Ankle-brachial index, high-sensitivity C-reactive protein and endothelial function in a cardiovascular risk population

Atherosclerotic vascular disease is the leading cause of death in the Western world. Its main three manifestations are coronary heart disease, cerebrovascular disease, and peripheral arterial disease. Asymptomatic peripheral arterial disease is usually diagnosed using the ankle brachial index, and values ≤ 0.90 are used to determine the diagnosis. The classical risk factors of peripheral arterial disease, such as smoking and diabetes, are well known and early interventions are mandatory to improve the prognosis. What is not well known is the role of inflammation as a risk factor. Yet, a novel approach to cardiovascular diseases is the measurement of endothelial function. In this thesis, we studied the ankle-brachial index, C-reactive protein and endothelial function in a cardiovascular risk population. A total of 2856 subjects were invited to the study and 2085 (73%) responded. From these subjects, a cohort of 1756 risk persons was screened. We excluded the subjects with previously known cardiovascular disease or diabetes, because they were already under systematic follow-up. Out of the study subjects, 983 (56%) were women and 773 (44%) men. The ankle brachial index and high-sensitivity C-reactive protein were measured from 1047 subjects. Endothelial function was assessed by measuring reactive hyperemia pulse amplitude tonometry from 66 subjects with borderline peripheral arterial disease. In this study, smoking was a crucial risk factor for peripheral arterial disease. Subclinical peripheral arterial disease seems to be more common in hypertensive patients even without comorbidities. The measurement of the ankle brachial index is an efficient method to identify patients at an increased cardiovascular risk. High-sensitivity C-reactive protein did not correlate with the ankle brachial index or peripheral arterial disease. Instead, it correlated with measures of obesity. In a cardiovascular risk population with borderline peripheral arterial disease, nearly every fourth subject had endothelial dysfunction. This might point out a subgroup of individuals in need of more intensive treatment for their risk factors.

Levels of C-reactive protein in serum samples from healthy children and adults in São Paulo, Brazil

C-reactive protein (CRP) was measured by ELISA in the sera of 165 healthy blood donors and 125 normal children 1 to 14 years old. The serum levels of blood donors ranged from 0.05 to 57.6 mg/l with median and mean values of 1.8 mg/l and 4.86 mg/l, respectively. CRP levels ranged from 0.02 to 14.4 mg/l in the children's sera, the median being 0.45 mg/l and the mean 1.65 mg/l. No individual lacking CRP was detected. The high CRP levels observed in the present study suggest that the population of the State of São Paulo may usually be exposed to subacute infections and/or inflammation without presenting clinical symptoms

c-Myc protein expression is not an independent prognostic predictor in cervical squamous cell carcinoma

The c-myc protein is known to regulate the cell cycle, and its down-regulation can lead to cell death by apoptosis. The role of c-myc protein as an independent prognostic determinant in cervical cancer is controversial. In the present study, a cohort of 220 Brazilian women (mean age 53.4 years) with FIGO stage I, II and III (21, 28 and 51%, respectively) cervical squamous cell carcinomas was analyzed for c-myc protein expression using immunohistochemistry. The disease-free survival and relapse-rate were analyzed using univariate (Kaplan-Meier) survival analysis for 116 women who completed the standard FIGO treatment and were followed up for 5 years. Positive c-myc staining was detected in 40% of carcinomas, 29% being grade 1, 9% grade 2, and 2% grade 3. The distribution of positive c-myc according to FIGO stage was 19% (17 women) in stage I, 33% (29) in stage II, and 48% (43) in stage III of disease. During the 60-month follow-up, disease-free survival in univariate (Kaplan-Meier) survival analysis (116 women) was lower for women with c-myc-positive tumors, i.e., 60.5, 47.5 and 36.6% at 12, 36, and 60 months, respectively (not significant). The present data suggest that immunohistochemical demonstration of c-myc does not possess any prognostic value independent of FIGO stage, and as such is unlikely to be a useful prognostic marker in cervical squamous cell carcinoma.

