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Aims: Despite vast clinical experience linking diabetes and atherosclerosis, the molecular mechanisms leading to accelerated vascular damage are still unclear. Here, we investigated the effects of nuclear factor of activated T-cells inhibition on plaque burden in a novel mouse model of type 2 diabetes that better replicates human disease. Methods & Results: IGF-II/LDLR-/-ApoB100/100 mice were generated by crossbreeding low-density lipoprotein receptor-deficient mice that synthesize only apolipoprotein B100 (LDLR-/-ApoB100/100) with transgenic mice overexpressing insulin-like growth factor-II in pancreatic β cells. Mice have mild hyperglycaemia and hyperinsulinaemia and develop complex atherosclerotic lesions. In vivo treatment with the nuclear factor of activated T-cells blocker A-285222 for 4 weeks reduced atherosclerotic plaque area and degree of stenosis in the brachiocephalic artery of IGF-II/LDLR-/- ApoB100/100 mice, as assessed non-invasively using ultrasound biomicroscopy prior and after treatment, and histologically after termination. Treatment had no impact on plaque composition (i.e. muscle, collagen, macrophages). The reduced plaque area could not be explained by effects of A-285222 on plasma glucose, insulin or lipids. Inhibition of nuclear factor of activated T-cells was associated with increased expression of atheroprotective NOX4 and of the anti-oxidant enzyme catalase in aortic vascular smooth muscle cells. Conclusion: Targeting the nuclear factor of activated T-cells signalling pathway may be an attractive approach for the treatment of diabetic macrovascular complications. [ABSTRACT FROM AUTHOR]

Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria. [ABSTRACT FROM AUTHOR]

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone with extrapancreatic effects beyond glycemic control. Here we demonstrate unexpected effects of GIP signaling in the vasculature. GIP induces the expression of the proatherogenic cytokine osteopontin (OPN) in mouse arteries via local release of endothelin-1 and activation of CREB. Infusion of GIP increases plasma OPN concentrations in healthy individuals. Plasma endothelin-1 and OPN concentrations are positively correlated in patients with critical limb ischemia. Fasting GIP concentrations are higher in individuals with a history of cardiovascular disease (myocardial infarction, stroke) when compared with control subjects. GIP receptor (GIPR) and OPN mRNA levels are higher in carotid endarterectomies from patients with symptoms (stroke, transient ischemic attacks, amaurosis fugax) than in asymptomatic patients, and expression associates with parameters that are characteristic of unstable and inflammatory plaques (increased lipid accumulation, macrophage infiltration, and reduced smooth muscle cell content). While GIPR expression is predominantly endothelial in healthy arteries from humans, mice, rats, and pigs, remarkable upregulation is observed in endothelial and smooth muscle cells upon culture conditions, yielding a "vascular disease-like" phenotype. Moreover, the common variant rs10423928 in the GIPR gene is associated with increased risk of stroke in patients with type 2 diabetes. [ABSTRACT FROM AUTHOR]

Aims: Sight-threatening diabetic retinopathy has been treated with photocoagulation for decades but the mechanisms behind the beneficial clinical effects are poorly understood. One target of irradiation and a potential player in this process is the retinal pigment epithelium (RPE). Here we establish an in vitro model for photocoagulation of human RPE cells. Methods: ARPE-19 cells were exposed to photocoagulation and studied at various time points up to 168h. Lesion morphology, necrosis and apoptosis were investigated by light microscopy; LIVE/DEAD staining and measurements of lactate dehydrogenase activity; and TUNEL- and ELISA-based quantification of DNA fragments, respectively. Cell migration and proliferation were explored using docetaxel and mitomycin C; temporal and spatial changes in proliferation were assessed by confocal immunofluorescence of proliferating cell nuclear antigen. Gene expression was measured by qPCR. Results: Photocoagulation of ARPE-19 resulted in denaturation of proteins and reproducible lesion formation. A transient peak in necrosis, followed by a peak in apoptosis was observed in cells within the lesions at 6h and 24h, respectively after photocoagulation. Cell proliferation was depressed during the first hours after photocoagulation, back to control levels at 24h and augmented in the following days. These effects were not limited to cells in the lesions, but also evident in neighbouring cells. Changes in cell proliferation during lesion repair were preceded by changes in cell migration. Altered mRNA expression of genes previously implicated in the regulation of cell proliferation (FOS, IL-1β, IL-8, HMGA2), migration and tissue repairing (TGFBR2, ADAMTS6, TIMP3, CTGF) was observed, as well as increased expression of the alarmin IL33 and the cytoprotective gene HSPA6. Conclusions: Using a laser system and experimental settings that comply with standards used in clinical practice, we have established a suitable model for in vitro photocoagulation of human RPE cells to isolate their contribution to the beneficial effects of laser treatment. [ABSTRACT FROM AUTHOR]

