a decrease in IMT in men, but not in women (Table 2 and Fig. 3). The effect of triglycenides and physical activity on IMT was stronger with increasing age (Table 2 and Fig.

444 E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 (P=0.07 for trend), but was similar across age strata in women (Fig. 3). Effects of other risk factors on IMT did not differ depending on age. These results were not altered notably when we excluded from the analysis subjects with a history of diabetes mellitus or CVD or who were taking antihypertensive or lipidlowering drugs. 3.3. Effects of a family history of cardio ascular disease on IMT Fig. 5. Age-adjusted values of mean IMT by quartiles of HDL cholesterol stratified by quartiles of triglycerides in men (top panel) and women (bottom panel). The Tromsø Study. 3). The age-trends were similar in men and women, although for physical activity the age-trend was stronger in men than in women. Effects of systolic blood pressure tended to increase with age in men Fig. 4. Age and sex-adjusted and multivariate-adjusted values of mean IMT ( SE) in subjects with no family history of CVD, subjects with a family history in brother only, sister only, father only, mother only, and a family history of CVD in two or more family members. Variables adjusted for were age, sex, systolic blood pressure, total cholesterol, HDL cholesterol, triglycerides, body mass index, smoking, and physical activity. P-values for overall F-test among the six categories of family history (Analysis of covariance). The Tromsø Study. Men with a family history of stroke, and women with a family history of myocardial infarction or stroke, had significantly thicker IMT than subjects with no family history (Table 3). Also, an additive effect of familial clustering was apparent since the thickest IMT-values were observed among subjects who had a family history of both stroke and myocardial infarction. Interestingly, there was a greater effect of a family history of CVD on IMT with increasing age (P=0.01 for interaction with age in pooled ANCOVA with IMT as dependent variable and age, sex, family history and family history by age as predictor variables). Having a sister who had suffered a stroke or myocardial infarction was a significantly stronger predictor for IMT than having a brother or a father who had suffered from cardiovascular disease (Fig. 4). 3.4. Multi ariate analyses of cardio ascular risk factors and IMT Table 4 shows determinants of IMT in a multiple linear regression model stratified by sex. Age was by far the strongest predictor for IMT in both sexes, but was stronger in men than in women (P 0.0001 for interaction). The strength of the association between systolic blood pressure, total cholesterol, HDL cholesterol, and smoking and IMT was similar in men and women. Female smokers had significantly lower systolic blood pressure than female nonsmokers (137 mmhg vs. 141 mmhg; P 0.0001). Whereas the association between smoking and IMT in women was non-significant in univariate analyses (Table 2), smoking was a significant predictor for IMT in women when controlling for systolic blood pressure (Table 4). Body mass index, fibrinogen, and physical activity had greater effects on IMT in men than in women (P=0.0002, P=0.006 and P=0.005, respectively, for interaction with sex), whereas triglycerides were independently associated with IMT in women only when controlling for HDL cholesterol level (Table 4). Fig. 5 shows that the triglyceride level is a significant predictor of IMT in women but not in men also when stratifying for HDL cholesterol level. Interaction terms of age by

E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 445 triglycerides and age by physical activity were significant in a pooled analysis of men and women (P 0.0001 and P=0.026, respectively). Diastolic blood pressure was not included in the model because of colinearity with systolic blood pressure (r=0.74). However, when systolic blood pressure was exchanged with diastolic blood pressure in the model, diastolic blood pressure was strongly associated with IMT in women, but not in men (data not shown). Mean values of IMT were significantly greater in subjects with cardiovascular disease than in subjects with no such diseases (Table 5). 4. Discussion The data presented here point to sex differences both in the distribution and the determinants of carotid IMT, and shows that a family history of cardiovascular disease is a strong predictor of subclinical atherosclerosis. In accordance with previous studies [6,11] we observed significant associations between carotid IMT and prevalent coronary heart disease, providing further evidence that carotid IMT reflects abnormalities in other vascular territories and may be used for in vivo quantification of early atherosclerosis. We studied a large population based sample of men and women covering a broad age-range from young adulthood through old age. The study is strengthened by the high participation rate and the standardised ultrasound methods [1]. Its primary limitation is the use of cross sectional data. Due to the cross-sectional study design, selective survival may have influenced the associations among the elderly since subjects whose arteries are less susceptible to elevated risk factor levels may be overrepresented in the study. The effect of this selection bias is, however, probably