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The pathogenesis of diabetic retinopathy (DR) remains unclear but hyperglycemia is an established risk factor. Endothelial dysfunction and changes in Ca2+ signaling have been shown to precede the onset of DR. We recently demonstrated that high extracellular glucose activates the Ca2+/calcineurin-dependent transcription factor NFAT in cerebral arteries and aorta, promoting the expression of inflammatory markers. Here we show, using confocal immunofluorescence, that NFAT is expressed in the endothelium of retinal microvessels and is readily activated by high glucose. This was inhibited by the NFAT blocker A-285222 as well as by the ectonucleotidase apyrase, suggesting a mechanism involving the release of extracellular nucleotides. Acute hyperglycemia induced by an IP-GTT (intraperitoneal glucose tolerance test) resulted in increased NFATc3 nuclear accumulation and NFAT-dependent transcriptional activity in retinal vessels of NFAT-luciferase reporter mice. In both Akita (Ins2+/− ) and streptozotocin- (STZ-) induced diabetic mice, NFAT transcriptional activity was elevated in retinal vessels. In vivo inhibition of NFAT with A-285222 decreased the expression of OPN and ICAM-1 mRNA in retinal vessels, prevented a diabetes driven downregulation of anti-inflammatory IL-10 in retina, and abrogated the increased vascular permeability observed in diabetic mice. Results identify NFAT signaling as a putative target for treatment of microvascular complications in diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Nitro-fatty acids represent endogenously occurring products of oxidant-induced nitration reactions. We have previously synthesized a mixture of four isomers of nitroarachidonic acid, a novel anti-inflammatory signaling mediator. In this study, we synthesized and chemically and biologically characterized for the first time an esterified nitroalkene derived from the nitration of methylarachidonate (AAMet): 6-methylnitroarachidonate (6-AAMetNO2). Synthesis was performed by reacting AAMet with sodium nitrite under acidic conditions. Analysis by mass spectrometry (positive-ion ESI-MS) showed an [M+H]+ ion of m/z 364, characteristic of AAMetNO2. Fragmentation of this ion yielded a daughter ion at m/z 317, corresponding to the neutral loss of the nitro group ([M+H−HNO2]+). Furthermore, IR signal at 1378cm−1 and NMR data confirmed the structure of a 6-nitro-positional isomer. This novel esterified nitroalkene was capable of promoting vascular protective actions including: (a) the induction of vasorelaxation via endothelium-independent mechanisms, associated with an increase in smooth muscle cell cGMP levels, and (b) a potent dose-dependent inhibition of human platelet aggregation. We postulate that 6-AAMetNO2 could be a potential drug for the prevention of vascular and inflammatory diseases, and the presence of the methyl group may increase its pharmacological potential. [ABSTRACT FROM AUTHOR]

Abstract: Synthesis, physicochemical, and biological characterization of a series of α-tocopherol mimetics with NO-releasing capacity are reported. The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities, vasodilating properties and mammal cytotoxic activities. The lipophilic–hydrophilic balance of all products was also evaluated. A new hybrid furoxan, phenol derivative 17, possesses adequate profile of the studied properties. [Copyright &y& Elsevier]

Perivascular pericytes are key regulators of the blood–brain barrier, vascular development, and cerebral blood flow. Deciphering pericyte roles in health and disease requires cellular tracking; yet, pericyte identification remains challenging. A previous study reported that the far‐red fluorophore TO‐PRO‐3 (642/661), usually employed as a nuclear dye in fixed tissue, was selectively captured by live pericytes from the subventricular zone. Herein, we validated TO‐PRO‐3 as a specific pericyte tracer in the nervous system (NS). Living pericytes from ex vivo murine hippocampus, cortex, spinal cord, and retina robustly incorporated TO‐PRO‐3. Classical pericyte immunomarkers such as chondroitin sulphate proteoglycan neuron‐glial antigen 2 (NG2) and platelet‐derived growth factor receptor beta antigen (PDGFrβ) and the new pericyte dye NeuroTrace 500/525 confirmed cellular specificity of dye uptake. The TO‐PRO‐3 signal enabled quantification of pericytes density and morphometry; likewise, TO‐PRO‐3 labeling allowed visualization of pericytes associated with other components of the neurovascular unit. A subset of TO‐PRO‐3 stained cells expressed the contractile protein α–SMA, indicative of their ability to control the capillary diameter. Uptake of TO‐PRO‐3 was independent of connexin/pannexin channels but was highly sensitive to temperature and showed saturation, suggesting that a yet unidentified protein‐mediated active transport sustained dye incorporation. We conclude that TO‐PRO‐3 labeling provides a reliable and simple tool for the bioimaging of pericytes in the murine NS microvasculature. [ABSTRACT FROM AUTHOR]

Objective of the Study: Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis. Methodology and Principal Findings: Streptozotocin (STZ)-induced diabetes in apolipoprotein E−/− mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice. Conclusions: Targeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications. [ABSTRACT FROM AUTHOR]

Abstract: Synthesis and biological characterization of a series of α-tocopherol analogs with NO-releasing capacity are reported. The selected NO-donor moieties were nitrooxy and furoxan. All products were tested for their in vitro NO-releasing capacities, vasodilating properties, and antiplatelet activity. They were also capable of preventing LDL oxidation. [Copyright &y& Elsevier]

Nitrated fatty acids (nitroalkenes) have been recently detected and quantified in cell membranes and human plasma. However, nitration of arachidonate (AA), that could redirect AA-dependent cell signaling pathways, has not been studied in detail. Herein, we synthesized and determined for the first time the isomer distribution of nitroarachidonate (AANO2) and demonstrate its ability to modulate inflammation. Synthesis of AANO2 was achieved by AA treatment with sodium nitrite in acidic conditions following HPLC separation. Mass spectrometry (MS) analysis showed the characteristic MS/MS transition of AANO2 (m/z 348/301). Moreover, the IR signal at 1378.3 cm-1 and NMR studies confirmed the presence of mononitrated nitroalkenes. Positional isomer distribution was determined by NMR and MS fragmentation with lithium; four major isomers (9-, 12-, 14-, and 15-AANO2) were identified, which exhibited key anti-inflammatory properties. These include their ability to release biologically relevant amounts of nitric oxide, induce cGMP-dependent vasorelaxation, and down-regulate inducible nitric oxide synthase (NOS2) expression during macrophage activation, providing unique structural evidence and novel regulatory signaling properties of AANO2. [ABSTRACT FROM AUTHOR]