Birgit Agerholm-Larsen

ACE gene polymorphism in cardiovascular disease meta-analyses of small and large studies in whites

The objective of this study was to assess the influence of the ACE gene insertion (I)/deletion (D) polymorphism on plasma ACE activity; blood pressure; and risk of myocardial infarction, ischemic heart disease, and ischemic cerebrovascular disease by comparing small and large studies. The meta-analyses are based on a literature search of MEDLINE up until April 1998 and assessment of bibliographies of published studies and reviews. Forty-six studies were selected, including a total of 32 715 white individuals. Plasma ACE activity was increased 40% and 71% for ID and DD versus II in small studies and 21% and 48% in large studies (small versus large: P<0.001 and P<0.001). Blood pressure was not influenced by genotype. Risk of myocardial infarction and ischemic heart disease was increased by 47% and 29%, respectively, for DD versus ID and II genotypes in small studies but not in large studies (small versus large: P<0.001 for risk of myocardial infarction and P=0.01 for risk of ischemic heart disease). Risk of ischemic cerebrovascular disease was not increased either in the small or in the largest study. In conclusion, the ACE gene polymorphism affects plasma ACE activity but not blood pressure and is not associated with increased risk of myocardial infarction, ischemic heart disease, or ischemic cerebrovascular disease in the largest studies.

Elevated HDL Cholesterol Is a Risk Factor for Ischemic Heart Disease in White Women When Caused by a Common Mutation in the Cholesteryl Ester Transfer Protein Gene

BACKGROUND The level of HDL cholesterol is inversely related to the risk of ischemic heart disease. METHODS AND RESULTS In 9168 women and men from a general population and 946 women and men with ischemic heart disease (all white), we tested the hypothesis that the Ile405Val mutation in the cholesteryl ester transfer protein gene (CETP) affects HDL cholesterol levels and the risk of ischemic heart disease. The relative frequencies of Ile/Ile, Ile/Val, and Val/Val carriers were 0.46, 0.43, and 0.11 for both women and men. Women with these 3 genotypes had mean HDL cholesterol levels of 1.68, 1.75, and 1.82 mmol/L, respectively (P<0.001, ANOVA), as well as a significant decrease in the ratio of total to HDL cholesterol (P=0. 002, ANOVA). On multiple logistic regression analysis, women not treated with hormone replacement therapy who were heterozygous or homozygous for Val405 had a 1.4-fold (95% CI 1.0 to 1.9) to 2.1-fold (95% CI 1.3 to 3.4) increase in the risk of ischemic heart disease. No significant associations were found in men. CONCLUSIONS Increased HDL cholesterol levels caused by mutations in CETP are associated with an increased risk of ischemic heart disease in white women.

ACE Gene Polymorphism: Ischemic Heart Disease and Longevity in 10 150 Individuals A Case-Referent and Retrospective Cohort Study Based on the Copenhagen City Heart Study

BACKGROUND Homozygosity for the deletion allele (D) of the angiotensin-converting enzyme (ACE) gene insertion-deletion polymorphism has been suggested to be a potent risk factor for myocardial infarction. With one exception, the samples studied so far have been small and/or ethnically heterogeneous, and most investigators have studied men only. METHODS AND RESULTS We investigated the association between ACE genotype and myocardial infarction as well as other manifestations of ischemic heart disease for both women and men in a case-referent study (n = 10,150) as well as in a retrospective cohort study (n = 7263). The cohort was from the ethnically homogeneous Danish population. Case subjects were from the same geographic area and had ischemic heart disease. Irrespective of the assumed degree of relative penetrance of the D allele, the odds ratios were not significantly different from 1.0 (P > .05) for ischemic heart disease, severe stenosis on coronary angiography, or myocardial infarction. There was also no association between ACE genotype and phenotypic variation in recognized risk factors for ischemic heart disease. Finally, the relative frequency of the D allele did not change as a function of age in subjects aged from 20 to > or = 80 years. CONCLUSIONS In two large studies, a case-referent study and a retrospective cohort study in an ethnically homogeneous white population, there was no evidence for a statistically significant difference in the development of myocardial infarction or any other manifestations of ischemic heart disease between genotype classes of the ACE gene polymorphism in either women or men.

