Marie-Louise M. Grønholdt

Ultrasound and Lipoproteins as Predictors of Lipid-Rich, Rupture-Prone Plaques in the Carotid Artery

The aim of this review is to summarize present knowledge of the ultrasonic detection and determinants of carotid atherosclerosis with lipid-rich cores and to review the evidence that these measures of plaque type may predict cerebral events. With the use of high-resolution ultrasound B-mode imaging, carotid plaques evaluated as only weakly reflecting the ultrasound beam (echolucent) have been associated with a higher risk of neurological events than are plaques reflecting the ultrasound signal strongly (echorich). Histologically, these echolucent plaques have a higher content of lipid and hemorrhage than do echorich plaques, which contain more calcification and fibrous tissue. Findings in the coronary arteries indicate that a lipid-rich plaque with a thin, fibrous cap is more vulnerable, is more prone to rupture, and cause symptoms compared with fibrous plaques. A search for determinants in the blood for these vulnerable plaques suggests that low density lipoprotein (LDL) cholesterol is the best lipid predictor for the extent of atherosclerosis, whereas triglyceride-rich lipoproteins in particular seem to predict an echolucent plaque. Lowering of LDL cholesterol and triglyceride-rich lipoproteins in plasma is associated with reduced progression of coronary atherosclerosis and coronary events. LDL cholesterol reduction is also associated with a reduced stroke rate. These improvements in the prognosis are thought to be the result of a reduction in the lipid content of the plaques, making them more stable and resistant to rupture rather than an actual reduction in plaque volume and degree of stenosis. In conclusion, it appears that ultrasound B-mode imaging as well as lipoproteins presumably may predict dangerous and rupture-prone, lipid-rich plaques in the carotid arteries, thereby being potential diagnostic tools in the prevention of neurological events.

Echo-Lucency of Computerized Ultrasound Images of Carotid Atherosclerotic Plaques Are Associated With Increased Levels of Triglyceride-Rich Lipoproteins as Well as Increased Plaque Lipid Content

BACKGROUND Echo-lucency of carotid atherosclerotic plaques on computerized ultrasound B-mode images has been associated with a high incidence of brain infarcts as evaluated on CT scans. We tested the hypotheses that triglyceride-rich lipoproteins in the fasting and postprandial state predict carotid plaque echo-lucency and that echo-lucency predicts a high plaque lipid content. METHODS AND RESULTS The study included 137 patients with neurological symptoms and > or = 50% stenosis of the relevant carotid artery. High-resolution B-mode ultrasound images of carotid plaques were computer processed to yield a measure of echogenicity (gray-scale level). Lipoproteins were measured before and hourly for 4 hours after a standardized fatty meal. A subgroup of 58 patients underwent endarterectomy. On linear regression analysis, echo-lucency (low gray-scale level) was associated with elevated levels of fasting and postprandial plasma triglycerides (P=.0002 and P=.002), IDL cholesterol (P=.0009 and P=.006), and VLDL/chylomicron remnant cholesterol (P=.0003 and P=.0004) and triglycerides (P=.0003 and P=.003), the area under the plasma triglyceride curve 0 to 4 hours after a fatty meal (P=.001), and body mass index (P=.0001). On ANCOVA, body mass index, fasting IDL cholesterol, and fasting plasma triglycerides were independent predictors of echo-lucency. Echo-lucency was associated with increased relative plaque lipid content (P=.02). CONCLUSIONS Increased plasma levels of triglyceride-rich lipoproteins predict echo-lucency of carotid plaques, which is associated with increased plaque lipid content. Because echo-lucency has been associated with a high incidence of brain infarcts on CT scans, triglyceride-rich lipoproteins may predict a plaque type particularly vulnerable to rupture.

