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Aims: Platelets are pivotal in arterial thrombosis, and platelet hyperresponsiveness may contribute to the increased incidence of cardiovascular events in diabetes mellitus. Consequently, we hypothesized that increased in vitro platelet aggregation responses exist in men with diabetes mellitus. Methods: The Danish Cardiovascular Screening Trial (DANCAVAS) is a community-based cardiovascular screening trial including men aged 65–74 years. Platelet aggregation was tested using 96-well light transmission aggregometry with thrombin receptor-activating peptide (TRAP), adenosine diphosphate, collagen type 1, arachidonic acid and protease-activated receptor-4 in three concentrations. Further, cardiovascular risk factors and coronary artery calcification (CAC), estimated by CT scans and ankle–brachial index, were obtained. Results: Included were 720 men aged 65–74 years, 110 with diabetes mellitus. Overall, there was no difference in platelet aggregation among men with versus without diabetes mellitus when adjusting for or excluding platelet inhibitor treatment and men with established cardiovascular disease (CVD). This was true for all agonists, e.g., 10 µM TRAP-induced platelet aggregation of median 69% (IQR 53–75) versus 70% (IQR 60–76) in men with versus without diabetes mellitus. Platelet aggregation did not correlate with HbA1c or CAC. Men with diabetes mellitus displayed higher CAC, median 257 Agatston units (IQR 74–1141) versus median 111 Agatston units (IQR 6–420) in the remaining individuals, p < 0.0001. Conclusions: Among outpatients with diabetes mellitus, but no CVD and no platelet inhibitor treatment, neither are platelets hyperresponsive in diabetes mellitus, nor is platelet aggregation associated with glycemic status or with the degree of coronary atherosclerosis. Trial Registration: ISRCTN12157806.

AIMS/HYPOTHESIS: Accelerated migration and proliferation of vascular smooth muscle cells (VSMCs) enhances arterial restenosis after angioplasty in insulin resistance and diabetes. Elevation of Src homology 2-containing protein tyrosine phosphatase 1 (SHP-1) induces apoptosis in the microvasculature. However, the role of SHP-1 in intimal hyperplasia and restenosis has not been clarified in insulin resistance and diabetes.METHODS: We used a femoral artery wire injury mouse model, rodent models with insulin resistance and diabetes, and patients with type 2 diabetes. Further, we modulated SHP-1 expression using a transgenic mouse that overexpresses SHP-1 in VSMCs (Shp-1-Tg). SHP-1 agonists were also employed to study the molecular mechanisms underlying the regulation of SHP-1 by oxidised lipids.RESULTS: Mice fed a high-fat diet (HFD) exhibited increased femoral artery intimal hyperplasia and decreased arterial SHP-1 expression compared with mice fed a regular diet. Arterial SHP-1 expression was also decreased in Zucker fatty rats, Zucker diabetic fatty rats and in patients with type 2 diabetes. In primary cultured VSMCs, oxidised LDL suppressed SHP-1 expression by activating Mek-1 (also known as Map2k1) and increased DNA methylation of the Shp-1 promoter. VSMCs from Shp-1-Tg mice exhibited impaired platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation with a concomitant decrease in PDGF-stimulated VSMC proliferation and migration. Similarly, HFD-fed Shp-1-Tg mice and mice treated with the SHP-1 inducer, Icariside II, were protected from the development of intimal hyperplasia following wire injury.CONCLUSIONS/INTERPRETATION: Suppression of SHP-1 by oxidised lipids may contribute to the excessive VSMC proliferation, inflammatory cytokine production and intimal hyperplasia observed in arteries from diabetes and insulin resistance. Augmenting SHP-1 levels is a potential therapeutic strategy to maintain stent patency in patients with insulin resistance and diabetes.

