The HMG-CoA reductase inhibitor rosuvastatin and the angiotensin receptor antagonist candesartan attenuate atherosclerosis in an apolipoprotein E-deficient mouse model of diabetes via effects on advanced glycation, oxidative stress and inflammation.

AIMS/HYPOTHESIS: We evaluated the anti-atherosclerotic effect of the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor, rosuvastatin, and the angiotensin II receptor blocker (ARB), candesartan, alone and in combination, in the streptozotocin-induced diabetic apolipoprotein E-deficient (Apoe (-/-)) mouse. METHODS: Control and streptozotocin-induced diabetic Apoe (-/-) mice received rosuvastatin (5 mg kg(-1) day(-1)), candesartan (2.5 mg kg(-1) day(-1)), dual therapy or no treatment for 20 weeks. Aortic plaque deposition was assessed by Sudan IV staining and subsequent visual quantification. The abundance of proteins was measured using immunohistochemistry. RESULTS: Diabetes was associated with a fourfold increase in total plaque area. Rosuvastatin attenuated plaque area in diabetic mice in the absence of lipid-lowering effects. The anti-atherosclerotic effect of rosuvastatin was comparable to that observed with candesartan. A similar beneficial effect was seen with dual therapy, although it was not superior to monotherapy. Rosuvastatin treatment was associated with attenuated accumulation of AGE and AGE receptor (RAGE) in plaques. Similar beneficial effects on markers of oxidative stress were seen with the ARB and statin. Candesartan was more effective at reducing macrophage accumulation and collagen I abundance in plaques compared with rosuvastatin. The combined effect of candesartan and rosuvastatin was superior in reducing macrophage infiltration, monocyte chemoattractant protein-1 level, vascular AGE accumulation and RAGE abundance in the vascular wall. Furthermore, the combination tended to be more effective in reducing smooth muscle cell infiltration and connective tissue growth factor abundance in plaques. CONCLUSIONS/INTERPRETATION: Rosuvastatin has direct anti-atherosclerotic effects in diabetic macrovascular disease. These effects are independent of effects on lipids and comparable to the effects observed with candesartan.

Monocyte chemoattractant protein-1 has prosclerotic effects both in a mouse model of experimental diabetes and in vitro in human mesangial cells.

AIMS/HYPOTHESIS: Diabetic nephropathy is characterised by mesangial extracellular matrix accumulation. Monocyte chemoattractant protein-1 (MCP-1), a chemokine promoting monocyte infiltration, is upregulated in the diabetic glomerulus. We performed in vitro and in vivo studies to examine whether MCP-1 may have prosclerotic actions in the setting of diabetes, presumably via its receptor, chemokine (C-C motif) receptor 2 (CCR2), which has been described in mesangial cells. METHODS: Human mesangial cells were exposed to recombinant human (rh)-MCP-1 (100 ng/ml) for 12, 24 and 48 h and to rh-MCP-1 (10, 100 and 200 ng/ml) for 24 h. Fibronectin, collagen IV and transforming growth factor, beta 1 (TGF-beta1) protein levels were measured by ELISA and pericellular polymeric fibronectin levels by western blotting. The intracellular mechanisms were investigated using specific inhibitors for CCR2, nuclear factor kappa B (NF-kappaB), p38 mitogen-activated protein kinase and protein kinase C, and an anti-TGF-beta1 blocking antibody. In both non-diabetic and streptozotocin-induced diabetic mice that were deficient or not in MCP-1, glomerular fibronectin accumulation was examined by immunohistochemistry, while cortical Tgf-beta1 (also known as Tgfb1) and fibronectin mRNA and protein levels were examined by real-time PCR and western blotting. RESULTS: In mesangial cells, MCP-1 binding to CCR2 induced a 2.5-fold increase in fibronectin protein levels at 24 h followed by a rise in pericellular fibronectin, whereas no changes were seen in collagen IV production. MCP-1-induced fibronectin production was TGF-beta1- and NF-kappaB-dependent. In diabetic mice, loss of MCP-1 diminished glomerular fibronectin protein production and both renal cortical Tgf-beta1 and fibronectin mRNA and protein levels. CONCLUSIONS/INTERPRETATION: Our in vitro and in vivo findings indicate a role for the MCP-1/CCR2 system in fibronectin deposition in the diabetic glomerulus, providing a new therapeutic target for diabetic nephropathy.

