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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. Comisión Sectorial de Investigación Científica. Proyecto de Investigación y Desarrollo CSIC I+D 2014. Agencia Nacional de Investigación e Innovación FCE_1_2017_1_136103

Synthesis, physicochemical, and biological characterization of a series of alpha-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.

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-AAMetNO 2 ). 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 AAMetNO 2 . Fragmentation of this ion yielded a daughter ion at m/z 317, corresponding to the neutral loss of the nitro group ([M + H − HNO 2 ] + ). Furthermore, IR signal at 1378 cm − 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-AAMetNO 2 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.

Postprint Synthesis and biological characterization of a series of 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 to prevent LDL oxidation.