Izabella Lipinska

Obesity and Systemic Oxidative Stress. Clinical Correlates of Oxidative Stress in The Framingham Study

Objective—To determine the clinical conditions associated with systemic oxidative stress in a community-based cohort. Information regarding cardiovascular risk factors associated with systemic oxidative stress has largely been derived from highly selected samples with advanced stages of vascular disease. Thus, it has been difficult to evaluate the relative contribution of each cardiovascular risk factor to systemic oxidative stress and to determine whether such risk factors act independently and are applicable to the general population. Methods and Results—We examined 2828 subjects from the Framingham Heart Study and measured urinary creatinine–indexed levels of 8-epi-PGF2&agr; as a marker of systemic oxidative stress. Age- and sex-adjusted multivariable regression models were used to assess clinical correlates of oxidative stress. In age- and sex-adjusted models, increased urinary creatinine–indexed 8-epi-PGF2&agr; levels were positively associated with female sex, hypertension treatment, smoking, diabetes, blood glucose, body mass index, and a history of cardiovascular disease. In contrast, age and total cholesterol were negatively correlated with urinary creatinine–indexed 8-epi-PGF2&agr; levels. After adjustment for several covariates, decreasing age and total/HDL cholesterol ratio, sex, smoking, body mass index, blood glucose, and cardiovascular disease remained associated with urinary 8-epi-PGF2&agr; levels. Conclusions—Smoking, diabetes, and body mass index were highly associated with systemic oxidative stress as determined by creatinine-indexed urinary 8-epi-PGF2&agr; levels. The effect of body mass index was minimally affected by blood glucose, and diabetes and may suggest an important role of oxidative stress in the deleterious impact of obesity on cardiovascular disease.

Visceral and Subcutaneous Adipose Tissue Volumes Are Cross-Sectionally Related to Markers of Inflammation and Oxidative Stress The Framingham Heart Study

Background— Excess adiposity is associated with greater systemic inflammation. Whether visceral adiposity is more proinflammatory than subcutaneous abdominal adiposity is unclear. Methods and Results— We examined the relations of abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT), assessed by multidetector computerized tomography, to circulating inflammatory and oxidative stress biomarkers in 1250 Framingham Heart Study participants (52% women; age 60±9 years). Biomarkers were examined in relation to increments of SAT and VAT after adjustment for age, sex, smoking, physical activity, menopause, hormone replacement therapy, alcohol, and aspirin use; additional models included body mass index and waist circumference. SAT and VAT were positively and similarly (with respect to strength of association) related to C-reactive protein, fibrinogen, intercellular adhesion molecule-1, interleukin-6, P-selectin, and tumor necrosis factor receptor-2 (multivariable model R2 0.06 to 0.28 [SAT] and 0.07 to 0.29 [VAT]). However, compared with SAT, VAT was more highly associated with urinary isoprostanes and monocyte chemoattractant protein-1 (SAT versus VAT comparison: isoprostanes, R2 0.07 versus 0.10, P=0.002; monocyte chemoattractant protein-1, R2 0.07 versus 0.08, P=0.04). When body mass index and waist circumference were added to the models, VAT remained significantly associated with only C-reactive protein (P=0.0003 for women; P=0.006 for men), interleukin-6 (P=0.01), isoprostanes (P=0.0002), and monocyte chemoattractant protein-1 (P=0.008); SAT only remained associated with fibrinogen (P=0.01). Conclusions— The present cross-sectional data support an association between both SAT and VAT with inflammation and oxidative stress. The data suggest that the contribution of visceral fat to inflammation may not be completely accounted for by clinical measures of obesity (body mass index and waist circumference).