Diabetic nephropathy: from mechanisms to rational therapies.

Diabetic nephropathy is a microvascular complication of diabetes. Specifically, it represents a major cause of morbidity and mortality in type 1 and type 2 diabetic subjects and has become the leading cause of end-stage renal disease in the Western world. Diabetic nephropathy appears to develop as a result of interactions between environmental insults and genetic susceptibility. Indeed, hyperglycemia is a clinical prerequisite for this complication, but it should be noted that only a subset of diabetic subjects will ultimately develop nephropathy. Over recent decades, cellular and molecular mechanisms underlying diabetic nephropathy have been increasingly delineated. In particular, diabetic kidney disease appears to occur as a result of the deleterious effects of both metabolic and hemodynamic insults, which at the cellular level lead to the activation of intracellular signaling pathways and transcription factors, thus triggering the production/release of cytokines, chemokines and growth factors, which mediate and/or amplify the renal damage. This ultimately leads to the structural and functional features characteristic of diabetic kidney disease. In the present review we summarize the evidence for key mediators of injury, which appear to be excellent treatment targets in diabetic nephropathy. The targets include various vasoactive hormones, the biochemical processes of the advanced glycation and protein kinase C. Furthermore, we review current and potentially new renoprotective therapies in the setting of diabetes.

Comparison between 24-h proteinuria, urinary protein/creatinine ratio and dipstick test in patients with nephropathy: patterns of proteinuria in dipstick-negative patients.

OBJECTIVE: Three main tests are commonly employed for the measurement of proteinuria: the dipstick test, the urinary protein/creatinine ratio (P/C) and the 24-h urine collection. The aim of this study was to evaluate the correlation between these methods, comparing linear regression and ROC curve data. MATERIAL AND METHODS: A total of 297 consecutive outpatients with different renal diseases were included in the study. Twenty-four-hour proteinuria was considered the reference test. RESULTS: A high degree of correlation was observed between all the tests (p<0.0001), the highest regression coefficient being between 24-h proteinuria and P/C (R=0.82), and the lowest between P/C and the dipstick test (R=0.72). The dipstick test failed to detect pathological proteinuria in 94 patients (31.6%). Therefore, in these subjects, the patterns of proteinuria were assessed by immunofixation and sodium dodecyl sulphate (SDS) electrophoresis. CONCLUSIONS: Our data strongly support the use of urinary P/C for the detection of proteinuria, at least in nephrology units, where the prevalence of proteinuria is likely to be high.

The MCP-1/CCR2 system has direct proinflammatory effects in human mesangial cells.

Both inflammatory and haemodynamic factors have been implicated in the pathogenesis of diabetic and other progressive glomerulopathies. Mesangial cell exposure to mechanical stretch induces both intercellular adhesion molecule 1 (ICAM-1) and monocyte chemoattractant protein-1 (MCP-1) expression. CC Chemokine receptor 2 (CCR2), the cognate MCP-1 receptor, has been recently demonstrated in human mesangial cells (HMCs). We tested whether MCP-1 binding to CCR2 affects ICAM-1 expression in HMCs and, secondly, if stretch-induced ICAM-1 is mediated by MCP-1 via an autocrine mechanism. Serum-deprived HMCs were exposed to either rh-MCP-1 (0.1-1-10-50-100 ng/ml) or mechanical stretch in the presence and in the absence of RS102895, a specific CCR2 inhibitor. ICAM-1 expression was assessed both by immunofluorescence and cytofluorimetry. Monocyte-HMC interaction was tested by adhesion assay. CCR2 expression was studied by reverse transcriptase-polymerase chain reaction, immunoblotting, and flow cytometry. HMCs exposure to rh-MCP-1 induced a significant twofold increase in ICAM-1 expression at 24 h, leading to enhanced monocyte adhesion. This effect occurred via the CCR2 receptor as CCR2 was expressed in HMCs and CCR2 blockade prevented ICAM-1 upregulation. Stretch-induced ICAM-1 expression was not altered by CCR2 blockade and stretch significantly reduced CCR2 mRNA and protein expression via an MCP-1-independent mechanism. In conclusion, stretch and MCP-1 independently induce ICAM-1 expression in HMCs. Stretch-induced CCR2 downregulation may favour MCP-1 paracrine activity.

