VAS2870|722456-31-7|安捷凯

Background

VAS2870 is a selective inhibitor of the NADPH oxidases.^1,2,3,4 Pre-incubation of rat vascular smooth muscle cells with VAS2870 abolishes platelet-derived growth factor-dependent chemotaxis without affecting cell cycle progression (IC₅₀ = 10 ?M).¹ At low (2.8 mM), but not high (16.7 mM), concentrations of glucose, treatment with VAS2870 (20 ?M) increases glucose-stimulated insulin secretion of rat pancreatic islets by 70%.⁵ It significantly reduces production of reactive oxygen species in mouse brain, rat vascular smooth muscle culture, and human umbilical vein endothelial cells.^1,6,7 Treatment with VAS2870 pre- or post-ischemia is neuroprotective in mouse brain.⁶ VAS2870 inhibits vasculogenesis of mouse embryonic stem cell cultures and inhibits cell proliferation of rat hepatoma cells.^8,9

1.ten Freyhaus, H., Huntgeburth, M., Wingler, K., et al.Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferationCardiovasc. Res.71(2)331-341(2006) 2.Altenh?fer, S., Kleikers, P.W.M., Radermacher, K.A., et al.The NOX toolbox: validating the role of NADPH oxidases in physiology and diseaseCell. Mol. Life Sci.69(14)2327-2343(2012) 3.Wingler, K., Altenhofer, S.A., Kleikers, P.W.M., et al.VAS2870 is a pan-NADPH oxidase inhibitorCell. Mol. Life Sci.69(18)3159-3160(2012) 4.Wind, S., Beuerlein, K., Eucker, T., et al.Comparative pharmacology of chemically distinct NADPH oxidase inhibitorsBr. J. Pharmacol.161(4)885-898(2010) 5.Munhoz, A.C., Riva, P., Sim?es, D., et al.Control of insulin secretion by production of reactive oxygen species: Study performed in pancreatic islets from fed and 48-hour fasted Wistar ratsPLoS One11(6)e0158166(2016) 6.Kleinschnitz, C., Grund, H., Wingler, K., et al.Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegenerationPLoS One8(9)e1000479(2010) 7.Stielow, C., Catar, R.A., Muller, G., et al.Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cellsBiochem. Biophys. Res. Commun.344(1)200-205(2006) 8.Lange, S., Heger, J., Euler, G., et al.Platelet-derived growth factor BB stimulates vasculogenesis of embryonic stem cell-derived endothelial cells by calcium-mediated generation of reactive oxygen speciesCardiovasc. Res.81(1)159-168(2009) 9.Sancho, P., and Fabregat, I.The NADPH oxidase inhibitor VAS2870 impairs cell growth and enhances TGF-β-induced apoptosis of liver tumor cellBiochem. Pharmacol.81(7)917-924(2011)

Protocol

Kinase experiment:

NADPH oxidase activity is measured by lucigenin-enhanced chemiluminescence in a 50 mM phosphate buffer (buffer A), pH 7.0, containing 1 mM EGTA, protease inhibitors, 150 mM sucrose, 5 μM lucigenin, and 250 μM NADPH as substrate. Quiescent cells are starved by serum deprivation for 24 h and treated as indicated, ished twice with ice-cold phosphate buffered saline (PBS), pH 7.4, and harvested. After low spin centrifugation, the pellet is re-suspended in ice-cold buffer A, lacking lucigenin and substrate. Then, the cells are lysed and total protein concentration is determined using a Bradford assay and adjusted to 1 mg/mL. 100 μL aliquots of the protein sample are measured over 6 min in quadruplicates using NADPH (100 μM) as substrate in a scintillation counter. Data are collected at 2 min intervals in order to measure relative changes in NADPH oxidase activity[1].

Cell experiment:

To test autocrine growth, cells are serum deprived at 40% confluence and, when indicated, the NADPH oxidase inhibitors Apocynin (300 mM) or VAS2870 are added 30 min before serum deprivation and maintained along the experiment. For TGF-b experiments, cells at 70% confluence are serum deprived for 16 h and treated with 2 ng/mL TGF-β in the presence or absence of the EGFR inhibitor AG1478 (20 mM) or VAS2870 (25 mM), which are added 30 min prior to TGF-β[2].

参考文献:

[1]. ten Freyhaus H, et al. Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation. Cardiovasc Res. 2006 Jul 15;71(2):331-41.
[2]. Sancho P, et al. The NADPH oxidase inhibitor VAS2870 impairs cell growth and enhances TGF-β-induced apoptosis of liver tumor cells. Biochem Pharmacol. 2011 Apr 1;81(7):917-24.

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VAS2870

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Background

Protocol

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