Serum levels of autoantibodies against the angiotensin II type I receptor are not associated with serum dicarbonyl or AGE levels in patients with an aldosterone-producing adenoma.

Patients with an aldosterone-producing adenoma (APA) carry a higher risk of cardiovascular disease and commonly have high levels of autoantibodies (AT1AA) that may activate the angiotensin II type 1 receptor (AT1R). AT1R activation is linked to an increase of the glucose metabolite methylglyoxal (MGO), a potential precursor of advanced glycation endproducts (AGEs) and driver of vascular inflammation. We investigated whether serum AT1AA levels are associated with serum MGO and AGE levels in APA patients. In a case series of 26 patients with APA we measured levels of dicarbonyls MGO, glyoxal (GO) and 3-deoxyglucosone (3-DG), and dicarbonyl-derived AGEs 5-hydro-5-methylimidazolone (MG-H1), Nε-(carboxyethyl)lysine (CEL) and Nε-(carboxymethyl)lysine (CML) with UPLC-MS/MS. We also measured AT1AA by ELISA. These measurements were repeated 1-month after adrenalectomy in a subset of 14 patients. Panels of inflammation and endothelial function were also measured by immunoassays. Although baseline higher AT1AA levels tended to be correlated with higher baseline serum MGO, GO and 3-DG levels (r = 0.18, p = 0.38; r = 0.20, p = 0.33; r = 0.23, p = 0.26; respectively), these correlations were not statistically significant. We observed no obvious correlations between higher AT1AA levels and protein-bound and free MG-H1, CEL and CML levels, and markers of inflammation and endothelial function. No decrease was observed in any of the dicarbonyls, protein-bound AGE levels and markers of inflammation and endothelial function after adrenalectomy. In patients with APA the serum levels of AT1AA were not significantly correlated with serum dicarbonyls, protein-bound and free AGE levels. Increased signalling of the AT1AA receptor may therefore be unlikely to overtly increase systemic dicarbonyl levels.

The hypoxia-sensor carbonic anhydrase IX affects macrophage metabolism, but is not a suitable biomarker for human cardiovascular disease.

Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.

Fasting and post-oral-glucose-load levels of methylglyoxal are associated with microvascular, but not macrovascular, disease in individuals with and without (pre)diabetes: The Maastricht Study.

AIMS: Reactive dicarbonyl compounds, such as methylglyoxal (MGO), rise during an oral glucose tolerance test (OGTT), particularly in (pre)diabetes. Fasting MGO levels are associated with chronic kidney disease (CKD) and cardiovascular disease (CVD) in patients with poorly controlled type 2 diabetes mellitus (T2DM). Yet, whether fasting or post-OGTT plasma MGO levels are associated with vascular disease in people with (pre)diabetes is unknown. METHODS: Subjects with normal glucose metabolism (n=1796; age: 57.9±8.2 years; 43.3% men), prediabetes (n=478; age: 61.6±7.6 years; 54.0% men) and T2DM (n=669; age: 63.0±7.5 years; 67.0% men) from the Maastricht Study underwent OGTTs. Plasma MGO levels were measured at baseline and 2h after OGTT by mass spectrometry. Prior CVD was established via questionnaire. CKD was reflected by estimated glomerular filtration rate (eGFR) and albuminuria; retinopathy was assessed using retinal photographs. Data were analyzed using logistic regression adjusted for gender, age, smoking, systolic blood pressure, total-to-HDL cholesterol ratio, triglycerides, HbA1c, BMI and medication use. Odd ratios (ORs) were expressed per standard deviation of LN-transformed MGO. RESULTS: Fasting and post-OGTT MGO levels were associated with higher ORs for albuminuria ≥30mg/24h [fasting: 1.12 (95% CI: 0.97-1.29); post-OGTT: 1.19 (1.01-1.41)], eGFR<60mL/min/1.73 m2 [fasting: 1.58 (95% CI: 1.38-1.82), post-OGTT: 1.57 (1.34-1.83)] and retinopathy [fasting: 1.59 (95% CI: 1.01-2.53), post-OGTT: 1.38 (0.77-2.48)]. No associations with prior CVD were found. CONCLUSION: Fasting and post-OGTT MGO levels were associated with microvascular disease, but not prior CVD. Thus, therapeutic strategies directed at lowering MGO levels may prevent microvascular disease.