OBJECTIVE--The incretin hormone GIP (glucose-dependent insulinotropic polypeptide) promotes pancreatic β-cell function by potentiating insulin secretion and β-cell proliferation. Recently, a combined analysis of several genome-wide association studies (Meta-analysis of Glucose and Insulin-Related Traits Consortium [MAGIC]) showed association to postprandial insulin at the GIP receptor (GIPR) locus. Here we explored mechanisms that could explain the protective effects of GIP on islet function. RESEARCH DESIGN AND METHODS--Associations of GIPR rs10423928 with metabolic and anthropometric phenotypes in both nondiabetic (N = 53,730) and type 2 diabetic individuals (N = 2,731) were explored by combining data from 11 studies. Insulin secretion was measured both in vivo in nondiabetic subjects and in vitro in islets from cadaver donors. Insulin secretion was also measured in response to exogenous GIP. The in vitro measurements included protein and gene expression as well as measurements of β-cell viability and proliferation. RESULTS--The A allele of GIPR rs10423928 was associated with impaired glucose- mid GIP-stimulated insulin secretion and a decrease in BMI, lean body mass, and waist circumference. The decrease in BMI almost completely neutralized the effect of impaired insulin secretion on risk of type 2 diabetes. Expression of GIPR mRNA was decreased in human islets from carriers of the A allele or patients with type 2 diabetes. GIP stimulated osteopontin (OPN) mRNA and protein expression. OPN expression was lower in carriers of the A allele. Both GIP and OPN prevented cytokine-induced reduction in cell viability (apoptosis). In addition, OPN stimulated cell proliferation in insulin-secreting cells. CONCLUSIONS--These findings support β-cell proliferative and antiapoptotic roles for GIP in addition to its action as an incretin hormone. Identification of a link between GIP and OPN may shed new light on the role of GIP in preservation of functional β-cell mass in humans. [ABSTRACT FROM AUTHOR]

Aims: Despite vast clinical experience linking diabetes and atherosclerosis, the molecular mechanisms leading to accelerated vascular damage are still unclear. Here, we investigated the effects of nuclear factor of activated T-cells inhibition on plaque burden in a novel mouse model of type 2 diabetes that better replicates human disease., Methods & Results: IGF-II/LDLR -/- ApoB 100/100 mice were generated by crossbreeding low-density lipoprotein receptor-deficient mice that synthesize only apolipoprotein B100 (LDLR -/- ApoB 100/100 ) with transgenic mice overexpressing insulin-like growth factor-II in pancreatic β cells. Mice have mild hyperglycaemia and hyperinsulinaemia and develop complex atherosclerotic lesions. In vivo treatment with the nuclear factor of activated T-cells blocker A-285222 for 4 weeks reduced atherosclerotic plaque area and degree of stenosis in the brachiocephalic artery of IGF-II/LDLR -/- ApoB 100/100 mice, as assessed non-invasively using ultrasound biomicroscopy prior and after treatment, and histologically after termination. Treatment had no impact on plaque composition (i.e. muscle, collagen, macrophages). The reduced plaque area could not be explained by effects of A-285222 on plasma glucose, insulin or lipids. Inhibition of nuclear factor of activated T-cells was associated with increased expression of atheroprotective NOX4 and of the anti-oxidant enzyme catalase in aortic vascular smooth muscle cells., Conclusion: Targeting the nuclear factor of activated T-cells signalling pathway may be an attractive approach for the treatment of diabetic macrovascular complications.

Objective: Vascular inflammation is a key feature of both micro- and macrovascular complications in diabetes. Several lines of evidence have implicated the cytokine tumor necrosis factor (TNF) alpha as an important mediator of inflammation in diabetes. In the present study we evaluated the role of TNF alpha in streptozotocin (STZ)-induced diabetes on vascular inflammation in C57BL/6 wild-type and apoE-/- mice., Methods and Results: Diabetes increased the expression of vascular cell adhesion molecule (VCAM)-1 in cerebral arteries 150 m in diameter as well as the macrophage accumulation in aortic root atherosclerotic plaques in apoE-/- mice. A more pronounced vascular inflammatory response was observed in diabetic TNF alpha-deficient apoE-/- mice. These mice were also characterized by increased accumulation of IgG and IgM autoantibodies in atherosclerotic lesions. Diabetes also increased VCAM-1 expression and plaque formation in apoE-competent TNF alpha -/- mice, whereas no such effects were observed in C57BL/6 wild-type mice., Conclusions: The present findings suggest that TNF alpha does not mediate diabetic-induced vascular inflammation in mice and reveal an unexpected protective role for TNF alpha. These effects are partly attributable to a direct antiinflammatory role of TNF alpha, but may also reflect a defective development of the immune system in these mice.