Background: Heart failure (HF) patients with diabetes (DM) have an adverse prognosis and reduced functional capacity, which could be associated with cardiac fibrosis, increased chamber stiffness and reduced left ventricular (LV) contractile reserve. Galectin-3 (Gal-3) and fibulin-1 are circulating biomarkers potentially reflecting cardiac fibrosis. We hypothesize that plasma levels of Gal-3 and fibulin-1 are elevated in HF patients with DM and are associated with reduced LV contractile reserve in these patients. Methods: A total of 155 patients with HF with reduced ejection fraction underwent a low-dose dobutamine echocardiography and blood sampling for biomarker measurements. Patients were classified according to history of DM and an oral glucose tolerance test (OGTT) as: normal glucose tolerance (NGT) (n = 70), impaired glucose tolerance (IGT) (n = 25) and DM (n = 60). Results: Galectin-3 levels were elevated in DM patients as compared to non-diabetic patients (P = 0.02), while higher fibulin-1 levels were observed in HF patients with IGF and DM (P = 0.07). Reduced LV contractile reserve was associated with increasing Gal-3 levels (β = -0.19, P = 0.03) although, this association was attenuated after adjustment for estimated glomerular filtration rate (P = 0.66). Fibulin-1 was not associated with LV contractile reserve (P = 0.71). Conclusions: Galectin-3 and fibulin-1 levels were elevated in HF patients with impaired glucose metabolism. However, reduced LV contractile reserve among HF patients with DM does not to have an independent impact on plasma Gal-3 and fibulin-1 levels.

Udgivelsesdato: 2009-Dec Calcification of the media of arterial walls is common in diabetes and is particularly associated with distal symmetrical neuropathy. Arterial calcification also complicates chronic kidney disease and is an independent risk factor for cardiovascular and all-cause mortality. The term calcification is not strictly accurate because the morphological changes incorporate those of new bone formation, i.e. ossification. The processes are complex, but are closely related to those involved in bone homeostasis, and it is relevant that calcification of the arterial wall and osteopenia often co-exist. One particular factor linked to the development of arterial calcification is distal symmetrical neuropathy; indeed, it has been suggested that neuropathy explains the distal distribution of arterial calcification in diabetes. It has also been suggested that the link with neuropathy results from loss of neuropeptides, such as calcitonin gene-related peptide, which are inherently protective. The association between distal symmetrical neuropathy and calcification of the arterial wall highlights the fact that neuropathy may be an independent risk factor for cardiovascular mortality.

Udgivelsesdato: 2007-Aug The bone-related protein osteoprotegerin (OPG) may be involved in the development of vascular calcifications, especially in diabetes, where it has been found in increased amounts in the arterial wall. Experimental studies suggest that members of the TGF-superfamily are involved in the transformation of human vascular smooth muscle cells (HVSMC) to osteoblast-like cells. In this study, we evaluated the effect of BMP-2, BMP-7 and transforming growth factor beta (TGF-beta1) on the secretion and mRNA expression of OPG and its ligands receptor activator of nuclear factor-kappabeta ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL) in HVSMC. All three growth factors decreased OPG protein production significantly; these results were paralleled by reduced OPG mRNA expression. TRAIL mRNA levels were also decreased. RANKL mRNA expression declined when treated with TGF-beta1 but were increased by both BMPs. Members of the TGF-superfamily, i.e. TGF-beta1, BMP-2 and BMP-7 exert effects on OPG and its ligands, indicating that these peptides may be involved in the development of vascular calcifications. The downregulation of OPG by these peptides does, however, not suggest that these factors are directly involved in OPG accumulation in diabetes.

Introduction: Arterial stiffening is associated with structural and biomechanical alterations in the aorta. However, there are still gaps in our understanding as to how the structure and properties of arteries across the vasculature are altered with high PWV.Objective: To determine whether altered ultrastructural and nanomechanical properties are exhibited in the internal mammary artery (IMA) in high PWV patients. Methods: Human IMA biopsies were obtained from patients with known carotid-femoral PWV. Patients were grouped as low PWV (8.5 ± 0.7 ms−1, n = 8) and high PWV (13.4 ± 3.0 ms−1, n = 9). With Peakforce QNM atomic force microscopy (AFM) the nanomechanical (elastic modulus) and morphological properties (collagen fibril diameter and D-Period) of the IMA were measured. Principal component analysis (PCA) was used to determine the relationship of nanomechanical and structural data with proteomics data (small leucine rich proteoglycans, SLRPs) [1] and patient metadata. Results: PCA analysis shows that the nano-scale elastic modulus was one of the key variables which separated low and high PWV groups and was correlated with PWV. Furthermore, nano-scale alterations in adventitial collagen fibrils were evident. D-Period and collagen fibril diameter were found to be negatively correlated. Most SLRPs were closely grouped in the PCA analysis. Conclusions: Although the IMA is not involved in the carotid-femoral pathway, patients with high PWV exhibited distinct alterations in the IMA at the nano-scale relative to those with low PWV. Our approach provides new insight into systemic structure-property changes in the vasculature, and also provides a novel method for characterizing small biopsy samples for arterial stiffening studies.