Dietary intake of advanced glycation end products (AGEs) and changes in body weight in European adults.

PURPOSE: Advanced glycation end products (AGEs) can be formed in foods by the reaction of reducing sugars with proteins, and have been shown to induce insulin resistance and obesity in experimental studies. We examined the association between dietary AGEs intake and changes in body weight in adults over an average of 5 years of follow-up. METHODS: A total of 255,170 participants aged 25-70 years were recruited in ten European countries (1992-2000) in the PANACEA study (Physical Activity, Nutrition, Alcohol, Cessation of smoking, Eating out of home in relation to Anthropometry), a sub-cohort of the EPIC (European Prospective Investigation into Cancer and Nutrition). Body weight was measured at recruitment and self-reported between 2 and 11 years later depending on the study center. A reference database for AGEs was used containing UPLC-MS/MS-measured Nε-(carboxymethyl)-lysine (CML), Nε-(1-carboxyethyl)-lysine (CEL), and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) in 200 common European foods. This reference database was matched to foods and decomposed recipes obtained from country-specific validated dietary questionnaires in EPIC and intake levels of CEL, CML, and MG-H1 were estimated. Associations between dietary AGEs intake and body weight change were estimated separately for each of the three AGEs using multilevel mixed linear regression models with center as random effect and dietary AGEs intake and relevant confounders as fixed effects. RESULTS: A one-SD increment in CEL intake was associated with 0.111 kg (95% CI 0.087-0.135) additional weight gain over 5 years. The corresponding additional weight gain for CML and MG-H1 was 0.065 kg (0.041-0.089) and 0.034 kg (0.012, 0.057), respectively. The top six food groups contributing to AGEs intake, with varying proportions across the AGEs, were cereals/cereal products, meat/processed meat, cakes/biscuits, dairy, sugar and confectionary, and fish/shellfish. CONCLUSION: In this study of European adults, higher intakes of AGEs were associated with marginally greater weight gain over an average of 5 years of follow-up.

Methylglyoxal, a Highly Reactive Dicarbonyl Compound, in Diabetes, Its Vascular Complications, and Other Age-Related Diseases.

The formation and accumulation of methylglyoxal (MGO), a highly reactive dicarbonyl compound, has been implicated in the pathogenesis of type 2 diabetes, vascular complications of diabetes, and several other age-related chronic inflammatory diseases such as cardiovascular disease, cancer, and disorders of the central nervous system. MGO is mainly formed as a byproduct of glycolysis and, under physiological circumstances, detoxified by the glyoxalase system. MGO is the major precursor of nonenzymatic glycation of proteins and DNA, subsequently leading to the formation of advanced glycation end products (AGEs). MGO and MGO-derived AGEs can impact on organs and tissues affecting their functions and structure. In this review we summarize the formation of MGO, the detoxification of MGO by the glyoxalase system, and the biochemical pathways through which MGO is linked to the development of diabetes, vascular complications of diabetes, and other age-related diseases. Although interventions to treat MGO-associated complications are not yet available in the clinical setting, several strategies to lower MGO have been developed over the years. We will summarize several new directions to target MGO stress including glyoxalase inducers and MGO scavengers. Targeting MGO burden may provide new therapeutic applications to mitigate diseases in which MGO plays a crucial role.

Association between bone metabolism regulators and arterial stiffness in type 2 diabetes patients.