Sex-differences in prevalence of electrocardiographic left ventricular hypertrophy in Type 2 diabetes: the Casale Monferrato Study.

UNLABELLED: AIMS Although left ventricular hypertrophy (LVH) defined by either standard 12-lead ECG or echocardiography strongly predicts cardiovascular mortality, its prevalence in Type 2 diabetes is largely unknown. We have assessed prevalence of ECG-LVH and its relationship with clinical and metabolic variables in an Italian population-based cohort of subjects with Type 2 diabetes. METHODS The study-base was 965 (61.3%) subjects with Type 2 diabetes of the population-based cohort living in Casale Monferrato (Italy). LVH was defined by ECG Cornell voltage-duration product. All measurements were centralized. RESULTS ECG-LVH was diagnosed in 165/965 subjects, giving a prevalence of 17.1% (95% CI 14.7-19.5). Large sex differences were found, with higher prevalence in women (23.5%, 19.9-27.0) than in men (8.4%, 5.6-11.0), even after adjustment for age, BMI and hypertension (OR 3.83, 95% CI 2.5-5.9). At the examination, subjects with ECG-LVH were older than those without it. Similar age- and sex-adjusted values of HbA(1c), plasma lipids, fibrinogen, uric acid and creatinine were found in the two subgroups. No differences in prevalence of hypertension, CHD, increased QT duration or dispersion, micro- and macro-albuminuria were found between subjects with ECG-LVH and those without it. In logistic regression analysis, variables independently associated with ECG-LVH, after age-adjustment, were sex and diastolic blood pressure. CONCLUSIONS: This population-based study shows: (i) a high prevalence of ECG-LVH in Type 2 diabetic subjects; (ii) 3-fold higher risk in women than in men, independently of age, BMI, and blood pressure; (iii) an independent association between ECG-LVH and diastolic blood-pressure. Screening for ECG-LVH in diabetic subjects is therefore recommended, particularly in diabetic women.

Autoantibody response to CD38 in Caucasian patients with type 1 and type 2 diabetes: immunological and genetic characterization.

Insulin secretion is one of the functions mediated by CD38, a nonlineage pleiotropic cell surface receptor. The molecule is the target of an autoimmune response, because serum autoantibodies (aAbs) to CD38 have been detected in diabetic patients. In the healthy Caucasian population, the CD38 gene is bi-allelic (86% CD38*B and 14% CD38*A), whereas an Arg140Trp mutation has been identified in Japanese diabetic patients. We investigated the relationship between CD38 and diabetes in Caucasian patients by characterizing anti-CD38 aAbs in terms of prevalence and function (agonistic/nonagonistic activity) and by exploring the potential influence of the CD38 genetic background. A novel enzymatic immunoassay, using recombinant soluble CD38 as the target antigen, was developed for the analysis of anti-CD38 aAb titers. Sera from 19.15% of type 1 and 16.67% of type 2 diabetic patients were positive. The majority of anti-CD38 aAbs (57.14%) displayed agonistic properties, i.e., they demonstrated the capability to trigger Ca2+ release in lymphocytic cell lines. In agreement with these functional features, the presence of anti-CD38 aAbs in type 2 diabetic patients was associated with significantly higher levels of fasting plasma C-peptide and insulin, as compared with anti-CD38-counterparts. No diabetic subject carrying the Arg140Trp mutation and no preferential association between diabetes or aAb status and the CD38*A allele was found in the study population. These results show the significance of anti-CD38 aAbs as a new diagnostic marker of beta-cell autoimmunity in diabetes. Moreover, the prevalent agonistic activity of these aAbs suggests that they could mediate relevant effects on target cells by means of Ca2+ mobilization.

Myocardial infarction before the age of 40 years is associated with insulin resistance.