Quantitative analysis of ultrasound B-mode images of carotid atherosclerotic plaque: correlation with visual classification and histological examination

This paper presents a quantitative comparison of three types of information available for 52 patients scheduled for carotid endarterectomy: subjective classification of the ultrasound images obtained during scanning before operation, first- and second-order statistical features extracted from regions of the plaque in still ultrasound images from three orthogonal scan planes and finally a histological analysis of the surgically removed plaque. The quantitative comparison was made with the linear model and with separation of the available data into training and test sets. The comparison of subjective classification with features from still ultrasound images revealed an overall agreement of 60% for classification of echogenicity and 70% for classification of structure. Comparison of the histologically determined relative volume of soft materials with features from the still images revealed a correlation coefficient of r=-0.42 (p=0.002), for mean echogenicity of the plaque region. The best performing feature was of second order and denoted contrast (r=-0.5). Though significant, the latter correlation is probably not strong enough to be useful for clinical prediction of relative volume of soft materials for individual patients. Reasons for this is discussed in the paper, together with suggestions for improvements.

Lipid-rich carotid artery plaques appear echolucent on ultrasound B-mode images and may be associated with intraplaque haemorrhage

OBJECTIVE To relate the histological composition of carotid artery plaques with morphology as evaluated by B-mode ultrasound. DESIGN Prospective study. MATERIAL AND METHODS Seventy-eight symptomatic patients underwent carotid endarterectomy after preoperative ultrasound Duplex scanning evaluating plaque morphology. Morphometric analysis of the removed specimen was performed in order to quantify content of lipid, haemorrhage, calcification and fibrous tissue. RESULTS Echolucent plaques contained more lipid (p = 0.01) and less calcification (p = 0.01) and fibrous tissue (p = 0.03) than echo-rich plaques. Intraplaque haemorrhage was directly related to lipid content (p = 0.004) and inversely related to amount of fibrous tissue in the plaque (p = 0.02). CONCLUSION The intensity of the reflected B-mode ultrasound signal appears related to the histological composition of the plaque. The association between intraplaque haemorrhage and a high lipid content may support the theory of the lipid-rich plaque being more prone to rupture.

Angiotensinogen Single Nucleotide Polymorphisms, Elevated Blood Pressure, and Risk of Cardiovascular Disease

Abstract— In this study of 10 690 individuals, associations with elevated blood pressure, ischemic heart disease, and ischemic cerebrovascular disease were determined for two noncoding [A(‐20)C, G(‐6)A] and two coding (T174M, M235T) single nucleotide polymorphisms, analyzed alone and in combination (haplotypes). Participants from the general population with (n=4950) and without (n=4234) elevated blood pressure were compared (study 1), as were participants from the general population without ischemic heart disease and ischemic cerebrovascular disease (n=7965) and cases with either ischemic heart disease (n=1850, study 2) or ischemic cerebrovascular disease (n=848, study 3). Finally, 22‐year follow‐up of 9184 individuals from the general population examined risk of ischemic heart disease (study 4) and ischemic cerebrovascular disease (study 5). Individuals with ‐6AA, 174TT, or 235TT had plasma angiotensinogen levels increased by 80 ng/mL (P =0.01 and 0.05 for women and men) compared with individuals with ‐6GG, 174TT, or 235 MM. In women, this difference was associated with an odds ratio of elevated blood pressure of 1.25 (1.03 to 1.51), which increased to 1.63 (1.05 to 2.51) in postmenopausal women receiving hormone replacement therapy. The promoter single nucleotide polymorphisms alone or as haplotypes did not predict the continuous variables of systolic, diastolic, or pulse pressure in cross section or the risk of ischemic heart disease or ischemic cerebrovascular disease in either gender in case‐control or prospective studies. Individuals with ‐6AA, 174TT, or 235TT in the angiotensinogen gene have increased plasma angiotensinogen levels and moderately increased risk of elevated blood pressure (women only) but unaltered blood pressure examined as a continuous variable and unaltered risk of ischemic heart disease and ischemic cerebrovascular disease.

