Effect of ACE insertion/deletion and 12 other polymorphisms on clinical outcomes and response to treatment in the LIFE study.

OBJECTIVE: This pharmacogenetics substudy from the Losartan Intervention for Endpoint reduction in Hypertension study in patients with hypertension and left ventricular hypertrophy (LVH) treated with the angiotensin receptor blocker losartan versus the beta-blocker atenolol for 4.8 years tested whether the insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene and 12 other previously well-characterized polymorphisms of hypertension susceptibility genes affected blood pressure reduction, heart rate reduction, cardiovascular events, and/or response to treatment. These polymorphisms were chosen because they could affect blood pressure control or the pharmacological action of losartan or atenolol. METHODS: We genotyped 3503 patients, 1774 on losartan and 1729 on atenolol. RESULTS: ACE and the 12 other genotypes did not affect the reduction in systolic blood pressure, diastolic blood pressure, pulse pressure, mean arterial pressure, or heart rate, or treatment differences between losartan and atenolol on these endpoints, as assessed by general linear models. Also, ACE and the 12 other genotypes did not affect risk of the primary composite endpoint or its components stroke, myocardial infarction, and cardiovascular death, or treatment differences between losartan and atenolol on these endpoints, as assessed by Cox proportional hazards models including baseline Framingham risk score and LVH. CONCLUSION: ACE insertion/deletion and 12 other polymorphisms of hypertension susceptibility genes did not affect blood pressure reduction, heart rate reduction, or cardiovascular events in patients with hypertension and LVH, or treatment differences between losartan and atenolol on these endpoints. These results suggest that the observed effects of losartan versus atenolol in the Losartan Intervention for Endpoint reduction in hypertension study do not depend on ACE and 12 other polymorphisms of hypertension susceptibility genes.

A twin study of nitric oxide levels measured by serum nitrite/nitrate.

Nitrite and nitrate are the stable end products of the L-arginine-NO pathway, and the sum of nitrite and nitrate (NOx) is a common way to measure nitric oxide (NO) production or secretion. To uncover any genetic influence on NO, we measured NOx in serum samples from monozygotic twin pairs after an overnight fast. Heritability was estimated as intraclass correlation coefficient. We arrived at a heritability estimate of .32 (95% confidence interval 0.17-0.45) for NOx. The numerical heritability estimate was higher for females than for males (0.38 vs. 0.21), and higher for nonsmokers than for smokers (0.36 vs. 0.22). The heritability estimate of NOx was lower than the heritability estimate for other cardiovascular risk factors. This study suggests a low degree of heredity for NOx levels.

Activity of peroxisomal enzymes, and levels of polyamines in LPA-transgenic mice on two different diets.

BACKGROUND: In man, elevated levels of plasma plipoprotein (a) (Lp(a)) is a cardiovascular risk factor, and oxidized phospholipids are believed to play a role as modulators of inflammatory processes such as atherosclerosis. Polyamines are potent antioxidants and anti-inflammatory agents. It was therefore of interest to examine polyamines and their metabolism in LPA transgenic mice. Concentration of the polyamines putrescine, spermidine and spermine as well as the activity of peroxisomal polyamine oxidase and two other peroxisomal enzymes, acyl-CoA oxidase and catalase were measured. The mice were fed either a standard diet or a diet high in fat and cholesterol (HFHC). Some of the mice in each feeding group were in addition given aminoguanidine (AG), a specific inhibitor of diamine oxidase, which catalyses degradation of putrescine, and also inhibits non-enzymatic glycosylation of protein which is implicated in the aetiology of atherosclerosis in diabetic patients. Non-transgenic mice were used as controls. RESULTS: Intestinal peroxisomal polyamine oxidase activity was significantly higher in LPA transgenic mice than in the non-transgenic mice, while intestinal peroxisomal catalase activity was significantly lower. Hepatic beta-oxidation increased in Lp(a) transgenic mice fed the HFHC diet, but not in those on standard diet. Hepatic spermidine concentration was increased in all mice fed the HFHC diet compared to those fed a standard diet, while spermine concentration was decreased. With exception of the group fed only standard diet, transgenic mice showed a lower degree of hepatic steatosis than non-transgenic mice. AG had no significant effect on hepatic steatosis. CONCLUSION: The present results indicate a connection between peroxisomal enzyme activity and the presence of the human LPA gene in the murine genome. The effect may be a result of changes in oxidative processes in lipid metabolism rather than resulting from a direct effect of the LPA construct on the peroximal gene expression.