Insulin resistance is associated with atherosclerosis, and hyperinsulinemia is predictive of coronary heart disease. However, a quantitative estimation of in vivo insulin sensitivity in juvenile myocardial infarction is still lacking and the mechanism of hyperinsulinemia is unknown. We estimated insulin sensitivity, beta-cell secretion, and hepatic insulin extraction using the minimal model analysis of a frequently sampled intravenous glucose tolerance test (FSIGT) in 25 normal-weight subjects without glucose intolerance and hypertension who had an acute myocardial infarction before the age of 40 years, and 10 control subjects comparable for age, sex, body mass index, and blood pressure. All patients underwent a coronary angiography. Insulin sensitivity was significantly lower in patients than in control subjects (mean +/- SEM, 4.6 +/- 0.6 v8.5 +/- 1.2 10(-4). min(-1)(microU/mL), P = .002). The basal C-peptide secretion rate (P = .02), total C-peptide secretion (P = .005), area under the curve (AUC) of insulin (P = .04) and C-peptide (P = .01), and hepatic insulin extraction (P = .04) were higher in patients versus control subjects. In conclusion, insulin resistance is evident in subjects with early myocardial infarction accurately selected to avoid the influence of other factors known to reduce insulin sensitivity, and hyperinsulinemia is due to an increase in beta-cell secretion rather than a decrease in hepatic insulin extraction.

Microvascular angina (cardiological syndrome X) per se is not associated with hyperinsulinaemia or insulin resistance.

BACKGROUND: Microvascular angina has been found to be associated with insulin resistance. However, many factors known to affect insulin sensitivity were not excluded in patient selection. We aimed to evaluate whether microvascular angina is per se associated with insulin resistance. MATERIALS AND METHODS: We performed a Frequently Sampled Intravenous Glucose Tolerance Test (0.33 g kg(-1) b.w.) in 10 normal weight and normotensive patients with microvascular angina, with normal glucose tolerance and normal plasma lipids. Ten healthy subjects, comparable for age, sex, body mass index, blood pressure and plasma lipids, were used as control group. RESULTS: Fasting serum glucose (4.49+/-0.2 SEM vs. 4.52+/-0.13 mmol L(-1), P = 0.9), insulin (39.46 +/-3.68 SEM vs. 47.12+/-4.6 pmol L(-1), P = 0.21) and C-peptide (0.56 +/-0.05 SEM vs. 0.53+/-0.05 nmol L(-1), P = 0. 68) values, as well as estimated parameters of insulin secretion and hepatic insulin extraction were similar in the two groups. Insulin sensitivity values (median, range) were also similar in the patients and control subjects (5.76 (3.39-12.30) vs. 7.54 (3.68-13.89. 10(-4) x min(-1)/(microU/mL), P = 0.97). CONCLUSION: Microvascular angina per se is not associated with hyperinsulinaemia or insulin resistance when other confounding factors are excluded in patient selection.

[Epidemiologic analysis of the incidence of new diabetic patients on dialysis as a basis for a clinical study]. / Analisi epidemiologica dell'incidenza dei nuovi ingressi diabetici in dialisi come base per uno studio clinico mirato.

BACKGROUND: Aim of this study is to analyse the incidence of diabetic patients starting dialysis in Piedmont (Italy) during the period 1981-1996 and to evaluate, in a subgroup of patients, the causes of uremia (diabetic nephropathy or other), and the type and seriousness of comorbid factors, in order to define the clinical conditions and try to explain the causes incidence increase. METHODS: Data are taken from the RPDT (Regional Registry of Dialysis and Transplantation of Piedmont). RESULTS: Total incidence of new patients starting dialysis in this Region increased from 65 pmp in 1981-1982 to 116 pmp in 1995-1996 and the mean age increased from 55.4 +/- 15.5 years in 1981-1982 to 61.5 +/- 15.6 in 1995-1996; 49% of patients had at least one of the 13 conditions of comorbidity considered by the Registry (including severe vascular, cardiac, systemic diseases and diabetes). In the same period the incidence of diabetic patients increased from 6 pmp (1981-1982) to 19 for males and 12 pmp for females (1995-1996); this increase is higher for males and limited to patients with age > or = 60 years (for example: group 70-79 years from 7 to 56 pmp). A study performed in a group of 64 patients (52 type 2 and 12 type 1) showed the incidence of multiple comorbid factors: the most important in type 2 are vascular diseases (44/52) and heart diseases (20/52); blindness and amputations are relatively rare (2 each). An important comorbid factor in type 1 diabetes is blindness (3/12) and in this group the most frequent cause of uremia is diabetic nephropathy (DN) (9/12); in the group of type 2 patients nephroangiosclerosis and a clinical picture of progressive renal failure, without nephrotic syndrome, represents 48% of all diagnoses. CONCLUSIONS: These results underline the necessity of a strict collaboration with diabetologists and of an improvement of dedicated structures in order to meet the increase of this critical population.