Effect of hepatitis C serology on C-reactive protein in a cohort of Brazilian hemodialysis patients

Hepatitis C (HCV) is not an uncommon feature in hemodialysis (HD) patients and may be a cause of systemic inflammation. Plasma cytokine interleukin-6 (IL-6) is mainly produced by circulating and peripheral cells and induces the hepatic synthesis of C-reactive protein (CRP), which is the main acute phase reactant. The aim of this study was to investigate the influence of HCV on two markers of systemic inflammation, serum CRP and IL-6, in HD patients. The study included 118 HD patients (47% males, age 47 ± 13 years, 9% diabetics) who had been treated by standard HD for at least 6 months. The patients were divided into two groups depending on the presence (HCV+) or absence (HCV-) of serum antibodies against HCV. Serum albumin (S-Alb), plasma high sensitivity CRP (hsCRP), IL-6, and alanine aminotransferase (ALT) were measured and the values were compared with those for 22 healthy controls. Median hsCRP and IL-6 values and hsCRP/IL-6 ratio were: 3.5 vs 2.1 mg/l, P < 0.05; 4.3 vs 0.9 pg/ml, P < 0.0001, and 0.8 vs 2.7, P < 0.0001, for patients and controls, respectively. Age, gender, S-Alb, IL-6 and hsCRP did not differ between the HCV+ and HCV- patients. However, HCV+ patients had higher ALT (29 ± 21 vs 21 ± 25 IU/l) and had been on HD for a longer time (6.1 ± 3.0 vs 4.0 ± 2.0 years, P < 0.0001). Moreover, HCV+ patients had a significantly lower median hsCRP/IL-6 ratio (0.7 vs 0.9, P < 0.05) compared to the HCV- group. The lower hsCRP/IL-6 ratio in HCV+ patients than in HCV- patients suggests that hsCRP may be a less useful marker of inflammation in HCV+ patients and that a different cut-off value for hsCRP for this population of patients on HD may be required to define inflammation.

Dialysis water treated by reverse osmosis decreases the levels of C-reactive protein in uremic patients

Atherosclerosis is a major complication of chronic renal failure. Microinflammation is involved in atherogenesis and is associated with uremia and dialysis. The role of dialysate water contamination in inducing inflammation has been debated. Our aim was to study inflammatory markers in patients on chronic dialysis, before and 3 to 6 months after switching the water purification system from deionization to reverse osmosis. Patients had demographic, clinical and nutritional information collected and blood drawn for determination of albumin, ferritin, C-reactive protein (CRP), interleukin-6, and tumor necrosis factor-alpha in both situations. Acceptable levels of water purity were less than 200 colony-forming units of bacteria and less than 1 ng/ml of endotoxin. Sixteen patients died. They had higher median CRP (26.6 vs 11.2 mg/dl, P = 0.007) and lower median albumin levels (3.1 vs 3.9 g/l, P < 0.05) compared to the 31 survivors. Eight patients were excluded because of obvious inflammatory conditions. From the 23 remaining patients (mean age ± SD: 51.3 ± 13.9 years), 18 had a decrease in CRP after the water treatment system was changed. Overall, median CRP was lower with reverse osmosis than with deionization (13.2 vs 4.5 mg/l, P = 0.022, N = 23). There was no difference in albumin, cytokines, subjective global evaluation, or clinical and biochemical parameters. In conclusion, uremic patients presented a clinically significant reduction in CRP levels when dialysate water purification system switched from deionization to reverse osmosis. It is possible that better water treatments induce less inflammation and eventually less atherosclerosis in hemodialysis patients.

Depressive symptoms and C-reactive protein in a Brazilian urban community

Psychological depression is an independent risk factor for coronary artery disease. C-reactive protein has been implicated as a mediator of the effect of psychological depression. Several studies have found that individuals, especially men, who report higher levels of psychological depression also have higher levels of C-reactive protein. The current study was undertaken to replicate these results in a Brazilian population, in which there is a much wider range of variation in both background characteristics (such as socioeconomic status) and coronary artery disease risk factors. A sample of 271 individuals was interviewed using the Center for Epidemiological Studies Depression Scale. Fasting blood samples were obtained and evaluated for C-reactive protein (assessed by a turbidimetric immunoassay using a Dade Behring kit) analysis in a subsample (N = 258) of individuals. The mean ± SD C-reactive protein for the entire sample was 0.43 ± 0.44, with 0.42 ± 0.48 for men and 0.43 ± 0.42 mg/L for women. Data were analyzed using multiple regression analysis, controlling for age, sex, body mass index, socioeconomic status, tobacco use, and both total cholesterol and low-density lipoprotein cholesterol. Higher reported depressive symptoms were correlated with higher C-reactive protein for men (partial r = 0.298, P = 0.004) and with lower C-reactive protein for women (partial r = -0.154, P = 0.059). The differences in the associations for men and women could be a result of differential effects of sex hormones on stress reactivity and immune response. On the other hand, this difference in the associations may be related to gender differences in the disclosure of emotion and the effect that self-disclosure has on physical health and immune response.