BACKGROUND AND AIM: Osteopontin (OPN), osteonectin (ON) and osteocalcin (OC) play an important role in the development of vascular calcifications, but it is unclear whether these bone metabolism regulators contribute to the development of arterial stiffness in type 2 diabetes patients. We therefore aim to determine the relationship between plasma concentrations of OPN, ON, OC and arterial stiffness in type 2 diabetes patients. METHODS: Cross-sectional study of 1003 type 2 diabetes patients included in the Second Manifestations of ARTerial disease (SMART)-cohort. Generalized linear models were used to evaluate the relation between plasma levels of OPN, ON and OC and arterial stiffness as measured by pulse pressure (PP), ankle-brachial index (ABI) (≥0.9), carotid artery distension and an arterial stiffness summary score. Analyses were adjusted for age, sex, kidney function, diabetes duration and diastolic blood pressure. Higher OPN plasma levels were significantly related to a lower ABI (ß-0.013; 95%CI -0.024 to -0.002) and a higher arterial stiffness summary score (OR1.24; 95%CI 1.03-1.49). OPN levels were not related to PP (ß 0.59; 95%CI -0.63-1.81) or absolute carotid artery distention (ß -7.03; 95%CI -20.00-5.93). ON and OC plasma levels were not related to any of the arterial stiffness measures. CONCLUSION: Only elevated plasma levels of OPN are associated with increased arterial stiffness in patients with type 2 diabetes as measured by the ankle-brachial index and arterial stiffness summary score. These findings indicate that OPN may be involved in the pathophysiology of arterial stiffness and call for further clinical investigation.

Abdominal subcutaneous and visceral adipocyte size, lipolysis and inflammation relate to insulin resistance in male obese humans.

Obesity is associated with a disturbed adipose tissue (AT) function characterized by adipocyte hypertrophy, an impaired lipolysis and pro-inflammatory phenotype, which contributes to insulin resistance (IR). We investigated whether AT phenotype in different AT depots of obese individuals with and without type 2 diabetes mellitus (T2DM) is associated with whole-body IR. Subcutaneous (SC) and visceral (V) AT biopsies from 18 lean, 17 obese and 8 obese T2DM men were collected. AT phenotype was characterized by ex vivo measurement of basal and stimulated lipolysis (mature adipocytes), adipocyte size distribution (AT tissue sections) and AT immune cells (flow cytometry). In VAT, mean adipocyte size, CD45+ leukocytes and M1 macrophages were significantly increased in both obese groups compared to lean individuals. In SCAT, despite adipocyte hypertrophy, no significant differences in immune cell populations between groups were found. In SCAT, multiple linear regression analysis showed that none of the AT phenotype markers independently contributed to HOMA-IR while in VAT, mean adipocyte size was significantly related to HOMA-IR. In conclusion, beside adipocyte hypertrophy in VAT, M1 macrophage- or B-cell-mediated inflammation, may contribute to IR, while inflammation in hypertrophic SCAT does not seem to play a major role in IR.

Alcohol and red wine consumption, but not fruit, vegetables, fish or dairy products, are associated with less endothelial dysfunction and less low-grade inflammation: the Hoorn Study.

PURPOSE: Endothelial dysfunction and low-grade inflammation are key phenomena in the pathobiology of cardiovascular disease (CVD). Their dietary modification might explain the observed reduction in CVD that has been associated with a healthy diet rich in fruit, vegetables and fish, low in dairy products and with moderate alcohol and red wine consumption. We investigated the associations between the above food groups and endothelial dysfunction and low-grade inflammation in a population-based cohort of Dutch elderly individuals. METHODS: Diet was measured by food frequency questionnaire (n = 801; women = 399; age 68.5 ± 7.2 years). Endothelial dysfunction was determined (1) by combining von Willebrand factor, and soluble intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule 1, endothelial selectin and thrombomodulin, using Z-scores, into a biomarker score and (2) by flow-mediated vasodilation (FMD), and low-grade inflammation by combining C-reactive protein, serum amyloid A, interleukin 6, interleukin 8, tumour necrosis factor α and sICAM-1 into a biomarker score, with smaller FMD and higher scores representing more dysfunction and inflammation, respectively. We used linear regression analyses to adjust associations for sex, age, energy, glucose metabolism, body mass index, smoking, prior CVD, educational level, physical activity and each of the other food groups. RESULTS: Moderate [ß (95% CI) -0.13 (-0.33; 0.07)] and high [-0.22 (-0.45; -0.003)] alcohol consumption, and red wine [-0.16 (-0.30; -0.01)] consumption, but none of the other food groups, were associated with a lower endothelial dysfunction biomarker score and a greater FMD. The associations for FMD were, however, not statistically significant. Only red wine consumption was associated with a lower low-grade inflammation biomarker score [-0.18 (-0.33; -0.04)]. CONCLUSIONS: Alcohol and red wine consumption may favourably influence processes involved in atherothrombosis.