Increased Platelet Aggregability Associated With Platelet GPIIIa PlA2 Polymorphism : The Framingham Offspring Study

The platelet glycoprotein IIb/IIIa (GP IIb/IIIa) plays a pivotal role in platelet aggregation. Recent data suggest that the Pl A2 polymorphism of GPIIIa may be associated with an increased risk for cardiovascular disease. However, it is unknown if there is any association between this polymorphism and platelet reactivity. We determined GPIIIa genotype and platelet reactivity phenotype data in 1422 subjects from the Framingham Offspring Study. Genotyping was performed using PCR based restriction fragment length polymorphism analysis. Platelet aggregability was evaluated by the Born method. The threshold concentrations of epinephrine and adenosine diphosphate (ADP) were determined. Allele frequencies of Pl A1 and Pl A2 were 0.84 and 0.16, respectively. The presence of one or two Pl A2 alleles was associated with increased platelet aggregability as indicated by incremen- tally lower threshold concentrations for epinephrine and ADP. For epinephrine, the mean concentrations were 0.9 µmol/L (0.9–1.0) for homozygous Pl A1 , 0.7 µmol/L (0.7–0.9) for the heterozygous Pl A1 / Pl A2 and 0.6 µmol/L (0.4–1.0) for homozygous Pl A2 individuals, p = 0.009. The increase in aggregability induced by epinephrine remained highly significant (p = 0.007) after adjustment for covariates. For ADP-induced aggregation, the respective mean concentrations were 3.1 µmol/L (3.0–3.2), 3.0 µmol/L (2.9–3.2), and 2.8 µmol/L (2.4–3.3), p = 0.19 after adjustment for covariates. Our findings indicate that molecular variants of the gene encoding GPIIIa play a role in platelet reactivity in vitro. Our observations are compatible with and provide an explanation for the reported association of the Pl A2 allotype with increased risk for cardiovascular disease.

Increased Platelet Aggregability Associated With Platelet GPIIIa PlA2 Polymorphism

The platelet glycoprotein IIb/IIIa (GP IIb/IIIa) plays a pivotal role in platelet aggregation. Recent data suggest that the PlA2 polymorphism of GPIIIa may be associated with an increased risk for cardiovascular disease. However, it is unknown if there is any association between this polymorphism and platelet reactivity. We determined GP IIIa genotype and platelet reactivity phenotype data in 1422 subjects from the Framingham Offspring Study. Genotyping was performed using PCR-based restriction fragment length polymorphism analysis. Platelet aggregability was evaluated by the Born method. The threshold concentrations of epinephrine and ADP were determined. Allele frequencies of PlA1 and PlA2 were 0.84 and 0.16, respectively. The presence of 1 or 2 PlA2 alleles was associated with increased platelet aggregability as indicated by incrementally lower threshold concentrations for epinephrine and ADP. For epinephrine, the mean concentrations were 0.9 micromol/L (0.9 to 1.0) for homozygous PlA1, 0.7 mmol/L (0.7 to 0.9) for the heterozygous PlA1/PlA2, and 0.6 micromol/L (0.4 to 1.0) for homozygous PlA2 individuals, P=0.009. The increase in aggregability induced by epinephrine remained highly significant (P=0.007) after adjustment for covariates. For ADP-induced aggregation, the respective mean concentrations were 3.1 micromol/L (3.0 to 3.2), 3.0 micromol/L (2.9 to 3.2), and 2.8 micromol/L (2.4 to 3.3); P=0.19 after adjustment for covariates. Our findings indicate that molecular variants of the gene encoding GP IIIa play a role in platelet reactivity in vitro. Our observations are compatible with and provide an explanation for the reported association of the PlA2 allotype with increased risk for cardiovascular disease.

Brachial Artery Vasodilator Function and Systemic Inflammation in the Framingham Offspring Study

Background—In experimental studies, traditional risk factors and proinflammatory processes alter the regulatory functions of the vascular endothelium to promote atherosclerosis. These alterations include expression of leukocyte adhesion molecules and decreased bioavailability of endothelium-derived nitric oxide, an important regulator of vascular homeostasis and tone. The precise relations among risk factors, inflammation, and nitric oxide bioavailability remain uncertain. Methods and Results—To test the hypothesis that inflammation impairs endothelial function in humans, we measured brachial artery flow-mediated dilation, reactive hyperemia, and serum concentrations of C-reactive protein (CRP), interleukin-6 (IL-6), soluble intracellular adhesion molecule-1 (sICAM-1), and monocyte chemotactic protein-1 (MCP-1) in 2701 participants from the Framingham Study (mean age 61 years, 53% women). There were modest unadjusted inverse correlations between flow-mediated dilation and CRP, IL-6, and sICAM-1 (P<0.001 for all) that were rendered nonsignificant after accounting for traditional coronary risk factors. For reactive hyperemia, we observed inverse correlations with markers of inflammation in unadjusted models that were attenuated 57% to 74% after accounting for risk factors. However, partial correlations of CRP, IL-6, and sICAM-1 with reactive hyperemia remained significant. Conclusions—Our observations are consistent with the hypothesis that risk factors induce a state of inflammation that impairs vascular function. For flow-mediated dilation, we found no evidence that inflammation has additional effects beyond those attributable to traditional risk factors. The incremental contribution of CRP, IL-6, and sICAM-1 to reactive hyperemia above and beyond known risk factors suggests that systemic inflammation may contribute to impaired vasomotor function in forearm microvessels.