Previous studies have shown that biomechanical analysis of aorta from diabetic subjects reveals a marked increase in stiffness compared to aorta from age-matched control subjects. In the present paper we have proposed that this increased stiffness can be attributed to glycation-induced inter-molecular cross-links based on a direct analysis of the two known glycation cross-links, the fluorescent pentosidine and the non-fluorescent NFC-1. There was a significant difference in the increase in concentration of both cross-links with increasing age for both the intima (p0.0025) and the media (p0.0005) from the diabetic compared to the control subjects, but no correlation with the mature enzymic cross-link hy droxylysyl-pyridinoline. Finally, we have obtained a significant correlation of stiffness with both glycation cross-links (NFC-1, r = 0.86; p0.005 and pentosidine r = 0.75, p0.05), but the concentration of NFC-1 is about 50 times greater than that of pentosidine, indicating that it is the major glycation cross-link responsible for the stiffening of the aorta.

An insertion(I)/deletion(D) polymorphism in the angiotensin I-converting enzyme (ACE) gene seems to be associated with clinical heart disease in patients with diabetes mellitus. It is not known whether increased atherosclerosis or other factors among individuals with certain ACE-gene subtypes form the basis for the increased prevalence of heart disease among these subjects. We measured, at autopsy, the extent of macroscopically visible aortic atherosclerosis in 22 diabetic and 39 non-diabetic subjects and determined the ACE-genotype of all individuals by the polymerase chain reaction. The percentage of aortic surface area covered with atherosclerotic lesions was 29 +/- 8 (n = 6), 71 +/- 7 (n = 9), and 65 +/- 7 (n = 5) in the II-, ID-, and DD-genotype subgroups, respectively, among diabetes patients (mean +/- SEM) (2 p0.01, when comparing values from the ID and DD groups to the II group). The values were 37 +/- 9 (n = 11), 40 +/- 5 (n = 14) and 37 +/- 6 (n = 11) in the II-, ID-, and DD-genotypes in the non-diabetic group. There were no differences in sex ratio or age in any of the ACE-gene subtypes. The previously described relationship between heart disease and the ACE-gene polymorphism in diabetes could thus be founded in an increased extent of atherosclerosis among patients with the ID- and DD-ACE-gene subtypes. Patients with diabetes have several alterations in the composition of the collagenous components in the arterial wall. We also analysed for associations between total collagen and type IV and type V collagen content in the aortic vessel wall and the ACE-gene subtypes. We were, however, not able to disclose correlations between the polymorphism and any of these parameters. In conclusion, our data show an association between the ACE-I/D polymorphism and the degree of aortic atherosclerosis in diabetes; however, we did not observe correlations between the polymorphism and data concerning arterial collagenous components.

Background: Arterial stiffening, occurring as part of the natural aging process of the artery, is well- established as a powerful predictor of cardiovascular disease. However, little is known about how localised changes in the extracellular matrix and mechanical properties of arterial tissue contribute to gross stiffening in the vasculature, particularly in the adventitia. The mechanical properties of the adventitia are attributed to the collagen fibrils which exhibit high tensile strength when an axial load is placed on the vessel. Objective: To determine the relationship between the adventitial collagen fibril properties and carotid-femoral pulse wave velocity (PWV). Methods: 16 patients were split into high PWV (13.6±1.1ms−1) and low (8.5±0.3ms−1) PWV groups (t-test, P < 0.001). Internal mammary arteries (IMAs) which were collected during coronary artery bypass grafting (CABG) were used to nano-scale characterisation of the tissue with atomic force microscopy (AFM). AFM was used to determine nanomechanical properties and collagen fibril morphology. Results: Abundant, highly oriented collagen fibrils were observed in the adventitial layer in both groups. The adventitia had high elastic modulus values in the high PWV group (Low PWV = 2298.64±75.38MPa; High PWV = 2734.63±95.52MPa, P < 0.001). The collagen fibril diameters were found to be higher in patients with high PWV (Low PWV = 117.23±22.19nm, High PWV = 119.18±21.96nm, P < 0.001). Conclusion: Nanomechanical properties and collagen fibril morphology in arterial tissue associated with carotid-femoral PWV. Nano-scale changes in the IMA are therefore indicative of systematic changes in arterial stiffness in the vasculature.