Diverging effects of diabetes mellitus in patients with peripheral artery disease and abdominal aortic aneurysm and the role of advanced glycation end-products: ARTERY study - protocol for a multicentre cross-sectional study.

INTRODUCTION: Diabetes mellitus is a well-defined risk factor for peripheral artery disease (PAD), but protects against the development and growth of abdominal aortic aneurysm (AAA). Diabetes mellitus is associated with arterial stiffening and peripheral arterial media sclerosis. Advanced glycation end-products (AGEs) are increased in diabetes mellitus and cardiovascular disease. AGEs are known to form cross-links between proteins and are associated with arterial stiffness. Whether AGEs contribute to the protective effects of diabetes mellitus in AAA is unknown. Therefore, the ARTERY (Advanced glycation end-pRoducts in patients with peripheral arTery disEase and abdominal aoRtic aneurYsm) study is designed to evaluate the role of AGEs in the diverging effects of diabetes mellitus on AAA and PAD. METHODS AND ANALYSIS: This cross-sectional multicentre study will compare the amount, type and location of AGEs in the arterial wall in a total of 120 patients with AAA or PAD with and without diabetes mellitus (n=30 per subgroup). Also, local and systemic vascular parameters, including pulse wave velocity, will be measured to evaluate the association between arterial stiffness and AGEs. Finally, AGEs will be measured in serum, urine, and assessed in skin with skin autofluorescence using the AGE Reader. ETHICS AND DISSEMINATION: This study is approved by the Medical Ethics committees of University Medical Center Groningen, Martini Hospital and Medisch Spectrum Twente, the Netherlands. Study results will be disseminated through peer-reviewed journals and scientific events. TRIAL REGISTRATION NUMBER: trialregister.nl NTR 5363.

Analysis of inflammatory cells and mediators in skin wound biopsies to determine wound age in living subjects in forensic medicine.

OBJECTIVE: In forensic medicine it is important to determine the age of skin wounds in living subjects. The aim of this study was to assess whether analysis of inflammatory cells and inflammatory mediators in skin biopsies of wounds from living subjects could improve wound age determination. METHODS: Biopsies (n=101), representing the superficial border area of a skin wound, were taken from skin injuries of known wound age (range: 4.5 hours to 25 days) of living subjects. All biopsies were analyzed for 3 inflammatory cell markers (MPO, CD45 and CD68) and 4 inflammatory mediators (MIP-1, IL-8, CML and vitronectin). For quantification, biopsies were subdivided in 4 different timeframes: 0.2-2 days, 2-4 days, 4-10 days and 10-25 days old wounds. Subsequently, a probability scoring system was developed. RESULTS: MPO, CD45, MIP-1, IL-8 (inflammatory cell markers) and N(epsilon)-(carboxymethyl)lysine (CML) positivity were maximal in wounds of 0.2-2 days old and then decreased in time. Remarkably, CD45, CD68 and CML showed a minor but non-significant increase again in 10-25 days old wounds. MPO and CD68 positivity was significantly lower in 4-25 days old wounds compared to 0.2-4 days old wounds. MPO positivity was also significantly lower in 10-25 days old wounds compared to 0.2-10 days old wounds. For CD45, MIP-1, IL-8 and CML no significant differences between the age groups were found. In case of vitronectin positivity in the extravasate or when the number of MIP-1 or IL-8-positive cells was more than 10 cells/mm(2) the probability that a wound was more than 10 days old was 0%. A probability scoring system of all analyzed markers can be used to calculate individual wound age probabilities in biopsies of skin wounds of living subjects. CONCLUSIONS: We have developed a probability scoring system of inflammatory cells and mediators that can be used to determine wound age in skin biopsies of living subjects.

Associations of advanced glycation end-products with cognitive functions in individuals with and without type 2 diabetes: the maastricht study.