Estrogen Receptor α Polymorphism and Risk of Cardiovascular Disease, Cancer, and Hip Fracture Cross-Sectional, Cohort, and Case–Control Studies and a Meta-Analysis

Background— We hypothesized that the estrogen receptor α (ESR1) IVS1-397T/C polymorphism affects high-density lipoprotein cholesterol response to hormone replacement therapy and risk of cardiovascular disease (CVD), cancer of reproductive organs, and hip fracture. Methods and Results— We studied cross-sectionally 9244 individuals from the Danish general population and followed them up for 23 to 25 years. End points were CVD (ischemic heart disease, myocardial infarction, angina pectoris, ischemic cerebrovascular disease, ischemic stroke, other ischemic cerebrovascular disease, venous thromboembolism, deep vein thrombosis, and pulmonary embolism), cancer of reproductive organs (breasts, ovaries, uterus, and prostate), and hip fracture. We also studied patients with ischemic heart disease (n=2495), ischemic cerebrovascular disease (n=856), and breast cancer (n=1256) versus general population controls. The CC, CT, and TT genotypes had general population frequencies of 21%, 50%, and 29%, respectively. Cross-sectionally, genotype did not influence high-density lipoprotein cholesterol response to hormone replacement therapy. In the cohort study, there were no differences in risks of CVD, cancer of reproductive organs, or hip fracture between genotypes. In case–control studies, risk of CVD did not differ between genotypes; however, the odds ratio for breast cancer in women with TT versus CC genotypes was 1.4 (95% CI, 1.1 to 1.7). Meta-analysis in men of 6 previous and the present 2 studies, including 4799 cases and 12 190 controls, showed odds ratios in CC versus CT and TT genotypes for fatal and nonfatal myocardial infarction of 0.81 (95% CI, 0.59 to 1.12) and 1.08 (95% CI, 0.97 to 1.21). Conclusions— ESR1 IVS1-397T/C polymorphism does not influence high-density lipoprotein cholesterol response to hormone replacement therapy or risk of CVD, most cancers of reproductive organs, or hip fracture.

Echolucent Carotid Artery Plaques Are Associated With Elevated Levels of Fasting and Postprandial Triglyceride-Rich Lipoproteins

BACKGROUND AND PURPOSE Echolucent carotid atherosclerotic plaques are associated with an increased risk of neurological symptoms. Elevated plasma triglycerides is a risk factor for cerebral and coronary infarction. This study examined these individual pathogenetic risk factors to determine whether they were related. METHODS We included 85 symptomatic patients with at least 40% carotid artery stenosis. Plaque morphology of the relevant artery was evaluated by high-resolution B-mode ultrasonography as echolucent, echo-rich, or intermediate. Fasting and postprandial lipids and lipoproteins were measured before and at hourly intervals for 4 hours after a fatty meal (1 g cream fat per kilogram body weight). RESULTS When we compared patients with echolucent plaques to patients with echo-rich or intermediate plaques, the former had higher fasting and postprandial plasma triglycerides (P < or = .006), higher chylomicron remnants/VLDL cholesterol (P = .02) and triglycerides (P < or = .004), a larger area under the plasma triglyceride curve 0 to 4 hours after a fatty meal, with (AUCTG-TG oh) or without (AUCTG) subtraction of fasting levels (P = .007 and P = .003), a larger body mass index (P = .03), and were younger (P = .01). Multiple logistic regression analysis found that when age and body mass index were taken into account, fasting plasma and VLDL triglycerides, postprandial chylomicron remnants/VLDL triglycerides, AUCTG-TG oh and AUCTG with odds ratios of 4.1, 3.8, 3.0, 2.7, and 4.3, respectively, were independent predictors of an echolucent plaque. CONCLUSIONS Echolucent carotid artery plaques are associated with elevated levels of triglyceride-rich lipoproteins in the fasting or postprandial state.

