Else M. Bladbjerg

Inflammation, thrombosis and atherosclerosis: results of the Glostrup study.

Summary.  Inflammation and thrombosis are important mechanisms in cardiovascular disease, as illustrated by the consistent association between inflammatory and hemostatic variables and the risk of cardiovascular events in epidemiological studies. However, the relationship between plasma concentrations of inflammatory and hemostatic markers and the severity of atherosclerosis is not yet well studied. We have evaluated 325 men and 370 women of 60 years, participating in the Danish Glostrup study. We diagnosed atherosclerosis by ultrasonographic measurement of intima‐media thickness (IMT) of the right carotid artery and the assessment of plaque occurrence. Plasma samples were analyzed for the concentration of C‐reactive protein (CRP), fibrinogen, d‐dimer, plasminogen activator inhibitor type‐1 (PAI‐1) antigen and activity, tissue‐type plasminogen activator (t‐PA) antigen and activity, factor VII (FVII) antigen, FVII coagulant activity (FVII:C) and activated FVII (FVIIa). DNA variations were determined for fibrinogen, PAI‐1, t‐PA, FVII, factor XIII and methylene tetrahydrofolate reductase (MTHFR). Subjects with high IMT (upper 10% of distribution, n = 63) had higher CRP levels [2.2 mg L−1 (SE 0.3)] than subjects with IMT in the lowest tertile (n = 217) [1.7 mg L−1 (SE 0.1), P = 0.04], whereas there was no association between the hemostatic variables and IMT. There was an association between fibrinogen and d‐dimer concentrations and number of plaques (P < 0.01), whereas there were no associations between CRP and the other hemostatic variables and the number of plaques. Genetic variation in the t‐PA and MTHFR gene was associated with IMT. In conclusion, in the Glostrup population study, thrombosis and inflammation are associated with the severity of atherosclerosis, as reflected by IMT and plaque occurrence.

Variations of cardiovascular disease associated genes exhibit sex-dependent influence on human longevity.

This article investigates the relationship between the polymorphic variations in genes associated with cardiovascular disease and longevity in the Danish population. A new procedure that combines both demographic and the individual genetic information in determining the relative risks of the observed genetic variations is applied. The sex-dependent influences can be found by introducing sex-specific population survival and incorporating the risk of gene-sex interaction. Three genetic polymorphisms, angiotensinogen M/T235, blood coagulation factor VII (FVII) R/Q353 and FVII-323ins10, manifest significant influences on survival in males, with reduced hazards of death for carriers of the angiotensinogen M235 allele, the F VII Q353 allele, and the FVII-323P10 allele. The results show that some of these genotypes associated with lower risk of CVD could also reduce the carriers death rate and contribute to longevity. However, the presence of sex-dependent effects and the fact that major CVD-associated genes failed to impose detrimental influence on longevity lead us to concur that the aging process is highly complicated.

Positive impact of hormone replacement therapy on the fibrinolytic system: a long‐term randomized controlled study in healthy postmenopausal women

Summary.  Background: The mechanisms by which postmenopausal hormone replacement therapy (HRT) may influence risk of cardiovascular disease are still unclear. Impaired fibrinolytic function is associated with an enhanced risk of cardiovascular disease and therefore the effect of HRT on fibrinolysis may be of importance. Objectives: To investigate the prolonged effect of HRT on the fibrinolytic system and to determine whether two common polymorphisms in the plasminogen activator inhibitor‐1 (PAI‐1) and tissue‐type plasminogen activator (t‐PA) genes modulate this effect. Methods: Healthy postmenopausal women (n = 248) were randomized to HRT (n = 122) or no substitution (n = 126) 5 years prior to investigation. Results: Significantly higher values of t‐PA activity and lower values of PAI‐1 activity and PAI‐1 antigen were found in the HRT group compared with the control group. This effect was independent of smoking and without influence from the two common polymorphisms PAI‐1 −675(4G/5G) and t‐PA intron8ins311. Furthermore, no difference between opposed estrogen (with norethisterone acetate as the gestagen component) and unopposed estrogen therapy was found. Both an intention‐to‐treat and a per‐protocol analysis were performed and similar results were obtained. Conclusions: Long‐term treatment with HRT in healthy postmenopausal women was found to be associated with a beneficial fibrinolytic profile. This effect was found independent of smoking status, opposed and unopposed estrogen therapy had equal effect, and no influence of the two common polymorphisms PAI‐1–675(4G/5G) and t‐PA intron8ins311 was found. This effect of HRT on fibrinolytic capacity may be one of the beneficial effects of HRT in relation to cardiovascular diseases.

