apyrase and Diabetic-Angiopathies

Diabetes is an important risk factor for the development of cardiovascular disease including atherosclerosis and ischemic heart disease. Vascular complications including macro- and micro-vascular dysfunction are the leading causes of morbidity and mortality in diabetes. Disease mechanisms at present are unclear and no ideal therapies are available, which urgently calls for the identification of novel therapeutic targets/agents. An altered nucleotide- and nucleoside-mediated purinergic signaling has been implicated to cause diabetes-associated vascular dysfunction in major organs. Alteration of both purinergic P1 and P2 receptor sensitivity rather than the changes in receptor expression accounts for vascular dysfunction in diabetes. Activation of P2X

Topics: 5'-Nucleotidase; Adenosine Triphosphate; Animals; Apyrase; Atherosclerosis; Diabetes Mellitus; Diabetic Angiopathies; Erythrocytes; GPI-Linked Proteins; Humans; Receptors, Purinergic P1; Receptors, Purinergic P2X7; Retinal Vessels; Signal Transduction

Hyperglycemia is a recognized risk factor for cardiovascular disease in diabetes. Recently, we reported that high glucose activates the Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in arteries ex vivo. Here, we sought to determine whether hyperglycemia activates NFAT in vivo and whether this leads to vascular complications.. An intraperitoneal glucose-tolerance test in mice increased NFATc3 nuclear accumulation in vascular smooth muscle. Streptozotocin-induced diabetes resulted in increased NFATc3 transcriptional activity in arteries of NFAT-luciferase transgenic mice. Two NFAT-responsive sequences in the osteopontin (OPN) promoter were identified. This proinflammatory cytokine has been shown to exacerbate atherosclerosis and restenosis. Activation of NFAT resulted in increased OPN mRNA and protein in native arteries. Glucose-induced OPN expression was prevented by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. The calcineurin inhibitor cyclosporin A or the novel NFAT blocker A-285222 prevented glucose-induced OPN expression. Furthermore, diabetes resulted in higher OPN expression, which was significantly decreased by in vivo treatment with A-285222 for 4 weeks or prevented in arteries from NFATc3(-/-) mice.. These results identify a glucose-sensitive transcription pathway in vivo, revealing a novel molecular mechanism that may underlie vascular complications of diabetes.

Topics: Animals; Apyrase; Arteries; Binding Sites; Blood Glucose; Calcineurin; Calcineurin Inhibitors; Cyclosporine; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Disease Models, Animal; Enzyme Inhibitors; Female; Glucose Tolerance Test; Humans; Hyperglycemia; Jurkat Cells; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth, Vascular; NFATC Transcription Factors; Osteopontin; Promoter Regions, Genetic; Pyrazoles; RNA, Messenger; Signal Transduction; Time Factors; Transcriptional Activation; Transfection; Uridine Triphosphate

Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) (also known as CD39) is the dominant vascular ectonucleotidase. By hydrolyzing ATP and ADP to AMP, ENTPD1 regulates ligand availability to a large family of P2 (purinergic) receptors. Modulation of extracellular nucleotide metabolism is an important factor in several acute and subacute models of vascular injury. We hypothesized that aberrant nucleotide signaling would promote chronic glomerular injury in diabetic nephropathy. Inducing diabetes in ENTPD1-null mice with streptozotocin resulted in increased proteinuria and more severe glomerular sclerosis compared with matched diabetic wild-type mice. Diabetic ENTPD1-null mice also had more glomerular fibrin deposition and glomerular plasminogen activator inhibitor-1 (PAI-1) staining than wild-type controls. In addition, ENTPD1-null mice showed increased glomerular inflammation, in association with higher levels of monocyte chemoattractant protein-1 (MCP-1) expression. Mesangial cell PAI-1 and MCP-1 mRNA expression were upregulated by ATP and UTP but not ADP or adenosine in vitro. The stable nucleotide analog ATPgammaS stimulated sustained expression of PAI-1 and MCP-1 in vitro, whereas the stable adenosine analog NECA [5'-(N-ethylcarboxamido)adenosine] downregulated expression of both genes. Extracellular nucleotide-stimulated upregulation of MCP-1 is, at least in part, protein kinase C dependent. We conclude that ENTPD1 is a vascular protective factor in diabetic nephropathy that modulates glomerular inflammation and thromboregulation.

Topics: Animals; Antigens, CD; Apyrase; Creatinine; Diabetes Mellitus, Experimental; Diabetic Angiopathies; Diabetic Nephropathies; Glomerular Mesangium; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Polymerase Chain Reaction; Serum Albumin; Streptozocin

We have investigated expression and function of the P2X7 receptor in fibroblasts from healthy subjects and patients with type 2 diabetes.. Fibroblasts were isolated from skin biopsies. P2X7 receptor expression in both cell populations was measured by functional assays, RT-PCR, fluorescence-activated cell sorter, and immunoblotting. We found that fibroblasts from diabetic subjects are characterized by enhanced P2X7-mediated responses as indicated by increased shape changes, microvesiculation, enhanced fibronectin and interleukin 6 secretion, and accelerated apoptosis. These responses were blocked by preincubation with the P2X blockers KN-62, oxidized ATP, or pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid). Furthermore, we also found a higher level of spontaneous fibronectin secretion and of apoptosis in fibroblasts from diabetic compared with healthy subjects. Both higher basal level of fibronectin secretion and spontaneous rate of apoptosis were likely attributable to the increased pericellular concentration of ATP because fibroblasts from diabetic subjects released 3x as much ATP into the supernatants compared with fibroblasts from healthy subjects.. We conclude that fibroblasts from type 2 diabetes patients are characterized by a hyperactive purinergic loop based either on a higher level of ATP release or on increased P2X7 reactivity.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Diphosphate; Adenosine Triphosphate; Apoptosis; Apyrase; Autocrine Communication; Cell Shape; Cytidine Triphosphate; Diabetes Mellitus, Type 2; Diabetic Angiopathies; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Interleukin-6; Membrane Potentials; Paracrine Communication; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Uridine Diphosphate; Uridine Triphosphate