Elevated matrix metalloproteinase-9 associated with stroke or cardiovascular death in patients with carotid stenosis.

Background— Matrix metalloproteinase-9 could exhibit an important role in the destabilization of atherosclerotic carotid plaques. We hypothesized that in patients with carotid stenosis, elevated levels of plasma matrix metalloproteinase-9 are associated with ipsilateral stroke or cardiovascular death. Methods and Results— We followed up 207 patients with ≥50% carotid stenosis initially for a mean of 4.4 years, during which time 53 patients developed ipsilateral stroke or died of cardiovascular causes. The cumulative incidence of ipsilateral stroke or cardiovascular death was higher in those with matrix metalloproteinase-9 above versus below the median of 41.9 ng/mL (log-rank P=0.002). Matrix metalloproteinase-9 above versus below the median had a hazard ratio for ipsilateral stroke or cardiovascular death of 1.9 (95% confidence interval [CI], 1.1 to 3.5); during extended follow-up, this remained significant until 10 years. The absolute risk of ipsilateral stroke or cardiovascular death at 4.4 years was 34% and 17% in those with matrix metalloproteinase-9 above and below the median, respectively. Elevated matrix metalloproteinase-9 and an echolucent plaque on B-mode ultrasound versus a low matrix metalloproteinase-9 and an echorich plaque had a hazard ratio for ipsilateral stroke or cardiovascular death of 4.4 (95% CI, 1.8 to 11.1) and for ipsilateral stroke of 3.3 (95% CI, 1.1 to 9.7). Conclusions— Elevated levels of matrix metalloproteinase-9 in patients with ≥50% carotid stenosis were associated with a 2-fold risk of ipsilateral stroke or cardiovascular death. Combining elevated matrix metalloproteinase-9 and plaque echolucency was associated with a 4-fold risk for ipsilateral stroke or cardiovascular death and a 3-fold risk for ipsilateral stroke.

Angiotensinogen Mutations and Risk for Ischemic Heart Disease, Myocardial Infarction, and Ischemic Cerebrovascular Disease: Six Case–Control Studies from the Copenhagen City Heart Study