The Göttingen minipig as a model for postprandial hyperlipidaemia in man: experimental observations

Postprandial hyperlipidaemia is believed to be atherogenic. This study aimed to establish a minipig model to investigate determinants of postprandial lipid metabolism. In a randomized cross-over design seven minipigs were subjected to six different feeding regimens: intragastric fat loads of 1, 2, and 4 g fat (Intralipid®, 20%) kg-1 in two fractions 1.5 h apart (1/3 first, 2/3 second), 2 g fat (Intralipid®) kg-1 in one fraction, and 2 g olive oil kg-1 in two fractions, all after pre-feeding with standard diet, and finally 2 g fat (Intralipid®) kg-1 in two fractions without pre-feeding. Blood was sampled before and hourly for 7 h after gavaging, and plasma triglycerides were measured. Triglycerides increased significantly in all the feeding regimens (P < 0.001), except when olive oil was used as the fat source. A borderline significant dose-response effect of the Intralipid® dose on the triglyceride response was observed. We found no significant differences in triglyceride response whether 2 g fat (Intralipid®) kg-1 was given in one or two fractions, with or without pre-feeding. We conclude that postprandial hyperlipidaemia in minipigs can be induced by gavaging an emulgated lipid solution (1-4 g fat/kg, Intralipid®), while olive oil is not applicable. There is no need to administer the fat fractionated or to withhold food prior to administration.

Effect of pre-analytical handling on haematological variables in minipigs

Pre-analytical handling may be an important determinant of haematological variables, if analysis is delayed. We investigated the effect of anticoagulants, i.e. tripotassium ethylenediamine-tetraacetic acid (EDTA) and citric acid, theophylline, adenosine, dipyridamole (CTAD), storage time (0.5, 1.5, 3.5, 5.5, 7.5, 25.5 and 27.5 h after blood sampling), and storage temperature (5°C and 20°C) on the variation in haemoglobin (HGB), red blood cell count (RBC), haematocrit (HCT ), white blood cell count (WBC), and platelet count (PLT) in minipigs. Medians of HGB, RBC, HCT, WBC and PLT were significantly higher in EDTA tubes than in CTAD tubes due to the dilution effect of the anticoagulant. We found a minor significant increase in HCT after 25.5 h in blood stored at 20°C, and at the same time a minor significant increase in WBC in EDTA tubes stored at 20°C. We found a significant decrease in PLT in blood stored at 5°C, especially in EDTA tubes. Minor variations were also observed in HGB and RBC. Our results indicate that PLT should only be measured in tubes placed at room temperature. If HCT or WBC analyses are to be performed on the day after blood sampling, the samples must be stored in a refrigerator until analysis. Our studies underline that time delay before analysis of haematological variables can cause increased variation, and should therefore be limited as far as possible in order to reduce the number of animals needed to make reliable conclusions.

Associations between inflammatory markers, candidate polymorphisms and physical performance in older Danish twins.

Inflammation may play an essential role in the decline of physical performance. In this study we investigated the associations between inflammatory markers, candidate polymorphisms and physical performance in elderly people. Plasma levels of TNF-α, IL-6, CRP, fibrinogen, sICAM-1 and candidate polymorphisms were measured in 600 twin individuals aged 73 years and older participating in the Longitudinal Study of Aging Danish Twins. Physical performance was assessed using a self-reported measure. The inclusion of twins allowed both traditional and within-twin-pair analysis which permitted control for shared environment and genetic factors. Higher levels of inflammatory markers were generally associated with a lower level of physical performance. The TNFα-238G/A polymorphism was significantly associated with physical performance in men, with A allele carriers having significantly better performance than GG homozygotes. However, this gene variation seems to have only a minor role in explaining the associations between the levels of inflammatory markers and physical performance. When using twin pair analysis to test whether genetic factors in general account for this association, results showed that the association between the level of fibrinogen and physical performance could be caused by genetic factors. Also the association between the level of TNF-α and physical performance in males could be caused by genetic factors. However, other gene variations than the candidate gene polymorphisms studied here seem to explain the major part of the genetic proportion of this association.

Plasma factor VII levels are determined by polymorphisms in the factor VII gene

Summary Elevated factor VII coagulant activity (FVII:C) is associated with increased risk of cardiovascular disease. FVII:C and FVII protein concentration (FVII:Ag) are influenced by environmental factors such as diet and by genetic factors. We examined the effects of FVII polymorphisms on 1) fasting and non-fasting values of FVII:C, FVII:Ag and activated FVII (FVIIa) and 2) the relation between total triglycerides and FVII measures. Fortyfour males (age 30–60 y) participated in the study, and had blood drawn in the fasting state. A subgroup (n=19) was also studied in the non-fasting state after consumption of two high-fat meals. FVII:C, FVII:Ag, FVIIa and total triglycerides were measured as well as genotypes of the promoter, HVR4 and Msp1 polymorphisms of the FVII gene. There was a marked effect of the promoter and Msp1 polymorphism on fasting FVII:C, FVII:Ag and FVIIa, and of the Msp1 polymorphism on the fasting FVIIa/FVIIag ratio, with about 15–40% lower levels in heterozygotes. The Msp1 polymorphism also had an effect on non-fasting FVII:C. A significant correlation between fasting total triglycerides and FVII measures was only seen in individuals homozygous for the common Msp1 and promoter alleles.