Inflammatory biomarkers are associated with total brain volume The Framingham Heart Study

Background: Systemic inflammation is associated with ischemia and Alzheimer disease (AD). We hypothesized that inflammatory biomarkers would be associated with neuroimaging markers of ischemia (i.e., white matter hyperintensities [WMH]) and AD (i.e., total brain volume [TCB]). Methods: MRI WMH and TCB were quantified on 1,926 Framingham Offspring participants free from clinical stroke, TIA, or dementia (mean age 60 ± 9 years; range 35 to 85 years; 54% women) who underwent measurement of a circulating inflammatory marker panel, including CD40 ligand, C-reactive protein, interleukin-6 (IL-6), soluble intracellular adhesion molecule-1, monocyte chemoattractant protein-1, myeloperoxidase, osteoprotegerin (OPG), P-selectin, tumor necrosis factor-alpha (TNFα), and tumor necrosis factor receptor II. To account for head size, both TCB (TCBV) and WMH (WMH/TCV) were divided by total cranial volume. We used multivariable linear regression to relate 10 log-transformed inflammatory biomarkers to brain MRI measures. Results: In multivariable models, inflammatory markers as a group were associated with TCBV (p < 0.0001) but not WMH/TCV (p = 0.28). In stepwise models adjusted for clinical covariates with backwards elimination of markers, IL-6 and OPG were inversely associated with TCBV; TNFα was inversely related to TCBV in a subset of 1,430 participants. Findings were similar in analyses excluding individuals with prevalent cardiovascular disease. The relations between TCBV and inflammatory markers were modified by both sex and age, and generally were more pronounced in men and in older individuals. Conclusions: Although our observational cross-sectional data cannot establish causality, they are consistent with the hypothesis that higher inflammatory markers are associated with greater atrophy than expected for age.

Association between obesity and a prothrombotic state: the Framingham Offspring Study

Although obesity is associated with increased cardiovascular risk, the mechanism has not been fully explained. Since thrombosis is a critical component of cardiovascular disease, we examined the relationship between obesity and hemostatic factors. We studied 3230 subjects (55% females, mean age 54 years) without a history of cardiovascular disease in cycle 5 of the Framingham Offspring Study. Obesity was assessed by body mass index and waist-to-hip ratio. Fasting blood samples were obtained for fibrinogen, plasminogen activator inhibitor (PAI-1) antigen, tissue plasminogen activator (tPA) antigen, factor VII antigen, von Willebrand factor (VWF), and plasma viscosity. Body mass index was directly associated with fibrinogen, factor VII, PAI-1 and tPA antigen in both men and women (p>0.001) and with VWF and viscosity in women. Similar associations were present between waist-to-hip ratio and the hemostatic factors. With minor exceptions for VWF and viscosity, all associations persisted after controlling for age, smoking, total and HDL cholesterol, triglycerides, glucose level, blood pressure, and use of antihypertensive medication. The association between increased body mass index and waist-to-hip ratio and prothrombotic factors and impaired fibrinolysis suggests that obesity is a risk factor whose effect is mediated in part by a prothrombotic state.