to attenuate any true association between risk factor levels and IMT. 4.1. Age and sex differences in the distribution of IMT Progression of IMT is an age-related process which affect both sexes, but the increase in IMT with age is greater in men than in women (Fig. 1). The distribution of IMT in women showed no inflection at the age of menopause that could be attributed to an effect of menopause on progression of atherosclerosis. Why is the intima-media complex thicker in men than in women? It has been suggested that the difference in IMT between men and women may be explained by sex differences in lumen diameter, and that sex differences in IMT merely reflect differences in physiology rather than differences in the extent of atherosclerosis [12]. In our study, the age-adjusted sex difference in common carotid IMT was 0.07 mm (P 0.0001). When controlling for the sex difference in lumen diameter the IMT difference between sexes was lowered to 0.05 mm, but remained highly significant (P 0.0001). Furthermore, if the difference in IMT between sexes could be attributed to differences in lumen diameter, one would expect that the difference was present at all ages. We found no difference between men and women under 50 years of age in common carotid artery IMT. Taken together, these observations indicate that physiological differences in lumen diameter cannot account for sex differences in IMT. We suggest that the observed sex differences in IMT reflect sex differences in the extent of atherosclerosis. Differences in cardiovascular risk factor levels may partly account for the thicker intimamedia complex in men than in women, however, our study also indicates that certain risk factors may have different effects in men and women. 4.2. Sex differences in risk factor associations with IMT Fig. 3. Estimated difference in IMT ( SE) for a 1 S.D. change in systolic blood pressure (top panel), triglycerides (middle panel) and physical activity (no/yes) (bottom panel) according to age. Men are shown in solid lines, women in dashed lines. The Tromsø Study. We observed that systolic blood pressure, total cholesterol, HDL cholesterol, body mass index, and smoking were independent predictors of IMT in both sexes. In contrast, the effects of fibrinogen, physical activity, and triglycerides differed in men and women. In the present study, fibrinogen was an independent risk factor for subclinical atherosclerosis in men only.

446 E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 In support of our findings, accumulating evidence indicates that fibrinogen may be a stronger cardiovascular risk factor in men than in women. Several large, prospective studies have documented fibrinogen as a predictor for cardiovascular disease in both sexes [13 15] but this association appears to be weaker in women than in men [13,15]. Fibrinogen has been identified as a significant predictor for IMT in pooled gender analyses [16] but in studies of middle aged [17] and elderly women [18], no significant association between fibrinogen and IMT has been shown. A study of peripheral atherosclerosis observed that men, but not women, were susceptible to increasing levels of fibrinogen and blood viscosity [19]. Smoking is the most important lifestyle determinant of fibrinogen [20]. In the present study the association between fibrinogen and IMT appeared slightly stronger in smokers than non-smokers, and the sex differences in the effect of fibrinogen on IMT were somewhat greater among non-smokers than smokers. Physical activity has been associated with a decreased risk of cardiovascular disease morbidity and mortality in men [21,22] while studies in women have produced mixed results [22 24]. The effect of physical activity on IMT has, to our knowledge, previously been examined in the ARIC study only [25]. That study showed that physical activity at work was a protective factor for IMT in both sexes, but appeared slightly stronger in men than women. We found a greater protective effect of leisure time physical activity on IMT in men than in women (Tables 2 and 4). Paffenbarger et al. suggested that the explanation for a lack of association between increased activity and decreased coronary heart disease risk in some studies was the low activity level in the so-called active group [26]. In our study, men were more physically active than women, and it is possible that the activity level among women was too low to show a benefit. Another possible explanation for the lack of protection of physical activity on IMT in women could be that the benefit of exercise is mediated by improving levels of something women already have, such as high levels of HDL cholesterol [27]. However, we found that the effect of physical activity on IMT was similar across HDL cholesterol strata in both men and women (data not shown). The atherogenicity of triglycerides and the role of serum triglycerides as a risk factor for coronary heart disease remains controversial, partly because multivariate analysis controlling for HDL cholesterol usually eliminates the role of triglycerides as a predictor for coronary heart disease. However, progression of IMT has been shown to be significantly correlated with levels of triglyceride-rich lipoproteins (intermediate density lipoproteins), whereas very-low density lipoproteins (VLDL), low-density lipoproteins (LDL), or HDL subfractions were not [28]. The present study indicates that triglycerides may be more strongly associated with subclinical atherosclerosis in women than in men, and shows that the association between IMT and triglycerides in women is independent of HDL cholesterol levels. A recent metaanalysis of prospective studies also showed a stronger association between plasma triglyceride levels and risk of clinical cardiovascular disease in women than in men [29]. The present study measured non-fasting triglyceride levels. Post-prandial hypertriglyceridemia has been found to be a stronger risk factor for IMT than fasting triglycerides [30]. Non-fasting triglycerides may therefore provide more information than fasting measurements. 