CONTEXT: Advanced glycation end-products (AGEs) are thought to be involved in the pathogenesis of Alzheimer's disease. AGEs are products resulting from nonenzymatic chemical reactions between reduced sugars and proteins, which accumulate during natural aging, and their accumulation is accelerated in hyperglycemic conditions such as type 2 diabetes mellitus. OBJECTIVE: The objective of the study was to examine associations between AGEs and cognitive functions. DESIGN, SETTING, AND PARTICIPANTS: This study was performed as part of the Maastricht Study, a population-based cohort study in which, by design, 215 participants (28.1%) had type 2 diabetes mellitus. MAIN OUTCOME MEASURES: We examined associations of skin autofluorescence (SAF) (n = 764), an overall estimate of skin AGEs, and specific plasma protein-bound AGEs (n = 781) with performance on tests for global cognitive functioning, information processing speed, verbal memory (immediate and delayed word recall), and response inhibition. RESULTS: After adjustment for demographics, diabetes, smoking, alcohol, waist circumference, total cholesterol/high-density lipoprotein cholesterol ratio, triglycerides, and lipid-lowering medication use, higher SAF was significantly associated with worse delayed word recall (regression coefficient, b = -0.44; P = .04), and response inhibition (b = 0.03; P = .04). After further adjustment for systolic blood pressure, cardiovascular disease, estimated glomerular filtration rate, and depression, associations were attenuated (delayed word recall, b = -0.38, P = .07; response inhibition, b = 0.02, P = .07). Higher pentosidine levels were associated with worse global cognitive functioning (b = -0.61; P = .04) after full adjustment, but other plasma AGEs were not. Associations did not differ between individuals with and without diabetes. CONCLUSION: We found inverse associations of SAF (a noninvasive marker for tissue AGEs) with cognitive performance, which were attenuated after adjustment for vascular risk factors and depression.

Endothelial dysfunction is associated with a greater depressive symptom score in a general elderly population: the Hoorn Study.

BACKGROUND: Endothelial dysfunction (ED), low-grade inflammation (LGI) and oxidative stress (OxS) may be involved in the pathobiology of depression. Previous studies on the association of these processes in depression have yielded contradictory results. We therefore investigated comprehensively, in a population-based cohort study, the association between ED, LGI and OxS on the one hand and depressive symptoms on the other. METHOD: We used data from the Hoorn Study and determined biomarkers of ED [flow-mediated dilatation (FMD), von Willebrand factor, soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1, soluble thrombomodulin and soluble endothelial selectin], LGI [C-reactive protein, tumour necrosis factor-α, interleukin 6, interleukin 8, serum amyloid A, myeloperoxidase (MPO) and sICAM-1] and OxS (oxidized low density lipoprotein and MPO). Depressive symptoms were quantified by the Center for Epidemiologic Studies Depression Scale (CES-D) questionnaire (n = 493; age 68 years; 49.9% female). Regression analyses were performed with the use of biomarker Z scores. Adjustments were made for age, sex and glucose metabolism status (cohort stratification variables) and prior cardiovascular disease, hypertension, waist-to-hip ratio, cholesterol levels, education level, physical activity, dietary habits, and the use of antihypertensive and/or lipid-lowering medication and/or metformin (potential confounders). RESULTS: After adjustment for age, sex and glucose metabolism status, one standard deviation increase in the ED Z score was associated with a 1.9 [95% confidence interval (CI) 0.7-3.1] higher CES-D score. Additional adjustments did not materially change this result. LGI and OxS were not associated with the CES-D score. CONCLUSIONS: ED, as quantified by an array of circulating biomarkers and FMD, was independently associated with depressive symptoms. This study supports the hypothesis that ED plays an important role in the pathobiology of depression.

Long-term effects of metformin on endothelial function in type 2 diabetes: a randomized controlled trial.

OBJECTIVES: We investigated whether metformin can improve endothelial function and decrease inflammatory activity, and thereby decrease the risk of atherothrombotic disease. SUBJECTS AND DESIGN: A randomized, placebo-controlled trial with a follow-up period of 4.3 years set in the outpatient clinics of three nonacademic hospitals (Hoogeveen, Meppel and Coevorden Hospitals, the Netherlands). A total of 390 patients with type 2 diabetes treated with insulin were included. Either metformin 850 mg or placebo (one to three times daily) was added to insulin therapy. Urinary albumin excretion and plasma levels of von Willebrand factor (vWf), soluble vascular adhesion molecule-1 (sVCAM-1), soluble E-selectin (sE-selectin), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), C-reactive protein (CRP) and soluble intercellular adhesion molecule-1 (sICAM-1) were measured at baseline and after 4, 17, 30, 43 and 52 months. RESULTS: Metformin significantly reduced levels of vWF, sVCAM-1, t-PA, PAI-1, CRP and sICAM-1, which, except for CRP, remained significant after adjustment for baseline differences in age, sex, smoking and severity of previous cardiovascular (CV) disease. No effects on urinary albumin excretion or sE-selectin were observed. The improvements in vWf and sVCAM-1 statistically explained about 34% of the reduction in the risk of CV morbidity and mortality associated with metformin treatment in this study. CONCLUSIONS: Metformin is associated with improvement in some (vWF and sVCAM-1) but not all markers of endothelial function, which may explain why it is associated with a decreased risk of CV disease in type 2 diabetes.