Reduced LPA expression after peroxisome proliferator-activated receptor alpha (PPARalpha) activation in LPA-YAC transgenic mice.

BACKGROUND:: Apolipoprotein(a) (apo(a)), which is part of the atherogenic lipoprotein Lp(a), shares structural homology with plasminogen (plg). Genes coding for plasminogen (PLG) and apo(a) (LPA) are linked and situated 40kb apart in the telomeric region of the long arm of chromosome 6. LPA is naturally expressed only in primates and hedgehogs. Thus, access to knowledge regarding the mechanism by which LPA expression is regulated is limited due to shortage of appropriate animal models. However, mice transgenic for the human LPA gene have been produced. Lp(a) levels in man are genetically determined and not altered significantly by dietary changes. In contrast, mice transgenic for LPA-yeast artificial chromosome (LPA-YAC) have markedly reduced apo(a) levels after maintenance on a high-fat diet. LPA-YAC carries the 40kb LPA-PLG intergenic region, which includes a putative binding site for peroxisome proliferator-activated receptor alpha (PPARalpha). Therefore, we examined if fibrates, which exert their effect via PPARalpha, could alter LPA expression in transgenic mice. METHODS:: Two LPA transgenic mouse lines with or without the LPA-PLG intergenic region we fed either PPARalpha agonist fenofibrate (FF) or 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid (WY 14643) containing diets for 3 weeks. For the study of serum apo(a) levels, blood were sampled prior the experiment and when the animals were sacrificed. For the study of gene expression pattern pieces of livers were collected and submerged in RNAlater buffer and stored at -70 degrees C until analysis by quantitative PCR. RESULTS AND CONCLUSIONS:: The results showed that fibrates reduce LPA expression in LPA-YAC transgenic mice, but have no impact on hepatic apo(a) mRNA or serum apo(a) protein levels in LPA-cDNA transgenic mice, which lack the LPA-PLG intergenic region. This suggests that the effect of fibrates on LPA expression is mediated upstream of the LPA gene. However, on the basis of current data it is not possible to conclude that PPARalpha is the primary factor that represses LPA expression in LPA-YAC transgenic mice. Negative correlation between FXR and apo(a) mRNA levels, in addition to putative FXR DNA binding sequence in LPA-PLG intergenic region, suggest that it is equally likely that reduced expression of LPA could be a secondary consequence of PPARalpha activation on other genes, such as FXR.

Atherogenesis and vascular calcification in mice expressing the human LPA gene.