Common Cholesteryl Ester Transfer Protein Mutations, Decreased HDL Cholesterol, and Possible Decreased Risk of Ischemic Heart Disease The Copenhagen City Heart Study

BackgroundCholesteryl ester transfer protein (CETP) mediates the transfer of cholesteryl ester from HDL in exchange for triglycerides in apolipoprotein B–containing lipoproteins. Methods and ResultsWe studied 2 common mutations in CETP, A373P and R451Q, in 8467 healthy women and men from the Danish general population and in 1636 Danish women and men with ischemic heart disease. The prevalence of 373P and 451Q was 0.10 and 0.07, respectively, for heterozygous carriers and 0.003 and 0.002, respectively, for homozygous carriers. All carriers of the 451Q allele also carried the 373P allele. HDL cholesterol in female noncarriers, heterozygotes, and homozygotes of 373P was 1.74±0.01 (mean±SE), 1.62±0.02, and 1.38±0.09 mmol/L, respectively (ANOVA, P <0.001). In men, equivalent values were 1.40±0.01, 1.26±0.02, and 1.19±0.09 mmol/L, respectively (ANOVA, P <0.001). HDL cholesterol decreased similarly as a function of 451Q genotypes and all 373P/451Q genotype combinations. Furthermore, apolipoprotein AI and the HDL cholesterol/apolipoprotein AI ratio was also lower in carriers of either of these mutations for both sexes. Finally, the CETP genotype was not associated with risk of ischemic heart disease unless we adjusted for HDL cholesterol: female heterozygous and homozygous carriers versus noncarriers had 36% lower risk of ischemic heart disease (95% CI 4% to 57%); in male carriers, we observed a similar trend. ConclusionsThe A373P/R451Q polymorphism in CETP is associated with decreases in HDL cholesterol of 0.12 to 0.36 mmol/L in women and 0.14 to 0.21 mmol/L in men and possibly with a paradoxical 36% decrease in the risk of ischemic heart disease in women.

Angiotensinogen Polymorphisms and Elevated Blood Pressure in the General Population: The Copenhagen City Heart Study

In the present study, we tested the hypothesis that the Met235Thr and Thr174Met mutations were associated or not with elevated blood pressure. We genotyped 9100 women and men from the Danish general population, of whom 54% had elevated blood pressure. Of the 9100, 41% and 12% carried the Thr235 and Met174 mutations, respectively; the Met174 mutation always occurred on the same allele as the Thr235 mutation. On multifactorial logistic regression analysis, women homozygous for Thr235 versus noncarriers had an odds ratio for elevated blood pressure of 1.29 (95% CI 1.05 to 1.58), which increased to 1.50 (1.15 to 1.96) if they also were homozygous for Thr174 (noncarrier of Met174). Women homozygous for Thr235 also had an increased risk of isolated elevated systolic blood pressure (1.37; 1.02 to 1.84) and of mild blood pressure elevation (1.40; 1.10 to 1.77). We found no statistically significant association between elevated blood pressure and genotype in men or among genotype and systolic blood pressure, diastolic blood pressure, or pulse pressure in either gender. Homozygosity for both Thr235 and Thr174 was associated with a 10% increase in plasma angiotensinogen levels in both genders compared with homozygosity for Met235 and Thr174; however, systolic and diastolic blood pressures were positively correlated to plasma angiotensinogen levels in women only. In conclusion, in this large-scale study of the general population, double homozygosity for Thr235 and Thr174 in the angiotensinogen gene is associated with a 10% increase in angiotensinogen levels and is a risk factor for elevated blood pressure in women but not in men.

Preclinical Validation of Electrochemotherapy as an Effective Treatment for Brain Tumors

Electrochemotherapy represents a strategy to enhance chemotherapeutic drug uptake by delivering electrical pulses which exceed the dielectric strength of the cell membrane, causing transient formation of structures that enhance permeabilization. Here we show that brain tumors in a rat model can be eliminated by electrochemotherapy with a novel electrode device developed for use in the brain. By using this method, the cytotoxicity of bleomycin can be augmented more than 300-fold because of increased permeabilization and more direct passage of drug to the cytosol, enabling highly efficient local tumor treatment. Bleomycin was injected intracranially into male rats inoculated with rat glia-derived tumor cells 2 weeks before the application of the electrical field (32 pulses, 100 V, 0.1 ms, and 1 Hz). In this model, where presence of tumor was confirmed by magnetic resonance imaging (MRI) before treatment, we found that 9 of 13 rats (69%) receiving electrochemotherapy displayed a complete elimination of tumor, in contrast to control rats treated with bleomycin only, pulses only, or untreated where tumor progression occurred in each case. Necrosis induced by electrochemotherapy was restricted to the treated area, which MRI and histology showed to contain a fluid-filled cavity. In a long-range survival study, treatment side effects seemed to be minimal, with normal rat behavior observed after electrochemotherapy. Our findings suggest that electrochemotherapy may offer a safe and effective new tool to treat primary brain tumors and brain metastases.