Angiotensinogen is a key protein in the reninangiotensin system, which influences vascular tone, renal sodium reabsorption, and blood pressure (1). Huge scientific interest was generated when one of two amino acidchanging mutations in the angiotensinogen gene, M235T but not T174M, was found to be associated with elevated plasma angiotensinogen levels (2), elevated blood pressure (2), ischemic heart disease (3), and ischemic cerebrovascular disease (4). The M235T mutation changes a nonpolar amino acid to a polar amino acid and thus potentially changes the tertiary structure of the protein. Because of this, it is likely that such a mutation may also influence protein function. A few studies (5-7), but not others (8-10), have confirmed the association of M235T with increased risk for ischemic cardiovascular disease. However, because these previous studies (3-10) included only 106 to 2250 participants, it is possible that the observed associations between the M235T angiotensinogen mutation and increased risk for ischemic heart and ischemic cerebrovascular disease represent chance observations rather than true associations. For example, the angiotensin-converting enzyme insertiondeletion polymorphism was associated with increased risk for ischemic cardiovascular disease in small studies, but not in large ones (11). Small studies with positive results are more likely to be published than those with negative results (12). In the Copenhagen City Heart Study, which has more than 9000 participants, we previously observed that single homozygosity (235TT/174TT) in the angiotensinogen gene was associated with a 10% increase in angiotensinogen levels and was a risk factor for elevated blood pressure in women but not in men (13). Of interest, however, blood pressure (systolic and diastolic) as a continuous variable was not associated with angiotensinogen mutations. These findings prompted us to explore whether angiotensinogen mutations influence risk for ischemic heart disease, myocardial infarction, or ischemic cerebrovascular disease, since elevated blood pressure is one of the most important risk factors for these diseases. Methods Overview To test the hypothesis that the M235T and T174M angiotensinogen mutations are associated with risk for ischemic heart disease, myocardial infarction, and ischemic cerebrovascular disease, we conducted six large casecontrol studies including both women and men in the ethnically homogeneous Danish population. Persons without ischemic heart disease, myocardial infarction, or ischemic cerebrovascular disease (n = 7975) were selected from the Copenhagen City Heart Study as controls (Figure 1) and were compared with other Copenhagen City Heart Study participants who had ischemic heart disease (n = 866), myocardial infarction (n = 519), or ischemic cerebrovascular disease (n = 489) (studies 1a, 1b, and 1c). Other case-patients who were referred to the same hospital with ischemic heart disease (n = 943), myocardial infarction (n = 493), or ischemic cerebrovascular disease (n = 434) were also compared with the controls (studies 2a, 2b, and 2c). Each casecontrol study examined M235T and T174M genotypes, separately and in combination. Finally, we investigated whether relative genotype frequencies changed as a function of age in both case-patients and controls. Figure 1. Study design. Participants Controls The Copenhagen City Heart Study (third examination, 19911994) is made up of women and men (age range, 20 to 93 years) stratified into 10-year age groups and sampled with the goal of obtaining a representative sample of the adult Danish general population (14-17). Each participant was randomly selected from the city of Copenhagen around Copenhagen University Hospital, Rigshospitalet, by using the Copenhagen Central Population Register. Of the 17 180 persons invited, 10 049 participated and 9259 gave blood for DNA isolation. In the 790 persons who declined to give blood for DNA, the distribution of ischemic heart disease and myocardial infarction was the same as that in our study sample (P>0.10 and P>0.20, respectively). However, this was not true for women who had ischemic heart disease, ischemic cerebrovascular disease, or myocardial infarction (P<0.001 for all three diseases) and for men with ischemic cerebrovascular disease (P<0.001). We performed genotyping for 7975 persons (4563 women and 3412 men) without ischemic heart disease, myocardial infarction, or ischemic cerebrovascular disease; these persons served as our control group. Controls reported smoking status and whether they had diabetes mellitus. Women reported menopausal status and use of hormone replacement therapy. Case-Patients from the Copenhagen City Heart Study In the Copenhagen City Heart Study cohort, information on development of ischemic heart disease, myocardial infarction, and ischemic cerebrovascular disease was collected and verified until 1997 by reviewing all hospital admissions and diagnoses entered in the Danish