Alterations in the connective tissue of the arterial wall have been suggested to play a role in the development of macrovascular disease in diabetes mellitus. The present study deals with changes in the content of GAG in aortic tunica media in human diabetes by separately analysing normal areas and areas with fibrous plaques. The thoracic aorta from 15 diabetic patients (7 with IDDM, 8 with NIDDM), and 30 sex- and age-matched non-diabetic subjects were collected at autopsy. Tunica intima was removed and GAG were isolated from the dried defatted and pulverized tunica media. GAG were quantified by uronic acid analysis and characterized by electrophoresis on cellulose acetate. Results showed that IDDM patients had a relative and absolute increase in hyaluronic acid in normal tunica media compared to non-diabetic subjects. There was a significant positive correlation between hyaluronic acid content of normal tunica media and duration of diabetes, but not between hyaluronic acid content and age. When tunica media from plaque areas was compared to normal areas the same pattern was evident in diabetic patients as in non-diabetic patients--significantly increased proportion of dermatan sulphate and reduced hyaluronic acid. The data agree with the notion that the arterial wall is subject to different pathological processes in diabetes, one of classical atherosclerosis with changes in GAG similar to non-diabetic subjects, and the other seen in areas without plaques with dissimilar alterations in GAG. These data therefore support the concept of the presence of a macrovascular disease in diabetes different from atherosclerosis.

Proteins are the main molecular components of the arterial wall. Alterations in the amounts of specific proteins in both the extracellular matrix and in vascular cells are believed to be associated with different arterial pathologies, however only sparse data is currently available, particularly in relation to human arteries. Proteome analysis is large scale analysis of the quantity of many proteins in a single analytical run from biological samples. Combining “state of the art” proteome analysis by LC-MS (liquid chromatography-mass spectrometry) with access to samples from a large human artery biobank, we have obtained knowledge about protein changes in arteries from patients with various cardiovascular conditions. Specific alterations in matrix proteins are for example present in relation to increased arterial stiffness and to diabetes, whereas alterations in non-matrix proteins are associated with the growth rate of aortic aneurysms. Such new knowledge about changes of arterial proteins in specific vascular conditions can direct our attention towards pathophysiological understandings and display routes to new potential treatment targets and novel biomarkers for arterial diseases.

Aim: Arterial stiffening occurs as part of the natural ageing process, and is thought to be related to the accumulation of collagen and degradation of elastin. However, little is known about how regional variations in arterial structure and mechanical properties contribute to arterial stiffening. This study compared localised differences in the nano-structure and mechanical properties in the internal mammary arteries (IMA) from patients with high and low PWV. Methods: 6 IMAs were collected from coronary bypass operations and the patients were grouped according to their carotid-femoral PWV; high (14.6 ± 1.4 m/s) and low (8.7 ± 0.5 m/s). The nano-topography and elastic modulus were determined by atomic force microscopy (AFM) using 5 μm cryosections. Data are presented as means ± SEMs. Results: Overall, IMAs in the high PWV group were significantly stiffer than in the low PWV group (High; 2234.7 ± 72.3 MPa, Low; 2015.3 ± 58.4 MPa), (p < 0.0001). Although no significant difference was found in the intimal or medial layers, the adventitia was stiffer in the high PWV group (High; 2597.6 ± 135.7 MPa, Low;2215.6 ± 110.2 MPa), (p < 0.001). Furthermore, the collagen fibrils in the adventitia of the high PWV group were found to have a smaller diameter (High; 118.44 ± 1.1 nm, Low; 123.81 ± 1.3 nm), (p < 0.01)). Conclusion: Overall, the nanomechanical data associates with PWV data. The high PWV group exhibited higher nanomechanical stiffness alongside morphological alterations within the adventitial layer.

Objectives: Fibulin-1 is an extracellular matrix protein that is increased in the arterial wall in diabetes. It is also present in plasma, where it is positively related to blood pressure and predicts mortality. In addition, plasma levels are associated with heart stiffness indices. The aim of this study was to evaluate the relation between plasma fibulin-1 and vascular diseases such as aortic abdominal aneurysm (AAA) and peripheral atherosclerotic disease (PAD). Methods: Plasma levels of fibulin-1 were measured by ELISA in patients from an ongoing population-based screening trial for AAA, PAD and hypertension in men aged 65–74 years. Samples were obtained consecutively from 477 patients with AAA, 120 patients with PAD and AAA and 197 age-matched controls. AAA is defined as having a maximal aortic diameter > 30 mm and PAD as an ankle-brachial index (ABI) < 0.90. Results: Plasma fibulin-1 in patients with AAA (86.2 μg/ml ± 1.01 μg/ml, mean±SEM) and controls (81.6 μg/ml. SEM ± 1.01 μg/ml) were significantly different (p=0.004), OR of 5.62 (95% C.I.: 2.29–13.79, p