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

Angiotensinogen and ACE gene polymorphisms and risk of atrial fibrillation in the general population.

Objectives The renin–angiotensin system may play a role in the pathogenesis of atrial fibrillation, and renin–angiotensin system blockers reduce the risk of atrial fibrillation. We hypothesized that polymorphisms in the angiotensinogen and angiotensin-converting enzyme (ACE) genes encoding proteins in this system predict risk of atrial fibrillation. Methods and results We genotyped 9235 individuals from the Danish general population, The Copenhagen City Heart Study, for the a-20c, g-6a, T174M, and M235T polymorphisms in the angiotensinogen gene and the insertion/deletion (I/D) polymorphism in the ACE gene; rare allele frequencies were 0.16, 0.40, 0.12, 0.41, and 0.49, respectively. Participants had sinus rhythm at inclusion. During 26 years of follow-up, 968 individuals developed atrial fibrillation. Multifactorially adjusted hazard ratios for atrial fibrillation for a-20c ac and cc versus aa genotype were 1.1(95% confidence interval: 1.0–1.3; P=0.05) and 1.5(1.1–2.1; P=0.01). Compared with double noncarriers (angiotensinogen −20aa and ACE II), double heterozygotes (ac-I/D genotype), and double homozygotes (cc-DD) had hazard ratios for atrial fibrillation of 1.2(0.9–1.6; P=0.06) and 2.4(1.4–4.1; P=0.001). a-20c cc homozygotes above 70 years of age who were overweight, severely hypertensive, and had heart failure, had an absolute 10-year risk of atrial fibrillation of 61%. Conclusion Angiotensinogen a-20c genotype alone and in combination with ACE I/D genotype predicts an increased risk of atrial fibrillation. Therefore, genetic variation in the renin–angiotensin system may influence effect of renin–angiotensin system blockers on atrial fibrillation.

Diffusion-Weighted MRI for Verification of Electroporation-Based Treatments

Clinical electroporation (EP) is a rapidly advancing treatment modality that uses electric pulses to introduce drugs or genes into, e.g., cancer cells. The indication of successful EP is an instant plasma membrane permeabilization in the treated tissue. A noninvasive means of monitoring such a tissue reaction represents a great clinical benefit since, in case of target miss, retreatment can be performed immediately. We propose diffusion-weighted magnetic resonance imaging (DW-MRI) as a method to monitor EP tissue, using the concept of the apparent diffusion coefficient (ADC). We hypothesize that the plasma membrane permeabilization induced by EP changes the ADC, suggesting that DW-MRI constitutes a noninvasive and quick means of EP verification. In this study we performed in vivo EP in rat brains, followed by DW-MRI using a clinical MRI scanner. We found a pulse amplitude–dependent increase in the ADC following EP, indicating that (1) DW-MRI is sensitive to the EP-induced changes and (2) the observed changes in ADC are indeed due to the applied electric field.

Efficiency of Cellular Delivery of Antisense Peptide Nucleic Acid by Electroporation Depends on Charge and Electroporation Geometry

Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, particularly relating to oligonucleotides and their analogs for genetic therapy. Using a sensitive and quantitative HeLa cell luciferase RNA interference mRNA splice correction assay with a functional luciferase readout, we demonstrate that parameters such as peptide nucleic acid (PNA) charge and the method of electroporation have dramatic influence on the efficiency of productive delivery. In a suspended cell electroporation system (cuvettes), a positively charged PNA (+8) was most efficiently transferred, whereas charge neutral PNA was more effective in a microtiter plate electrotransfer system for monolayer cells. Surprisingly, a negatively charged (-23) PNA did not show appreciable activity in either system. Findings from the functional assay were corroborated by pulse parameter variations, polymerase chain reaction, and confocal microscopy. In conclusion, we have found that the charge of PNA and electroporation system combination greatly influences the transfer efficiency, thereby illustrating the complexity of the electroporation mechanism.