Correlates of C-reactive protein levels in young adults: a population-based cohort study of 3827 subjects in Brazil

The socio-demographic, behavioral and anthropometric correlates of C-reactive protein levels were examined in a representative young adult Brazilian population. The 1982 Pelotas Birth Cohort Study (Brazil) recruited over 99% of births in the city of Pelotas that year (N = 5914). Individuals belonging to the cohort have been prospectively followed up. In 2004-2005, 77.4% of the cohort was traced, members were interviewed and 3827 individuals donated blood. Analyses of the outcome were based on a conceptual model that differentiated confounders from potential mediators. The following independent variables were studied in relation to levels of C-reactive protein in sex-stratified analyses: skin color, age, family income, education, parity, body mass index, waist circumference, smoking, fat/fiber/alcohol intake, physical activity, and minor psychiatric disorder. Geometric mean (95% confidence interval) C-reactive protein levels for the 1919 males and 1908 females were 0.89 (0.84-0.94) and 1.96 mg/L (1.85-2.09), respectively. Pregnant women and those using oral contraceptive therapies presented the highest C-reactive protein levels and all sub-groups of women had higher levels than men (P < 0.001). Significant associations between C-reactive protein levels were observed with age, socioeconomic indicators, obesity status, smoking, fat and alcohol intake, and minor psychiatric disorder. Associations were stronger at higher levels of C-reactive protein and some associations were sex-specific. We conclude that both distal (socio-demographic) and proximal (anthropometric and behavioral) factors exert strong effects on C-reactive protein levels and that the former are mediated to some degree by the latter.

C-reactive protein in acute coronary syndrome: association with 3-year outcomes

Inflammatory markers have been associated with clinical outcome in patients with acute coronary syndrome (ACS). The present study evaluated the role of high-sensitivity C-reactive protein (CRP) measurements as a predictor of late cardiovascular outcomes after ACS. One hundred and ninety-nine ACS patients in a Coronary Care Unit from March to November 2002 were included and were reassessed clinically after ~3 years. Clinical variables and CRP levels were evaluated as predictors of major cardiovascular events (MACE, defined as the occurrence of cardiac death, ischemic stroke or myocardial infarction) and mortality. Statistical analyses included Cox multivariable analysis and survival curves (Kaplan-Meier). Of the 199 patients, 11 died within 1 month (5.5%). Of the 188 remaining patients, 22 died after a mean follow-up of 2.9 ± 0.5 years. Baseline CRP levels for patients with MACE (N = 57) were significantly higher than those of patients with no events (median = 0.67 mg/L; 25th-75th percentiles = 0.32 and 1.99 mg/L vs median = 0.45 mg/L; 25th-75th percentiles = 0.24 and 0.83 mg/L; P < 0.001). Patients with CRP levels >3 mg/L had a significantly lower survival than the other two groups (1-3 and <1 mg/L; P = 0.001, log-rank test). The odds ratio for MACE was 7.41 (2.03-27.09) for patients with CRP >3 mg/L compared with those with CRP <1 mg/L. For death by any cause, the hazard ratio was 4.58 (1.93-10.86). High CRP levels predicted worse long-term outcomes (MACE and death by any cause) in patients with ACS.

Highly sensitive C-reactive protein and male gender are independently related to the severity of coronary disease in patients with metabolic syndrome and an acute coronary event

Patients with metabolic syndrome are at high-risk for development of atherosclerosis and cardiovascular events. The objective of this study was to examine the major determinants of coronary disease severity, including those coronary risk factors associated with metabolic syndrome, during the early period after an acute coronary episode. We tested the hypothesis that inflammatory markers, especially highly sensitive C-reactive protein (hsCRP), are related to coronary atherosclerosis, in addition to traditional coronary risk factors. Subjects of both genders aged 30 to 75 years (N = 116) were prospectively included if they had suffered a recent acute coronary syndrome (acute myocardial infarction or unstable angina pectoris requiring hospitalization) and if they had metabolic syndrome diagnosed according to the National Cholesterol Education Program/Adult Treatment Panel III. Patients were submitted to a coronary angiography and the burden of atherosclerosis was estimated by the Gensini score. The severity of coronary disease was correlated (Spearman’s or Pearson’s coefficient) with gender (r = 0.291, P = 0.008), age (r = 0.218, P = 0.048), hsCRP (r = 0.256, P = 0.020), ApoB/ApoA ratio (r = 0.233, P = 0.041), and carotid intima-media thickness (r = 0.236, P = 0.041). After multiple linear regression, only male gender (P = 0.046) and hsCRP (P = 0.012) remained independently associated with the Gensini score. In this high-risk population, male gender and high levels of hsCRP, two variables that can be easily obtained, were associated with more extensive coronary disease, identifying patients with the highest potential of developing new coronary events.