4.3. Age differences in risk factor associations with IMT Studies which examined whether risk factors have the same power to predict cardiovascular disease in old age as in middle-aged persons have produced inconsistent results [31 33]. We found that total cholesterol, HDL cholesterol, smoking and body mass index had a relatively stable association with IMT across age strata, whereas the impact of triglycerides and physical activity increased with age. Interestingly, none of the risk factors showed a decreasing impact with age. The increasing effect of physical activity with age is supported by findings from the Framingham Heart Study [22] and the Honolulu Heart Study [34], in which a stronger protective effect of physical activity on stroke risk in older than in younger men was found. Selection of men into various levels of activity is one possible source of bias by which physical impaired subjects may have been at the low end of the physical activity spectrum. However, in the present study, after excluding subjects with cardiovascular disease, the trend towards a greater protective effect of physical activity among elderly men persisted. It is possible that different risk factors are active in different stages of atherogenesis. Risk factors found to have a stable effect across age could be important in initiating atherosclerosis, whereas risk factors with increasing effect with age may be more associated with the progression of atherosclerosis. The increasing strength of the association between systolic blood pressure and IMT with age in men could represent stiffening of the arteries due to advanced atherosclerosis rather than an increasing impact of blood pressure on atherosclerosis [35,36]. 4.4. Association between family history of cardio ascular disease and IMT The present study shows that a family history of cardiovascular disease is a risk factor for subclinical atherosclerosis also when controlling for conventional

E. Stensland-Bugge et al. / Atherosclerosis 154 (2001) 437 448 447 risk factors. The family history can influence a person s risk of atherosclerosis by means of genetic or environmental factors or their combined effects. There is a well-known familial aggregation of established cardiovascular risk factors such as hyperlipemia and blood pressure [37,38]. Our findings indicate, however, that familial clustering of atherosclerosis cannot be fully explained by familial aggregation of the conventional risk factors measured in this study. It has been reported that family history may predict clinical cardiovascular disease independent of known risk factors [39,40], but the effect of a family history on subclinical atherosclerosis has to a little extent been examined previously. In a recent report from the EVA-study [41], parental history of premature death of coronary heart disease was associated with presence of carotid plaques, but not with carotid IMT in 1040 men and women 59 71 years old. However, the EVA Study did not include plaques in IMT measurements and had few subjects with a positive family history (23 men and 30 women). Interestingly, we observed that the effect of the family history increased with age. Among subjects younger than 60 years, 57% reported no family history of CVD. The corresponding number in subjects 60 years and older was 48%, suggesting that the stronger association between family history of CVD among the elderly may not be explained by higher rates of a positive family history among the elderly. Our results may imply a time-dependent expression of genes, a gene environment interaction or a familial aggregation of environmental factors. We found that the occurrence of cardiovascular disease in a sister was a particular powerful determinant for subclinical atherosclerosis. Similar findings have been reported from a case-control study of myocardial infarction [42], and indicate that women afflicted with cardiovascular disease have a particular strong familial susceptibility. This sex difference could be expected if the genetic component in liability to atherosclerosis is polygenic, and modified by sex in such a way that women require significantly stronger genetic liability before they develop atherosclerosis [43]. In conclusion, the data presented here points to significant sex differences both in the distribution and the determinants of subclinical atherosclerosis. The sex difference in IMT becomes more pronounced with age, maybe as a consequence of sex differences in the susceptibility for atherosclerosis or sex differences in the distribution and effect of cardiovascular risk factors. 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