National Hospital Discharge Register, all deaths entered in the Danish National Register of Cause of Death, and medical records from hospitals and general practitioners. Case-patients had prevalent (developed before 19911994) or incident (developed after 19911994) disease. Experienced cardiologists determined whether case-patients had ischemic heart disease (World Health Organization [WHO] International Classification of Diseases, Eighth Edition, codes 410414) on the basis of previous myocardial infarction or characteristic symptoms of stable angina pectoris according to the guidelines of the European Society of Cardiology (location, character, and duration of pain and the relation of pain to exercise [18]). Experienced cardiologists determined whether case-patients had myocardial infarction (WHO International Classification of Diseases, Eighth Edition, code 410) on the basis of presence of at least two of the following: characteristic chest pain, elevated levels of cardiac enzymes, and electrocardiographic results consistent with a myocardial infarction. Experienced neurologists determined whether case-patients had ischemic cerebrovascular disease (WHO International Classification of Diseases, Eighth Edition, codes 432435) on the basis of sudden onset of focal neurologic symptoms (ischemic stroke, transient ischemic attack, or amaurosis fugax). The diagnostic criteria for these three conditions were the same as for case-patients from Copenhagen University Hospital with ischemic cerebrovascular disease (focal neurologic symptoms lasting 24 hours when hemorrhage was excluded on computed tomography), transient ischemic attack (focal neurologic symptoms lasting <24 hours), or amaurosis fugax (transient monocular blindness). Patients with cerebral hemorrhage were excluded. We performed genotyping for 866 and 489 patients with ischemic heart disease and ischemic cerebrovascular disease, respectively (Figure 1). Five hundred nineteen of those with ischemic heart disease had myocardial infarction. Case-Patients from Copenhagen University Hospital During 19911993, 992 patients from the greater Copenhagen area were referred to Copenhagen University Hospital, Rigshospitalet, for coronary angiography (14-17). Among these consecutive incident case-patients, 948 received a diagnosis of ischemic heart disease from experienced cardiologists because of characteristic symptoms of stable angina pectoris according to the guidelines of the European Society of Cardiology (location, character, and duration of pain and the relation of pain to exercise, plus 1 of the following: severe stenosis on coronary angiography [>70% stenosis of 1 coronary vessel or >50% stenosis of the left main coronary artery], myocardial infarction, or a positive result on exercise electrocardiography) (18). We performed genotyping for 943 patients with ischemic heart disease, 493 of whom had myocardial infarction (Figure 1). Two of the patients with ischemic heart disease were also registered as participants with ischemic heart disease in the Copenhagen City Heart Study. These two patients were not excluded from the hospital sample. We identified patients referred from 1994 to 1999 for outpatient ultrasonography of the carotid artery at Copenhagen University Hospital, Rigshospitalet (19). Experienced neurologists and vascular surgeons diagnosed ischemic cerebrovascular disease on the basis of sudden onset of focal neurologic symptoms, together with carotid artery stenosis of at least 50% on the symptomatic or most stenotic side. Therefore, neurologic symptoms were most likely to indicate large-vessel atherothrombotic disease. At least one computed tomography scan was obtained to exclude cerebral hemorrhage. Patients had ischemic stroke (focal neurologic symptoms lasting 24 hours when hemorrhage was excluded on computed tomography [n = 259]), transient ischemic attack (focal neurologic symptoms lasting <24 hours [n = 126]), or amaurosis fugax (transient monocular blindness [n = 46]). We performed genotyping for 434 consecutive incident case-patients with ischemic cerebrovascular disease (Figure 1). Four of these case-patients were registered as participants in the Copenhagen City Heart Study; however, they were not excluded from the hospital sample. Study Designs CaseControl Studies 1a, 1b, and 1c (Copenhagen City Heart Study) Participants in the Copenhagen City Heart Study with ischemic heart disease (n = 866), myocardial infarction (n = 519), or ischemic cerebrovascular disease (n = 489) were compared with the 7975 controls (Figure 1). CaseControl Studies 2a, 2b, and 2c (Copenhagen University Hospital) Patients with ischemic heart disease (n = 943), myocardial infarction (n = 493), or ischemic cerebrovascular disease (n = 434) were compared with the 7975 controls (Figure 1). These casecontrol studies are stronger than casecontrol studies 2a, 2b, and 2c because case-patients underwent more rigorous diagnostic assessment. DNA Analyses The M235T and T174M