Association of Blood Pressure With Fibrinolytic Potential in the Framingham Offspring Population

BACKGROUND Hypertension is an established risk factor for acute coronary events. Because fibrinolytic and hemostatic factors are also associated with cardiovascular disease, we examined the relations of systolic and diastolic blood pressures (SBP and DBP) to levels of plasminogen activator inhibitor antigen, tissue plasminogen activator antigen, fibrinogen, factor VII, von Willebrand factor, fibrinogen, and plasma viscosity in subjects of the Framingham Offspring Study. METHODS AND RESULTS We studied 1193 men and 1459 women after the exclusion of subjects with known cardiovascular disease and those receiving anticoagulant or antihypertensive therapy. Linear regression models were used to evaluate SBP and DBP as predictors of fibrinolytic and hemostatic factor levels in separate sex models, with adjustment for age, body mass index, smoking, diabetes, total cholesterol, HDL, triglycerides, alcohol intake, and estrogen use (in women). In both sexes, levels of plasminogen activator inhibitor and tissue plasminogen activator antigen were positively related to SBP and DBP (P<0.001). Plasma viscosity was positively related to SBP (P=0.008) and DBP (P=0.001) in women only. There was no association between SBP or DBP and fibrinogen, factor VII, or von Willebrand factor in either sex. CONCLUSIONS These data suggest that impaired fibrinolysis may play an important role in the pathogenesis of cardiovascular disease in hypertensive patients.

Relations of Inflammatory Biomarkers and Common Genetic Variants With Arterial Stiffness and Wave Reflection

Inflammation causes vascular dysfunction and perpetuates proatherosclerotic processes. We hypothesized that a broad panel of inflammatory biomarkers and single nucleotide polymorphisms in inflammatory genes is associated with vascular stiffness. We assessed 12 circulating inflammatory biomarkers (C-reactive protein, fibrinogen, interleukin-6, intercellular adhesion molecule-1, lipoprotein-associated phospholipase-A2 [mass and activity], monocyte chemoattractant protein-1, myeloperoxidase, CD40 ligand, osteoprotegerin, P-selectin, and tumor necrosis factor receptor-II) in relation to tonometry variables (central pulse pressure, mean arterial pressure, forward pressure wave, reflected pressure wave, carotid-femoral pulse wave velocity, and augmentation index) measured in 2409 Framingham Heart Study participants (mean age: 60 years; 55% women; 13% ethnic/racial minorities). Single nucleotide polymorphisms (n=2195) in 240 inflammatory candidate genes were related to tonometry measures in 1036 white individuals. In multivariable analyses, biomarkers explained <1% of any tonometry measure variance. Applying backward elimination, markers related to tonometry (P<0.01) were as follows: tumor necrosis factor receptor-II (inversely) with mean arterial pressure; C-reactive protein (positively) and lipoprotein-associated phospholipase-A2 (inversely) with reflected pressure wave; and interleukin-6 and osteoprotegerin (positively) with carotid-femoral pulse wave velocity. In genetic association analyses, lowest P values (false discovery rate <0.50) were observed for rs10509561 (FAS), P=6.6×10−5 for central pulse pressure and rs11559271 (ITGB2), P=1.1×10−4 for mean arterial pressure. These data demonstrate that, in a community-based sample, circulating inflammatory markers tumor necrosis factor receptor-II (mean arterial pressure), C-reactive protein, lipoprotein-associated phospholipase-A2 activity (reflected pressure wave), interleukin-6, and osteoprotegerin (carotid-femoral pulse wave velocity) were significantly but modestly associated with measures of arterial stiffness and wave reflection. Additional studies are needed to determine whether variation in inflammatory marker genes is associated with tonometry measures.