Objective Plasma osteoprotegerin (P-OPG) is an independent predictor of cardiovascular disease in diabetic and other populations. OPG is a bone-related glycopeptide produced by vascular smooth muscle cells and increased P-OPG may reflect arterial damage. We investigated the correlation between P-OPG and coronary artery disease (CAD) in asymptomatic type 2 diabetic patients with microalbuminuria. Methods P-OPG was measured in 200 asymptomatic diabetic patients without known cardiac disease. Patients with P-NT-proBNP >45.2 ng/l and/or coronary calcium score (CCS) ≥400 were stratified as high risk of CAD (n = 133), and all other patients as low risk patients (n = 67). High risk patients were examined by myocardial perfusion imaging (MPI; n = 109), and/or CT-angiography (n = 20), and/or coronary angiography (CAG; n = 86). Significant CAD was defined by presence of significant myocardial perfusion defects at MPI and/or >70% coronary artery stenosis at CAG. Results Significant CAD was demonstrated in 70 of the high risk patients and of these 23 patients had >70% coronary artery stenosis at CAG. Among high risk patients, increased P-OPG was an independent predictor of significant CAD (adjusted odds ratio [CI] 3.11 [1.01-19.54] and 3.03 [1.00-9.18] for second and third tertile vs.first tertile P-OPG, respectively) and remained so after adjustments for NT-proBNP and CCS. High P-OPG was also associated with presence of >70% coronary artery stenosis(adjusted odds ratio 14.20 [1.35-148.92] for third vs. first tertile P-OPG), and 91% of patients with low (first tertile) P-OPG did not have >70% coronary artery stenosis. Conclusions Elevated P-OPG is an independent predictor of the presence of CAD in asymptomatic type 2 diabetic patients with microalbuminuria.

In 14 patients with arterial hypertension secondary to chronic renal parenchymal disease and impaired renal function, 24-h ambulatory and casual blood pressure readings plasma, angiotensin II, aldosterone, arginine vasopressin and atrial natriuretic peptide, creatinine clearance, plasma lipids and lipoproteins, and body weight were determined after consecutive 3-week periods on placebo and sustained release verapamil 240 mg/day. Verapamil reduced the mean 24-h ambulatory blood pressure from 152/104 to 142/97 mm Hg. Blood pressure was significantly reduced during the daytime and the evening, but not at night. Casual blood pressure was also significantly reduced from 176/106 mm Hg to 154/96 mm Hg. No significant changes were found in the hormones, creatinine clearance, plasma lipids and lipoproteins, heart rate or body weight. The atrial natriuretic peptide level was significantly correlated with the calculated creatinine clearance (r = -0.797). Thus, sustained release verapamil 240 mg as a single daily dose had a moderate hypotensive effect in patients with chronic renal disease without inducing tachycardia, activation of the renin-angiotensin-aldosterone system, or increasing body weight, and without altering renal function and plasma lipids and lipoproteins. The negative correlation between atrial natriuretic peptide and glomerular filtration rate supports the hypothesis that the extracellular volume increases during progression of renal disease.

The biomechanical properties of aortic samples from patients with Type 1 (insulin-dependent) diabetes mellitus and age- and sex-matched control subjects were analysed using a materials testing machine. The specimens were prepared from tissue outside areas of visible atherosclerosis in order to discriminate between primary Type 1 diabetic alterations in the aortae and secondary changes due to increased atherosclerosis. We paid special attention to the correction of biomechanical parameters for differences in wall thickness and registration of specimen length values. In the Type 1 diabetic aortae a marked reduction was found in the extensibility and an increase in their stiffness. The reduced extensibility was correlated significantly to the duration of Type 1 diabetes. The pronounced alterations in the mechanical properties could not be explained by the increase in the wall thickness which was observed among the Type 1 diabetic patients and the alterations could not be correlated to the grade of atherosclerosis in the thoracic aorta. The results of the present study, therefore, strongly suggest that Type 1 diabetic patients develop alterations in the arterial connective tissue independent of the presence of atherosclerosis. Such primary alterations in the vessel wall may play a role in the pathogenesis of large vessel disease among these patients.