Unhealthy dietary patterns associated with inflammation and endothelial dysfunction in type 1 diabetes: the EURODIAB study.

BACKGROUND AND AIMS: A healthy diet has been inversely associated with endothelial dysfunction (ED) and low-grade inflammation (LGI). We investigated the association between nutrient consumption and biomarkers of ED and LGI in type 1 diabetes. METHODS AND RESULTS: We investigated 491 individuals. Nutrient consumption and lifestyle risk factors were measured in 1989 and 1997. Biomarkers of ED (von Willebrand factor, soluble vascular cell adhesion molecule-1 and soluble endothelial selectin) and LGI (C-reactive protein, interleukin 6 and tumour necrosis factor α) were measured in 1997 and averaged into Z-scores. The nutrient residual method was used to adjust individual nutrient intake for energy intake. Data were analysed with generalised estimation equations. We report increments/decrements in nutrient consumption, averaged over time, per +1 standard deviation (SD) of 1997 ED or LGI Z-scores, after adjustment for sex, age, duration of diabetes, investigation centre, body mass index, energy intake, smoking behaviour, alcohol consumption, and each of the other nutrients. One SD elevation in ED Z-score was associated with a diet lower in fibre [ß(95%CI);-0.09(-0.18;-0.004)], polyunsaturated fat [-0.18(-0.31;-0.05)] and vegetable protein [-0.10(-0.20;-0.001)]. For the LGI Z-score results showed associations with fibre [-0.09(-0.17;-0.01)], polyunsaturated fat [-0.14(-0.24;-0.03)] and cholesterol [0.10(0.01; 0.18)]. CONCLUSION: In type 1 diabetes, consumption of less fibre, polyunsaturated fat and vegetable protein, and more cholesterol over the study period was associated with more ED and LGI. Following dietary guidelines in type 1 diabetes may reduce cardiovascular disease risk by favourably affecting ED and LGI.

Erythropoietin and vascular endothelial growth factor as risk markers for severe hypoglycaemia in type 1 diabetes.

OBJECTIVE: Circulating erythropoietin (EPO) and vascular endothelial growth factor (VEGF) increase during hypoglycaemia and may represent protective hormonal counter-regulatory responses. We tested the hypothesis that low levels of EPO and VEGF are associated with a higher frequency of severe hypoglycaemia in a cohort of patients with type 1 diabetes. DESIGN: Prospective observational follow-up study. METHODS: Totally 219 patients with type 1 diabetes (41% females, age 46+/-13 years (mean+/-s.d.), duration of diabetes 21+/-12 years, and HbAlc 8.5+/-1.1%) were followed in a 1-year observational study. Plasma EPO and serum VEGF levels were measured at baseline with ELISA. Events of severe hypoglycaemia defined by third party assistance were recorded and validated in telephone interviews within 24 h. RESULTS: Totally 235 episodes of severe hypoglycaemia (1.1 episodes per patient-year) were reported by 82 patients (37%). At baseline, plasma EPO was 8.6 (3.1-34.3) U/l (median (range)), and serum VEGF was 52.2 (6.6-337) pg/ml. The levels of EPO and VEGF were not associated with frequency of severe and mild hypoglycaemia. The levels of EPO were not associated with age, sex, duration of diabetes, body mass index, HbAlc, C-peptide level or hypoglycaemia awareness status. The levels of VEGF were positively associated with age and female sex. CONCLUSIONS: Although several studies suggest that VEGF and EPO may affect brain function during hypoglycaemia, this study does not support random VEGF or EPO levels to determine future risk of severe hypoglycaemia in people with type 1 diabetes.