Background: Lp(a) lipoprotein (Lp(a)) contains polymorphic glycoprotein, apolipoprotein(a) (apo(a)) and low density lipoprotein (LDL). The extensive homology between apo(a) and plasminogen is believed to contribute to the pathogenicity of apo(a), but the precise mechanisms by which Lp(a) participates in atherogenesis is still unknown. We used LPA-yeast artificial chromosome (LPA-YAC) transgenic mice with or without the human APOB (hAPOB) gene to study pathogenicity of apo(a)/Lp(a) and illucidate its role in regulation of serum lipid levels. Methods: Middle-aged (1-year-old) mice were fed a control (AIN-76), a high-cholesterol (HC) or a high-cholesterol/high-fat (HCHF) diet for 7 weeks. For the study of serum total apo(a) and lipid levels, mice were sampled prior to the experiment, at 2 weeks and at 7 weeks when the animals were sacrificed. Hearts with ascending aorta were fixed in formalin, embedded in gelatine and prepared for sections on a cryostat. Livers were washed in ice cold saline and submerged in RNAlater trade mark buffer and stored at -70 degrees C until mRNA analysis. Results: Wild type mice fed the control diet did not develop aortic lesions. Presence of the LPA gene was sufficient to induce development of aortic lesions, but neither coexpression of the hAPOB gene nor feeding the HC diet or the HCHF diet augmented the development of aortic lesions in LPA-YAC transgenic mice. On the control diet transgenic females had larger aortic lesion size than transgenic males. Furthermore, aortic lesions in transgenic females were associated with calcification more often than in transgenic males. Serum total cholesterol levels were higher both in wild type and LPA-YAC transgenic males than in females mainly because of higher serum high-density lipoprotein cholesterol levels. HC and HCHF feeding had more pronounced effect on total cholesterol levels in LPA-YAC/hAPOB transgenic mice than in either wild type or LPA-YAC transgenic mice, due to increased low density lipoprotein cholesterol levels. Furthermore, these diets reduced serum total apo(a) levels in both transgenic mouse lines. Conclusion: Expression of the human LPA gene in mice is sufficient to trigger development of aortic lesions. Similar frequency of calcified lesions in LPA-YAC transgenic mice with or without hAPOB gene may suggest that apo(a) is the part of the Lp(a) molecule that causes aortic calcification. The basis for reduced serum total apo(a) level in response to cholesterol feeding is not clear, but interplay between LPA and factors involved in cholesterol or bile acid homeostasis is worth of future studies.

Human apoB contributes to increased serum total apo(a) level in LPA transgenic mice.

BACKGROUND: The Lp(a) lipoprotein (Lp(a)) consists of the polymorphic glycoprotein apolipoprotein(a) (apo(a)), which is attached by a disulfide bond to apolipoprotein B (apoB). Apo(a), which has high homology with plasminogen, is present only in primates and hedgehogs. However, transgenic mice and rabbits with high serum apo(a) levels exist. Liver is the main site for apo(a) synthesis, but the site of removal is uncertain. To examine differences between transgenic mice expressing the LPA gene and mice capable of forming Lp(a) particles, LPA-YAC transgenic mice and hAPOB transgenic mice were crossed and their offspring examined. RESULTS: Comparison of LPA-YAC with LPA-YAC/hAPOB transgenic mice showed that LPA-YAC/hAPOB transgenic mice have higher serum total apo(a) and total cholesterol level than mice lacking the hAPOB gene. However, hepatic apo(a) mRNA level was higher in LPA-YAC transgenic mice than in LPA-YAC/hAPOB transgenic mice. Feeding of a high-cholesterol/high-fat diet to male LPA-YAC transgenic mice with or without the hAPOB gene resulted in reduced serum total apo(a) and hepatic apo(a) mRNA level. CONCLUSION: In conclusion, the higher serum total apo(a) level in LPA-YAC/hAPOB transgenic mice than in LPA-YAC transgenic mice is not caused by increased apo(a) synthesis. Lower hepatic apo(a) mRNA level in LPA-YAC/hAPOB than in LPA-YAC transgenic mice may suggest that the increase in total apo(a) level is a result of apo(a) accumulation in serum. Furthermore, observed higher serum total cholesterol level in LPA-YAC/hAPOB transgenic mice than either in wild type or LPA-YAC transgenic mice may further suggest that human APOB transgenicity is a factor that contributes to increased serum total apo(a) and cholesterol levels. Our results on reduced serum total apo(a) and hepatic apo(a) mRNA levels in HCHF fed male LPA-YAC transgenic mice confirm earlier findings in females, and show that there are no sex difference in mechanisms for lowering apo(a) level in response to HCHF feeding.