Apolipoprotein H is increased in type 2 diabetic patients with microalbuminuria.

BACKGROUND AND AIM: Serum apolipoprotein H (Apo H) levels are increased in hyperlipidemic subjects and type 2 diabetics, but unknown in microalbuminuria, another disorder with an increased cardiovascular risk. We looked to see whether increased Apo H levels are associated with microalbuminuria in type 2 diabetes. METHODS AND RESULTS: Serum Apo H was calculated in 20 normoalbuminuric and 17 microalbuminuric type 2 diabetics matched for age, sex, body mass index (BMI), glycosylated haemoglobin, plasma lipids and duration of diabetes, and also compared with 20 non-diabetic controls matched for age, sex, BMI and plasma lipids. Mean serum Apo H was significantly higher in the microalbuminuric patients (31.12 +/- 1.58 SEM vs 25.25 +/- 1.52 and 24.72 +/- 0.99 mg/dL, p = 0.003). CONCLUSION: Serum apo H levels are increased in type 2 diabetics with microalbuminuria.

Microalbuminuria in IDDM is associated with increased expression of monocyte procoagulant activity.

Microalbuminuria, the early phase of diabetic nephropathy, is associated with an increased risk of atherothrombosis. Monocytes play an important part in the pathogenesis of atherosclerosis and in the activation of haemostasis. However, procoagulant activity is poorly understood in Type I (insulin-dependent) diabetes mellitus, particularly in the presence of microalbuminuria. This study aimed to evaluate spontaneous and endotoxin-induced monocyte procoagulant activity in insulin-dependent diabetic patients with normoalbuminuria or microalbuminuria. Seventeen patients with microalbuminuria, 28 with normoalbuminuria and 26 healthy control subjects matched for age, sex, body mass index and smoking habit were studied. Mononuclear cells from peripheral venous blood were incubated with or without bacterial lypopolysaccharide. Spontaneous procoagulant activity and procoagulant activity after 3 h and 6 h of incubation were calculated. Spontaneous procoagulant activity values were similar in the three groups. After 3 h and 6 h incubation with bacterial lypopolysaccharide, procoagulant activity values were slightly, but not statistically significantly, higher in the normoalbuminuric diabetic group than in control group, and significantly higher in microalbuminuric diabetic group than in control group (p < 0.01). The increased endotoxin-induced monocyte procoagulant activity helps to explain the link between microalbuminuria and the increased risk of atherothrombosis in patients with Type I diabetes.

Single-sweep analysis using an autoregressive with exogenous input (ARX) model.

Single-sweep visual evoked potential analysis would be useful in clinical electrophysiology practice because it would make possible the evaluation of transient phenomena, but recording single-sweep visual evoked potentials is difficult because of the low signal-noise ratio. To increase this ratio we used a filter based on an autoregressive with exogenous input model. We studied a group of 12 diabetic patients matched with a control group of 14 normal subjects. The model, in most cases, allowed us to extrapolate the P100 component from each single sweep of visual evoked potential. The visual evoked potential values obtained by means of averaging were not significantly different in the groups studied, but single-sweep analysis showed different distribution of the P100 component amplitude. The preliminary results of our study evidenced differences in the amplitude and latency distribution of normal and diabetic subjects, thus confirming the power of this new technique and its ability to obtain some information that is masked by the averaging method.