Low-grade inflammation can partly explain the association between the metabolic syndrome and either coronary artery disease or severity of peripheral arterial disease: the CODAM study.

BACKGROUND: Low-grade inflammation has been hypothesized to underlie the coronary artery disease (CAD) risk associated with the metabolic syndrome, but the evidence is not conclusive. For peripheral arterial disease (PAD; as measured by the ankle-arm index), this association has not been studied before. The aim was to study whether the association between the metabolic syndrome and CAD or the severity of PAD can be explained by low-grade inflammation. METHODS: The Cohort study Diabetes and Atherosclerosis Maastricht population includes 574 subjects, with an increased risk of type 2 diabetes, of whom 560 were included in the analyses (343 males; age: 59.5 +/- 7.0 years). The inflammation markers that were measured were C-reactive protein, interleukin 6, soluble vascular cell adhesion molecule-1, soluble intercellular adhesion molecule-1 and serum amyloid A. All analyses were adjusted for age, sex and smoking. RESULTS: Logistic regression showed that the metabolic syndrome was significantly associated with CAD [odds ratio (OR) = 1.86, 95% CI: 1.21; 2.84, P = 0.004]. Further adjustment for inflammatory status, as captured in a combination of the inflammation markers (using an averaged Z-score), resulted in significant associations of both the metabolic syndrome and inflammatory status with CAD [OR(metabolic syndrome) (95% CI) = 1.58 (1.01; 2.46), P = 0.044; OR(inflammation) (95% CI) = 1.59 (1.14; 2.21), P = 0.007]. Linear regression analysis showed similar results for the ankle-arm index. CONCLUSIONS: The association between the metabolic syndrome, on the one hand, and prevalence of CAD or the severity of PAD, on the other, can be partly but not completely, 26% and 29% respectively, explained by low-grade inflammation.

Platelet and leukocyte activation, atherosclerosis and inflammation in European and South Asian men.

BACKGROUND: Increased platelet activation occurs in ischemic heart disease (IHD), but increased platelet activation is also seen in cerebrovascular atherosclerosis and peripheral artery disease. It is not clear therefore whether platelet activation is an indicator of IHD or a marker of generalized atherosclerosis and inflammation. South Asian subjects are at high risk of IHD, but little is known regarding differences in platelet and leukocyte function between European and South Asian subjects. METHODS: Fifty-four male subjects (age 49-79 years) had coronary artery calcification measured by multislice computed tomography (CT), aortic atherosclerosis assessed by measurement of carotid-femoral pulse wave velocity (aortic PWV), and femoral and carotid atherosclerosis measured by B-mode ultrasound. Platelet and leukocyte activation was assessed by flow cytometry of platelet-monocyte complexes (PMC), platelet expression of PAC-1 binding site and CD62P, and expression of L-selectin on leukocytes. RESULTS: Elevated circulating PMC correlated significantly with elevated aortic PWV and PMC were higher in subjects with femoral plaques. In contrast PMC did not differ by increasing coronary artery calcification category or presence of carotid plaques. Higher numbers of PMC were independently related to elevated levels of C-reactive protein (CRP), higher aortic PWV, hypertension and smoking in a multivariate model. Markers of platelet and leukocyte activation did not differ significantly by ethnicity. CONCLUSIONS: Increased PMC are related to the extent of aortic and femoral atherosclerosis rather than coronary or carotid atherosclerosis. The association between elevated CRP and increased PMC suggests that inflammation in relation to generalized atherosclerosis may play an important role in PMC activation.

Advanced glycation end products cause increased CCN family and extracellular matrix gene expression in the diabetic rodent retina.