Variations in transfection efficiency of VEGF165 and VEGF121-cDNA: its effects on proliferation and migration of human endothelial cells.

Little is known about the expression pattern of vascular endothelial growth factor (VEGF) among smooth muscle cells of different arterial regions. Therefore, we have conducted studies aimed at increasing expression of VEGF in cultured human smooth muscle cells (SMCs) from different sites: aorta, umbilical artery, and coronary artery. Two plasmids harboring human VEGF121 and VEGF165 isoforms, respectively, were constructed and lipotransfected into vascular SMCs, using the Fu-GENE 6. Extensive optimization of transfection conditions were performed prior to this. Different basal levels of VEGF were observed between cell types: from 0.51-0.95 pg/mL/micrograms protein in umbilical artery, through 2.32-2.39 pg/mL/micrograms protein in coronary artery, to 5.45-7.52 pg/mL/micrograms protein in aortic SMCs. Significant differences in responses to transfection were also observed: The increase in VEGF production was most pronounced in umbilical artery SMCs (e.g., with 4 micrograms VEGF121-cDNA /in the wells)- an approximate 600-fold as opposed to an 18-fold increase in aortic SMCs and a 29-fold increase in coronary artery SMCs. In addition, we observed significant increases in proliferation rate of aortic and coronary endothelial cells (ECs), after incubation with conditioned medium from VEGF-transfected SMCs. Observed changes differed in relation to cell origin and isoform.

The role of the children of twins design in elucidating causal relations between parent characteristics and child outcomes.

BACKGROUND: Determination of causal connections between parental measures and child outcomes using typical samples is limited by the inability to account for all confounds, both environmental and genetic. This paper discusses the strength of the Children of Twins (COT) design to highlight the role of specific environments. METHODS: A new analytical model is presented which helps differentiate and quantify the environmental and genetic processes underlying associations between family-level risk factors and child adjustment. In order to illustrate the COT design, the relation between smoking during pregnancy and child birth weight (BW) is examined in a sample of female twins and their children from Norway and the United States. RESULTS: The results illustrate that smoking during pregnancy is influenced by genetic factors. However, the Children of Twins model supports the claim that smoking during pregnancy has a direct environmental influence on BW and that genetic and shared environmental confounds cannot account for the association. CONCLUSIONS: An assessment of the strengths and limitations of the Children of Twins design and a comparison with other research strategies suggest that the design plays a unique role in the study of developmental psychology and psychopathology. Finally, the authors describe how methodological advances and future applications of the design will provide additional insight into the causal processes underlying children's adjustment to environmental stimuli.

A twin study of C-Reactive Protein compared to other risk factors for coronary heart disease.

Coronary heart disease (CHD) tends to cluster in families, and several established risk factors for the disease are to some extent inherited. Inflammation plays a key role in the development of atherosclerosis and CHD. A low-grade inflammation may be detected by highly sensitive C-Reactive Protein (CRP) determination, which is strongly associated to CHD. In order to uncover any role of genetics in low-grade inflammation, we measured CRP in healthy monozygotic twins. The within-pair correlation coefficient of CRP was 0.40, suggesting an important genetic contribution to the control of CRP level. CRP correlated significantly to other CHD risk factors like body mass index (BMI), systolic blood pressure, diastolic blood pressure, plasma fibrinogen, serum high-density lipoprotein cholesterol, plasma homocysteine, and serum triglycerides. Of these variables, BMI was most significantly associated to CRP in a linear multiple regression analysis. We conclude that CRP level (reflecting a low-grade inflammation) exhibits a moderate, but significant degree of heritability. The association between CRP and BMI, which has a larger degree of heritability, could partly explain the heritability of serum CRP level.

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system.

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).

Absence of enhanced systemic inflammatory response at 18 weeks of gestation in women with subsequent pre-eclampsia.