High sensitivity C-reactive protein distribution in the elderly: the Bambuí Cohort Study, Brazil

The measurement of the serum concentration of the acute-phase reactant C-reactive protein (CRP) provides a useful marker in clinical practice. However, the distribution of CRP is not available for all age and population groups. This study assessed the distribution of high sensitivity-CRP (hs-CRP) by gender and age in 1470 elderly individuals from a Brazilian community that participates in the Bambuí Cohort Study. Blood samples were collected after 12 h of fasting and serum samples were stored at -70°C. Measurements were made with a commercial hs-CRP immunonephelometric instrument. More than 50% of the results were above 3.0 mg/L for both genders. Mean hs-CRP was higher in women (3.62 ± 2.58 mg/L) than in men (3.03 ± 2.50 mg/L). This difference was observed for all ages, except for the over-80 age group. This is the first population-based study to describe hs-CRP values in Latin American elderly subjects. Our results indicate that significant gender differences exist in the distribution of hs-CRP, and suggest that gender-specific cut-off points for hs-CRP would be necessary for the prediction of cardiovascular risks.

Cardiorespiratory fitness, pulmonary function and C-reactive protein levels in nonsmoking individuals with diabetes

The objective of this study was to evaluate cardiorespiratory fitness and pulmonary function and the relationship with metabolic variables and C-reactive protein (CRP) plasma levels in individuals with diabetes mellitus (DM). Nineteen men with diabetes and 19 age- and gender-matched control subjects were studied. All individuals were given incremental cardiopulmonary exercise and pulmonary function tests. In the exercise test, maximal workload (158.3±22.3vs 135.1±25.2, P=0.005), peak heart rate (HRpeak: 149±12 vs 139±10, P=0.009), peak oxygen uptake (VO2peak: 24.2±3.2 vs18.9±2.8, P<0.001), and anaerobic threshold (VO2VT: 14.1±3.4 vs 12.2±2.2, P=0.04) were significantly lower in individuals with diabetes than in control subjects. Pulmonary function test parameters, blood pressure, lipid profile (triglycerides, HDL, LDL, and total cholesterol), and CRP plasma levels were not different in control subjects and individuals with DM. No correlations were observed between hemoglobin A1C (HbA1c), CRP and pulmonary function test and cardiopulmonary exercise test performance. In conclusion, the results demonstrate that nonsmoking individuals with DM have decreased cardiorespiratory fitness that is not correlated with resting pulmonary function parameters, HbA1c, and CRP plasma levels.

Influence of apolipoprotein E genotype and dietary alpha-tocopherol on redox status and C-reactive protein levels in apolipoprotein E3 and E4 targeted replacement mice

The molecular basis of the positive association between apoE4 genotype and CVD remains unclear. There is direct in vitro evidence indicating that apoE4 is a poorer antioxidant relative to the apoE3 isoform, with some indirect in vivo evidence also available. Therefore it was hypothesised that apoE4 carriers may benefit from alpha-tocopherol (alpha-Toc) supplementation. Targeted replacement mice expressing the human apoE3 and apoE4 were fed with a diet poor (0 mg/kg diet) or rich (200 mg/kg diet) in alpha-Toc for 12 weeks. Neither apoE genotype nor dietary alpha-Toc exerted any effects on the antioxidant defence system, including glutathione, catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase activities. In addition, no differences were observed in mitogen-induced lymphocyte proliferation. alpha-Toc concentrations were modestly higher in plasma and lower in tissues of apoE4 compared with apoE3 mice, with the greatest differences evident in the lung, suggesting that an apoE4 genotype may reduce alpha-Toc delivery to tissues. A tendency towards increased plasma F-2-isoprostanes in apoE4 mice was observed, while liver thiobarbituric acid-reactive substances did not differ between apoE3 and apoE4 mice. In addition, C-reactive protein (CRP) concentrations were reduced in apoE4 mice indicating that this positive effect on CRP may in part negate the increased CVD risk associated with an apoE4 genotype.