AIMS/HYPOTHESIS: Referred to as CCN, the family of growth factors consisting of cystein-rich protein 61 (CYR61, also known as CCN1), connective tissue growth factor (CTGF, also known as CCN2), nephroblastoma overexpressed gene (NOV, also known as CCN3) and WNT1-inducible signalling pathway proteins 1, 2 and 3 (WISP1, -2 and -3; also known as CCN4, -5 and -6) affects cellular growth, differentiation, adhesion and locomotion in wound repair, fibrotic disorders, inflammation and angiogenesis. AGEs formed in the diabetic milieu affect the same processes, leading to diabetic complications including diabetic retinopathy. We hypothesised that pathological effects of AGEs in the diabetic retina are a consequence of AGE-induced alterations in CCN family expression. MATERIALS AND METHODS: CCN gene expression levels were studied at the mRNA and protein level in retinas of control and diabetic rats using real-time quantitative PCR, western blotting and immunohistochemistry at 6 and 12 weeks of streptozotocin-induced diabetes in the presence or absence of aminoguanidine, an AGE inhibitor. In addition, C57BL/6 mice were repeatedly injected with exogenously formed AGE to establish whether AGE modulate retinal CCN growth factors in vivo. RESULTS: After 6 weeks of diabetes, Cyr61 expression levels were increased more than threefold. At 12 weeks of diabetes, Ctgf expression levels were increased twofold. Treatment with aminoguanidine inhibited Cyr61 and Ctgf expression in diabetic rats, with reductions of 31 and 36%, respectively, compared with untreated animals. Western blotting showed a twofold increase in CTGF production, which was prevented by aminoguanidine treatment. In mice infused with exogenous AGE, Cyr61 expression increased fourfold and Ctgf expression increased twofold in the retina. CONCLUSIONS/INTERPRETATION: CTGF and CYR61 are downstream effectors of AGE in the diabetic retina, implicating them as possible targets for future intervention strategies against the development of diabetic retinopathy.

Anti-inflammatory agents and monoHER protect against DOX-induced cardiotoxicity and accumulation of CML in mice.

Cardiac damage is the major limiting factor for the clinical use of doxorubicin (DOX). Preclinical studies indicate that inflammatory effects may be involved in DOX-induced cardiotoxicity. Nepsilon-(carboxymethyl) lysine (CML) is suggested to be generated subsequent to oxidative stress, including inflammation. Therefore, the aim of this study was to investigate whether CML increased in the heart after DOX and whether anti-inflammatory agents reduced this effect in addition to their possible protection on DOX-induced cardiotoxicity. These effects were compared with those of the potential cardioprotector 7-monohydroxyethylrutoside (monoHER).BALB/c mice were treated with saline, DOX alone or DOX preceded by ketoprofen (KP), dexamethasone (DEX) or monoHER. Cardiac damage was evaluated according to Billingham. Nepsilon-(carboxymethyl) lysine was quantified immunohistochemically. Compared to saline, a 21.6-fold increase of damaged cardiomyocytes was observed in mice treated with DOX (P<0.001). Addition of KP, DEX or monoHER before DOX significantly reduced the mean ratio of abnormal cardiomyocytes in comparison to mice treated with DOX alone (P

No increase in C-reactive protein levels during intranasal compared to oral hormone therapy in healthy post-menopausal women.

BACKGROUND: Inflammation plays an important role in the development of atherosclerotic disease. Oral post-menopausal hormone therapy increases serum C-reactive protein (CRP) levels. This study compared the effects of intranasal and oral administration of 17beta-estradiol (E2) combined with norethisterone acetate (NETA) on markers of inflammation in healthy post-menopausal women. METHODS: Ninety healthy post-menopausal women (age 56.6 +/- 4.7 years) participated in this 1-year trial. After computerized block randomization, they daily received, in a double-blind fashion, either intranasal E2/NET [175 microg/275 microg (n = 47)] or oral E2/NETA [1 mg/0.5 mg (n = 43)]. Concentrations of high sensitivity CRP and adhesion molecules were measured at baseline and after 12, 24 and 52 weeks of treatment. RESULTS: CRP levels were increased (P = 0.001) in the oral but not in the intranasal group. The increase in the oral group was highest at week 12 (64.9%) and was larger (P < 0.01) compared with the non-significant increase (8.6%) found in the intranasal group. Both groups showed decreases (P < 0.001) in soluble vascular cell adhesion molecule (sVCAM), soluble intracellular adhesion molecule (sICAM) and sE-selectin. The decreases were larger (P < 0.01) in the oral than in the intranasal group. CONCLUSION: Intranasal E2/NET therapy did not significantly increase CRP levels, in contrast to the increase observed in the oral E2/NETA treatment group. Both intranasal and oral therapy lowered plasma concentrations of adhesion molecules, however, more so in the oral group.