Effect of Marathon Running on Inflammatory and Hemostatic Markers

A lthough increasing levels of regular physical activity are incrementally cardioprotective, prolonged strenuous exercise such as marathon running may trigger acute myocardial infarction and sudden cardiac death. The mechanism of such events is not well understood but may be due to hemodynamic, vasoconstrictive, and prothrombotic effects with disruption of unstable coronary plaques leading to acute coronary thrombosis. Although several studies have demonstrated exercise-induced activation of fibrinolysis and coagulation, the effect of marathon running on hemostatic balance has not been well studied. We therefore measured changes in C-reactive protein (CRP), von Willebrand factor (vWF), D-dimer, fibrinogen, fibrinolytic activity, white blood cell (WBC) counts, and platelet activation in middle-aged runners before and after the Boston Marathon. An imbalance in prothrombotic and fibrinolytic factors after strenuous physical exertion may transiently increase the risk for intravascular—including coronary—thrombosis and trigger acute ischemic events. • • • Subjects were attendees at the prerace Scientific Symposia of the American Medical Athletic Association as entrants in the 100th to 105th Boston Athletic Association Marathons from 1966 to 2001 (mean age 47.6 years), who reported no smoking or known coronary heart disease by yearly questionnaire. Blood samples were drawn without stasis from an antecubital vein using a 21-gauge butterfly needle the morning before, within 4 hours after the race, and the next morning. Samples were drawn at the same time points for platelet studies in the year 2000 from subjects reporting no use of anti-inflammatory drugs in the prior 2 weeks and for complete blood counts in the year 2001. Whole blood was collected in 3.2% sodium citrate (9:1, vol/vol), and centrifuged at 2,500 g for 20 minutes at 4°C to obtain platelet-poor plasma for the measurement of CRP, vWF, D-dimer, fibrinolytic activity, and fibrinogen. Whole blood was similarly collected and processed in 15% (K3) ethylenediaminetetraacetic acid at a final concentration of 1 mg/ml for determination of vWF. CRP and D-dimer levels were determined using commercially available enzymelinked immunosorbant assays by TintEliza Biopool (Ventura, California) and Diagnostica Stago (Persippany, New Jersey), respectively. The vWF was determined by an enzyme-linked immunoassay procedure described by Penny et al, fibrinolytic activity by a fibrin plate assay as described by Brackman, and fibrinogen levels by the Clauss method. Whole blood for platelet aggregation studies was collected in 3.2% sodium citrate (9:1, vol/vol), and centrifuged within 1 hour of collection at 125 g for 10 minutes at room temperature to obtain platelet-rich plasma for testing after 30 minutes. Platelet aggregation testing was performed using a platelet aggregometer (PACKS-4 Platelet Aggregation Chromogenic Kinetic System, Helena Laboratories, Beaumont, Texas) in response to 10 ug/ml collagen, 10 uM adenosine diphosphate, 10 uM epinephrine, 500 ug/ml arachidonate, and 1.5 mg/ml ristocetin. Complete blood counts were performed in an ADVIA 120 automated hematology autoanalyzer (Bayer Diagnostics, Tarrytown, New York). Total and high-density lipoprotein cholesterol levels were measured using an Abbot Diagnostics ABA-200 biochromatic analyzer and Abbot A-Gent enzymatic reagents (Abbott Lab., Chicago, Illinois). Creatinine, blood urea nitrogen, and electrolytes were determined using a Hitachi 917 chemistry autoanalyzer (Roche Diagnostics, Indianapolis, Indiana). The Student paired t test was used to compare results from pooled data before and after the 1996 and 1997 marathons becausea there were no significant differences in baseline values between the 2 races. In comparing prerace values with those within 4 hours after the race in 55 finishers in 1996 to 1997, marathon running resulted in a twofold or greater increase in CRP (343 611 to 762 973 ng/ml [p 0.001]), vWF (109 47% to 233 65% [p 0.001]), D-dimer (177 137 to 529 279 ng/ml [p 0.001]), and fibrinolytic activity (75 48 to 213 95 mm [p 0.001]), whereas fibrinogen levels decreased (278 52 to 260 45 mg/dl [p 0.001]; Table 1). When comparing prerace specimens with those within 4 hours after the marathon in 32 runners in 2001, WBC and platelet counts increased (5.5 0.2 to 17.4 1.5 th/mm [p 0.005] and 226 25 to 253 27 th/mm [p 0.05]), respectively, whereas hematocrit and hemoglobin levels were unchanged. Platelet studies showed a shortened lag time to aggregation with collagen and an increase with epinephrine (n 5, year 2000). When comparing prerace values with those the morning after the race in 13 runners in 1997, vWF and From the Department of Medicine, McLean Hospital, Belmont; Institute for Prevention of Cardiovascular Disease; Massachusetts General Hospital and Brigham and Women’s Hospital, Boston, Massachusetts; Royal North Shore Hospital, Sydney, Australia; and Harvard Medical School, Boston, Massachusetts. Dr. Siegel’s address is: McLean Hospital, Department of Internal Medicine, 115 Mill Street, Belmont, Massachusetts 02478. E-mail: ajsiegel@mclean.harvard.edu. Manuscript received December 29, 2000; revised manuscript received and accepted May 23, 2001.