Targeting C-reactive protein for the treatment of cardiovascular disease

Complement-mediated inflammation exacerbates the tissue injury of ischaemic necrosis in heart attacks and strokes, the most common causes of death in developed countries. Large infarct size increases immediate morbidity and mortality and, in survivors of the acute event, larger non-functional scars adversely affect long-term prognosis. There is thus an important unmet medical need for new cardioprotective and neuroprotective treatments. We have previously shown that human C-reactive protein (CRP), the classical acute-phase protein that binds to ligands exposed in damaged tissue and then activates complement(1), increases myocardial and cerebral infarct size in rats subjected to coronary or cerebral artery ligation, respectively(2,3). Rat CRP does not activate rat complement, whereas human CRP activates both rat and human complement(4). Administration of human CRP to rats is thus an excellent model for the actions of endogenous human CRP2,3. Here we report the design, synthesis and efficacy of 1,6-bis(phosphocholine)-hexane as a specific small-molecule inhibitor of CRP. Five molecules of this palindromic compound are bound by two pentameric CRP molecules, crosslinking and occluding the ligand-binding B-face of CRP and blocking its functions. Administration of 1,6-bis(phosphocholine)-hexane to rats undergoing acute myocardial infarction abrogated the increase in infarct size and cardiac dysfunction produced by injection of human CRP. Therapeutic inhibition of CRP is thus a promising new approach to cardioprotection in acute myocardial infarction, and may also provide neuroprotection in stroke. Potential wider applications include other inflammatory, infective and tissue-damaging conditions characterized by increased CRP production, in which binding of CRP to exposed ligands in damaged cells may lead to complement-mediated exacerbation of tissue injury.

Both tumour-promoting and non-promoting phorbol esters inhibit 125I-EGF binding and stimulate the phosphorylation of a 80kd protein kinase C substrate protein in intact Swiss 3T3 cells

Sapintoxin A (SAP A) and 12-deoxyphorbol 13-phenylacetate (DOPP), are two biologically active but non-turnour-promoting phorbol esters that potently bind to and activate the phorbol ester receptor, protein kinase C (PKC). SAP A and DOPP cause a dose-dependent increase in the phosphorylation of an 80 kd (80K) substrate protein for PKC in Swiss 3T3 cells. A similar dose—response effect was seen with sapintoxin D (SAP D), the stage 2 promoting analogue of 12-O-tetradecanoylphorbol-13-acetate and the complete promoter phorbol 12,13-dibutyrate (PDB). The doses resulting in a half maximal phosphorylation of this protein (Ka were 20 nM (SAP A), 45 nM (DOPP), 23 nM (SAP D) and 37 nM (PDB). Both non-promoting and phorbol esters induced a dose-dependent inhibition of [125I]epidermal growth factor (EGF) binding to its receptor in Swiss 3T3 cells. The doses required for 50% inhibition of binding (Ki) were: 8 nM (SAP A), 16 nM (DOPP), 14 nM (SAP D) and 17 nM (PDB). The results clearly demonstrate that induction of phosphorylation of the Pu 80K phosphoprotein and inhibition of [125I]EGF binding in Swiss 3T3 cells following exposure to phorbol esters is independent of the tumour-promoting activity of these compounds. The fact that SAP A, DOPP, SAP D and PDB are mitogenic for a variety of cell types and that exposure to these compounds leads to 80K phosphorylation and inhibition of [125I]EGF binding, suggests that these early biological events may play a role in the mitogenic response induced by these compounds.

APOE genotype influences triglyceride and C-reactive protein responses to altered dietary fat intake in UK adults

Background: The response of plasma lipids to dietary fat manipulation is highly heterogeneous, with some indications that APOE genotype may be important. Objective: The objective was to use a prospective recruitment approach to determine the effect of dietary fat quantity and composition on both lipid and nonlipid cardiovascular disease biomarkers according to APOE genotype. Design: Participants had a mean (±SD) age of 51 ± 9 y and a BMI (in kg/m2) of 26.0 ± 3.8 (n = 44 E3/E3, n = 44 E3/E4) and followed a sequential dietary intervention (the SATgenϵ study) in which they were assigned to a low-fat diet, a high-fat high-SFA (HSF) diet, and the HSF diet with 3.45 g DHA/d (HSF-DHA), each for 8 wk. Fasting blood samples were collected at the end of each intervention arm. Results: An overall diet effect was evident for all cholesterol fractions (P < 0.01), with no significant genotype × diet interactions observed. A genotype × diet interaction (P = 0.033) was evident for plasma triglycerides, with 17% and 30% decreases in APOE3/E3 and APOE3/E4 individuals after the HSF-DHA diet relative to the low-fat diet. A significant genotype × diet interaction (P = 0.009) was also observed for C-reactive protein (CRP), with only significant increases in concentrations after the HSF and HSF-DHA diets relative to the low-fat diet in the APOE3/E4 group (P < 0.015). Conclusions: Relative to the wild-type APOE3/E3 group, our results indicate a greater sensitivity of fasting triglycerides and CRP to dietary fat manipulation in those with an APOE3/E4 genotype (25% population), with no effect of this allelic profile on cholesterol concentrations. The SATgenϵ study was registered at clinicaltrials.gov as NCT01384032.