ACE gene polymorphism as a risk factor for ischemic cerebrovascular disease.

Stroke and myocardial infarction are major causes of death in the western world. Hormonal regulation of the cardiovascular system includes the renin-angiotensin system. As part of this system, angiotensin-converting enzyme (ACE) is involved in bradykinin metabolism and converts the physiologically inactive angiotensin I to the active angiotensin II; the latter plays a major role in the regulation of vasoconstriction and sodium retention secondary to aldosterone secretion [1]. The ACE gene is located on chromosome 17q23 and consists of 26 exons and 25 introns; an insertion (I)-deletion (D) polymorphism of 287 base pairs has been identified in intron 16 [2]. The D allele of this polymorphism has been associated with elevated plasma ACE levels [2] and ACE activity [3-7] in a codominant pattern. Homozygosity for the D allele has also been found to be associated with myocardial infarction [8], although the two largest studies to date did not confirm this association [9, 10]. Nevertheless, a recent meta-analysis of studies that examined the association between ACE gene polymorphism and myocardial infarction (the studies included a total of 8873 persons) supported the hypothesis that ACE gene polymorphism represents a susceptibility mutation for myocardial infarction [11]. This metaanalysis, however, suggested publication bias for the positive results in the smaller studies. Associations between the D allele and stroke have also been found [12-15]. We tested the hypothesis that ACE gene polymorphism may represent a susceptibility mutation for ischemic cerebrovascular disease. We performed genotyping on 9495 persons from the ethnically homogeneous Danish population; 452 of these persons had ischemic cerebrovascular disease. Our study was approved by the Danish ethical committee for the City of Copenhagen and Frederiksberg, and all participants gave informed consent. Methods Participants Case-Referent Study 1 Case-patients were 35 women and 38 men from the greater Copenhagen area in whom focal neurologic symptoms due to ischemic cerebrovascular disease developed suddenly before 50 years of age (Figure 1). The case-patients were consecutively referred for outpatient examination of defects in the coagulation system at Rigshospitalet in Copenhagen from 1989 to 1995. The criteria for diagnosis of ischemic cerebrovascular disease were ischemic stroke (focal neurologic symptoms that lasted for more than 24 hours and for which computed tomography excluded intracerebral and extracerebral hemorrhage), transient ischemic attack (focal neurologic symptoms that lasted less than 24 hours), or amaurosis fugax (transient blindness in one eye only). Seventy-one case-patients had Danish parents, one was from France, and one was not white. The referent group comprised 1454 women and 1737 men from the general population sample who were within the same age range as the case-patients (women >30 years and <54 years of age; men >24 years and <56 years of age). In a case-referent study, case-patients are compared with a sample of the general population; the sample is roughly within the same age range as the case-patients, but participants are not exactly matched for age. Figure 1. Study designs. Case-Referent Study 2 Case-patients were 82 women and 137 men from the greater Copenhagen area who had sudden onset of focal neurologic symptoms due to ischemic cerebrovascular disease (Figure 1). The case-patients were consecutively referred for outpatient ultrasonography of the carotid artery at Rigshospitalet from 1994 to 1996. The overlap in time between the two groups of case-patients (1989 to 1995 and 1994 to 1996) reflects the different periods in which we could recruit the two groups of case-patients. It is unlikely that the small difference in recruitment period is important for the questions asked in our study: Neither genotype frequencies nor the context in which the genotypes act would change significantly in such a short period. The criteria for a diagnosis of ischemic cerebrovascular disease were ischemic stroke, transient ischemic attack, or amaurosis fugax, all diagnosed according to the criteria described in the preceding section. Only patients with carotid stenosis greater than 40% on the symptomatic side were included in this study; thus, case-patients had ischemic cerebrovascular disease and clinically significant atherosclerosis. All patients were white, and more than 98% had Danish parents. The referent group comprised 4273 women and 3091 men from the general population sample who were within the same age range as the case-patients (women >36 years and <80 years of age; men >42 years and <80 years of age). Cross-Sectional Study A general population sample comprising 9203 persons was assembled during the Copenhagen City Heart Study from 1991 to 1994. The sample, which consisted of an almost equal number of women and men stratified for age (from 20 to 80 years), was drawn randomly from the