A novel diselenide attenuates the carrageenan-induced inflammation by reducing neutrophil infiltration and the resulting tissue damage in mice.

Selenium-containing compounds have emerged as promising treatment for redox-based and inflammatory diseases. This study aimed to investigate the in vitro and in vivo anti-inflammatory activity of a novel diselenide named as dibenzyl[diselanediyIbis(propane-3-1diyl)] dicarbamate (DD). DD reacted with HOCl (k = 9.2 x 107 M-1s-1), like glutathione (k = 1.2 x 108 M-1s-1), yielding seleninic and selenonic acid derivatives, and it also decreased HOCl formation by activated human neutrophils (IC50=4.6 µM) and purified myeloperoxidase (MPO) (IC50=3.8 µM). However, tyrosine, MPO-I and MPO-II substrates, did not restore HOCl formation in presence of DD. DD inhibited the oxidative burst in dHL-60 cells with no toxicity up to 25 µM for 48h. Next, an intraperitoneal administration of 25, 50, and 75 mg/kg DD decreased total leukocyte, neutrophil chemotaxis, and inflammation markers (MPO activity, lipid peroxidation, albumin exudation, nitrite, TNF-α, IL-1ß, CXCL1/KC, and CXCL2/MIP-2) on a murine model of carrageenan-induced peritonitis. Likewise, 50 mg/kg DD (i.p.) decreased carrageenan-induced paw edema over 5h. Histological and immunohistochemistry analyses of the paw tissue showed decreased neutrophil count, edema area, and MPO, carbonylated, and nitrated protein staining. Furthermore, DD treatment decreased the fMLP-induced chemotaxis of human neutrophils (IC50=3.7 µM) in vitro with no toxicity. Lastly, DD presented no toxicity in a single-dose model using mice (50 mg/kg, i.p.) over 15 days and in Artemia salina bioassay (50 to 2000 µM), corroborating findings from in silico toxicological study. Altogether, these results demonstrate that DD attenuates carrageenan-induced inflammation mainly by reducing neutrophil migration and the resulting damage from MPO-mediated oxidative burst.

Uric Acid Reacts with Peroxidasin, Decreases Collagen IV Crosslink, Impairs Human Endothelial Cell Migration and Adhesion.

Uric acid is considered the main substrate for peroxidases in plasma. The oxidation of uric acid by human peroxidases generates urate free radical and urate hydroperoxide, which might affect endothelial function and explain, at least in part, the harmful effects of uric acid on the vascular system. Peroxidasin (PXDN), the most recent heme-peroxidase described in humans, catalyzes the formation of hypobromous acid, which mediates collagen IV crosslinks in the extracellular matrix. This enzyme has gained increasing scientific interest since it is associated with cardiovascular disease, cancer, and renal fibrosis. The main objective here was to investigate whether uric acid would react with PXDN and compromise the function of the enzyme in human endothelial cells. Urate decreased Amplex Red oxidation and brominating activity in the extracellular matrix (ECM) from HEK293/PXDN overexpressing cells and in the secretome of HUVECs. Parallelly, urate was oxidized to 5-hydroxyisourate. It also decreased collagen IV crosslink in isolated ECM from PFHR9 cells. Urate, the PXDN inhibitor phloroglucinol, and the PXDN knockdown impaired migration and adhesion of HUVECs. These results demonstrated that uric acid can affect extracellular matrix formation by competing for PXDN. The oxidation of uric acid by PXDN is likely a relevant mechanism in the endothelial dysfunction related to this metabolite.

Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence.

BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

Distinct metabolic patterns during microglial remodeling by oleate and palmitate.

Microglial activation by oleate and palmitate differentially modulates brain inflammatory status. However, the metabolic reprogramming supporting these reactive phenotypes remains unknown. Employing real-time metabolic measurements and lipidomic analysis, we show that both fatty acids promote microglial oxidative metabolism, while lipopolysaccharide (LPS) enhances glycolytic rates. Interestingly, oleate treatment was followed by enrichment in storage lipids bound to polyunsaturated fatty acids (PUFA), in parallel with protection against oxidative imbalance. Palmitate, in turn, induced a distinct lipid distribution defined by PUFA linked to membrane phospholipids, which are more susceptible to lipid peroxidation and inflammatory signaling cascades. This distribution was mirrored by LPS treatment, which led to a strong pro-inflammatory phenotype in microglia. Thus, although both oleate and palmitate preserve mitochondrial function, a contrasting lipid distribution supports differences in fatty acid-induced neuroinflammation. These data reinforce the concept that reactive microglial profiles are achieved by stimulus-evoked remodeling in cell metabolism.