OBJECTIVE: To compare indicators of systemic inflammatory response in the second trimester in women who developed pre-eclampsia with normal pregnancies. DESIGN: Prospective nested case control study derived from a cohort of 2190 pregnant women. Blood samples were obtained at 18 weeks of gestation. The following inflammatory parameters were measured: tumour necrosis factor-alpha (TNF-alpha), plasminogen activator inhibitor-1 (PAI-1), interleukin-1beta (IL-1beta), IL-6, IL-10, microCRP and tissue factor (TF). SETTING: Institute of Medical Genetics, University of Oslo, and Department of Medical Genetics, Ullevål University Hospital and Departments of Obstetrics and Gynecology, Aker University Hospital, Oslo, Norway. SAMPLE: The cases were 71 women who subsequently developed pre-eclampsia. The controls were 71 healthy, pregnant women matched for age, parity and first trimester body mass index (BMI). METHODS: Venous blood was drawn from fasting subjects into 5 mL test tubes containing EDTA. Samples were analysed for inflammatory parameters: IL-1-beta, IL-6, IL-10, TNF-alpha, PAI-1, TF (ELISA-technique) and CRP (latex-enhanced immunonephelometric assay), strictly according to the manufacturer's recommendation. MAIN OUTCOME MEASURES: The matched case and control subjects were compared by the paired two-tailed Wilcoxon signed rank test. All P values were two-tailed and P < 0.05 was deemed statistically significant. RESULTS: We found no differences in plasma concentrations of PAI-1, IL-1beta, IL-6,IL-10, microCRP, TNF-alpha or TF at 18 weeks of gestation between women who subsequently developed pre-eclampsia and matched control women. CONCLUSION: In contrast to findings from women with overt pre-eclampsia, the present study indicates that there are no indications of intensified systemic inflammatory response at 18 weeks of gestation in women who later develop pre-eclampsia.

Spontaneous atherosclerosis in the proximal aorta of LPA transgenic mice on a normal diet.

Serum levels of Lp(a) lipoprotein are under genetic control and a high level is a risk factor for atherosclerotic disease. We have examined the aorta of LPA transgenic mice and their non-transgenic litter mates who had all been given a regular, not lipid fortified diet. When sacrificed, the animals had an average age of 66 weeks. Lipid lesions were observed in the aorta of 13 out of 18 LPA transgenic mice and in five out of 21 non-transgenic animals. The difference is statistically significant. We conclude that LPA transgenic mice develop lipid lesions in aorta more frequently than non-transgenic animals, even on a diet with a low fat content. LPA transgenic mice on a normal diet could be a useful animal model for the study of spontaneous human atherosclerosis, its treatment and prevention.

C-reactive protein predicts death in patients with previous premature myocardial infarction--a 10 year follow-up study.

Atherosclerosis is an inflammatory disease. C-reactive protein (CRP), a marker of inflammation, is associated with coronary heart disease (CHD). We measured CRP in a cohort of 247 patients (193 males and 54 females) who had had their first myocardial infarction (MI) at age < or = 55 (males) or < or = 60 (females). The cut-off values of the 25th, 50th and 75th centiles of CRP were 1.20, 2.37 and 4.20 mg/l. After 10 years, a total of 44 patients (17.8%) had died, 36 (81.8%) of cardiac causes. Unadjusted and adjusted (i.e. for age, ejection fraction (EF), serum total cholesterol (TC), fibrinogen, smoking and hypertension) relative risks (RRs) for total and cardiac mortality were generated. CRP was a strong predictor of death of all causes due to its strength as predictor of cardiac death. The RR of cardiac death was doubled with increasing CRP quartiles, and patients in the top quartile had six times as high risk of cardiac death as patients in the lowest quartile. The RRs were moderately attenuated after adjustment, but still significant. We conclude that CRP is a strong predictor of mortality in patients with premature MI. Thus, inflammation appears to be a critical prognostic factor in patients with previous premature MI.