Copenhagen Central Population Register with the aim of obtaining a representative sample of the adult Danish general population (Figure 1) [16]. Less than 1% of the sample was nonwhite, and 98.8% had Danish citizenship (that is, they were essentially of Danish parentage). Persons with previous sudden onset of focal neurologic symptoms due to ischemic cerebrovascular disease were identified by an experienced neurologist on the basis of history and by review of all hospital admissions and diagnoses (obtained from the Danish National Hospital Discharge Register) and, if necessary, medical records from hospitals or general practitioners. The criteria for diagnosis of ischemic cerebrovascular disease were ischemic stroke, transient ischemic attack, or amaurosis fugax [17]; the diagnostic criteria for these conditions were almost identical to those described in the section on case-referent study 1. Of 107 patients with stroke, 70 had definite ischemic stroke, 9 had intracerebral hemorrhage, and 28 had strokes that could not be classified because of the lack of a computed tomographic scan. However, given the ratio of cases of intracerebral hemorrhage to all cases with a definite diagnosis (9 of 79), 3 ([9/79] 28) of the 160 patients with ischemic cerebrovascular disease probably had intracerebral hemorrhage. Information on ischemic cerebrovascular disease was available for 7393 persons; of these, 67 women and 93 men had ischemic cerebrovascular disease and 4077 women and 3156 men did not have disease. Laboratory Methods Cholesterol and triglyceride levels were determined enzymatically (CHOD-PAP, GPO-PAP, Boehringer Mannheim, Mannheim, Germany). High-density lipoprotein (HDL) cholesterol was measured in the supernatant after precipitation of apolipoprotein B-containing lipoproteins (Boehringer Mannheim). Lipoprotein(a), apolipoprotein A1, and apolipoprotein B levels were measured by using end-point turbidimetry with commercially available antisera (rabbit antihuman lipoprotein[a], DAKO A/S, Glostrup, Denmark; sheep antihuman apolipoprotein A1 and apolipoprotein B, Boehringer Mannheim). Plasma fibrinogen levels were measured kinetically (Fibrinogen Kinetic, Boehringer Mannheim). All analyses in all patients were performed by use of identical diagnostic kits and very similar autoanalyzers and were done at the examination facility of the Copenhagen City Heart Study, Rigshospitalet; the Department of Clinical Biochemistry, Rigshospitalet; or the Department of Clinical Biochemistry, Herlev University Hospital. Lipoprotein(a), apolipoprotein A1, apolipoprotein B, and fibrinogen levels were measured at only one of the departments on only one autoanalyzer. Precision and accuracy of all analyses were continually tested by using internal departmental controls; accuracy of cholesterol, triglyceride, and HDL cholesterol levels was also monitored by a nationwide external quality control program. Persons who read the biochemical results were blinded to the disease status of all participants. Other Analyses Body mass index was calculated as weight in kilograms divided by height in squared meters. Blood pressure was considered elevated if 1) the systolic blood pressure in mm Hg was larger than both 145 mm Hg and the combination of 110 and the participants age, 2) the diastolic blood pressure (phase 5) was greater than 100 mm Hg regardless of age [16], or 3) the participant was receiving treatment with at least one antihypertensive drug. Participants were classified as diabetic if they received treatment for diabetes. Smokers were considered to be persons who currently smoked; former smokers had stopped smoking at least the day before the examination. The presence of hypertension and diabetes mellitus and data on smoking habits were based on cross-sectional information and measurements at the time of examination; no information on the duration of these risk factors was used. ACE Gene Polymorphism Total genomic DNA was extracted from whole blood, as described elsewhere [18]. The insertion-deletion polymorphism of 287 base pairs in intron 16 of the ACE gene was identified by conventional polymerase chain reaction (PCR) by using two primers flanking the site of the insertion [19]. Fragments of about 190 base pairs (D allele) and 490 base pairs (I allele) were separated on a 2% agarose gel. All samples that seemed homozygous for the D allele were subjected to a second PCR amplification with an insertion-specific primer to check for misclassification resulting from a potential preferential amplification of the smaller D allele [10]. Four percent to 5% of persons with the ID genotype were initially misclassified as having the DD genotype, but this error was corrected before statistical analysis. All PCR analyses were performed at the Department of Clinical Biochemistry, Herlev University Hospital, by the same scientist and two technicians, both of whom were supervised by the scientist. Persons who read the PCR results were blinded to the disease status of the participants fr