and G.E.M. ischemic stroke, bEnd.3?cells were adapted to 18 or 5?kPa O2 and subjected to hypoxia (1?kPa O2, 1?h) and reoxygenation. In cells adapted to 18?kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5?M). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX? Red and FeRhoNox?-1, was diminished in cells cultured under 5?kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling PLX5622 pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require studies conducted under well-defined O2 levels. findings. We recently reported that SFN mediated induction of select Nrf2 Rabbit Polyclonal to CAMK5 target genes in umbilical vein endothelial cells (HUVEC) is usually attenuated under physiological normoxia (5?kPa O2) compared to atmospheric O2 levels [22]. Moreover, we reported that adaptation of HUVEC to 5?kPa O2 enhances nitric oxide bioavailability, modulates agonist-induced Ca2+ signaling [24] and protects against Ca2+ overload due to increased SERCA activity [25]. In this study, we further PLX5622 explore the mechanisms underlying SFN afforded protection in ischemic stroke by investigating redox signaling in mouse brain microvascular endothelial cells (bEnd.3) subjected to hypoxia-reoxygenation following adaptation PLX5622 to defined O2 levels. Our findings demonstrate that SFN induces Nrf2-regulated defense enzymes in bEnd.3?cells to protect against reoxygenation induced reactive oxygen species generation. These findings together with our study in of ischemic stroke [17,18] suggest that SFN may be a prophylactic therapeutic for targeting the Keap1-Nrf2 defense pathway in stroke and potentially coronary and renal disease. 2.?Methods and materials 2.1. Culture and adaptation of bEnd.3?cells under defined O2 levels Endothelialpolyoma middle T antigen transformed mouse brain microvascular endothelial cells (bEnd.3) were obtained from ATCC-LGC (Teddington, UK). Cells were cultured in phenol red free DMEM (Sigma, UK), supplemented with fetal calf serum (10%), l-glutamine (4?mM) and penicillin (100U/ml)/streptomycin (100?g/ml). Cell monolayers were maintained for at least 5 days (d) in an O2-regulated dual workstation (Scitive, Baker-Ruskinn, USA), gassed to 18?kPa (hyperoxia), 5?kPa (physiological normoxia) or 1?kPa (hypoxia) O2 under 5% CO2 at 37?C. This experimental protocol ensures adaptation of the cell proteome [20] and obviates re-exposure of cells to room air, as all cell culture, treatments and experiments are conducted within the O2-regulated workstation and/or plate reader (CLARIOstar, BMG Labtech, Germany). All experiments were conducted using bEnd.3?cells in passages 7C15. 2.2. Phosphorescence lifetime measurements of O2 levels in bEnd.3?cell cytosol and medium Intracellular O2 levels were monitored in live cells using a cell-penetrating phosphorescent platinumCporphyrin based nanoparticle probe, MitoXpress?-INTRA (Agilent, USA) [26]. A time-resolved fluorescence plate reader (CLARIOstar, BMG Labtech), equipped with an atmospheric control unit, enabled us to measure cytosolic O2 levels under defined ambient O2 levels. PLX5622 bEnd.3?cells were seeded into 96-well black microtitre plates and loaded with MitoXpress?-INTRA (10?g/ml) for 16?h in complete DMEM. The probe emits a phosphorescence signal at 655??55?nm when excited at 355??55?nm [22,24]. Molecular oxygen quenches the phosphorescence signal, and the signal decay is usually inversely proportional to the concentration of O2. Phosphoresence intensity after excitation was measured after 30?s?(ambient O2 levels in the plate reader were fit by exponential analysis. 18?kPa O2: 20.7??1.7?nmol/mg.protein) (Fig. 2D). Intracellular GSH (Fig. 2E) and catalase (Fig. 2F) levels were significantly lower in bEnd.3?cells adapted to 5?kPa O2, consistent with our previous findings in airway epithelial cells [23] and other studies in epidermoid carcinoma cells [40]. Total intracellular GSH.developed the methodology, T.P.K. HO-1 and GCLM by SFN (2.5?M) was significantly attenuated in cells adapted to 5?kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3?cells were adapted to 18 or 5?kPa O2 and subjected to hypoxia (1?kPa O2, 1?h) and reoxygenation. In cells adapted to 18?kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5?M). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX? Red and FeRhoNox?-1, was diminished in cells cultured under 5?kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require studies conducted under well-defined O2 levels. findings. We recently reported that SFN mediated induction of select Nrf2 target genes in umbilical vein endothelial cells (HUVEC) is usually attenuated under physiological normoxia (5?kPa O2) compared to atmospheric O2 levels [22]. Moreover, we reported that adaptation of HUVEC to 5?kPa O2 enhances nitric oxide bioavailability, modulates agonist-induced Ca2+ signaling [24] and protects against Ca2+ overload due to increased SERCA activity [25]. In this study, we further explore the mechanisms underlying SFN afforded protection in ischemic stroke by investigating redox signaling in mouse brain microvascular endothelial cells (bEnd.3) subjected to hypoxia-reoxygenation following adaptation to defined O2 levels. Our findings demonstrate that SFN induces Nrf2-regulated defense enzymes in bEnd.3?cells to protect against reoxygenation induced reactive oxygen species generation. These findings together with our study in of ischemic stroke [17,18] suggest that SFN may be a prophylactic therapeutic for targeting the Keap1-Nrf2 defense pathway in stroke and potentially coronary and renal disease. 2.?Methods and materials 2.1. Culture and adaptation of bEnd.3?cells under defined O2 levels Endothelialpolyoma middle T antigen transformed mouse brain microvascular endothelial cells (bEnd.3) were obtained from ATCC-LGC (Teddington, UK). Cells were cultured in phenol red free DMEM (Sigma, UK), supplemented with fetal calf serum (10%), l-glutamine (4?mM) and penicillin (100U/ml)/streptomycin (100?g/ml). Cell monolayers were maintained for at least 5 days (d) in an O2-regulated dual workstation (Scitive, Baker-Ruskinn, USA), gassed to 18?kPa (hyperoxia), 5?kPa (physiological normoxia) or 1?kPa (hypoxia) O2 under 5% CO2 at 37?C. This experimental protocol ensures adaptation of the cell proteome [20] and obviates re-exposure of cells to room air, as all cell culture, treatments and experiments are conducted within the O2-regulated workstation and/or plate reader (CLARIOstar, BMG Labtech, Germany). All experiments were conducted using bEnd.3?cells in passages 7C15. 2.2. Phosphorescence lifetime measurements of O2 levels in bEnd.3?cell cytosol and medium Intracellular O2 levels were monitored in live cells using a cell-penetrating phosphorescent platinumCporphyrin based nanoparticle probe, MitoXpress?-INTRA (Agilent, USA) [26]. A time-resolved fluorescence plate reader (CLARIOstar, BMG Labtech), equipped with an atmospheric control unit, enabled us to measure cytosolic O2 levels under defined ambient O2 levels. bEnd.3?cells were seeded into 96-well black microtitre plates and loaded with MitoXpress?-INTRA (10?g/ml) for 16?h in complete DMEM. The probe emits a phosphorescence signal at 655??55?nm when excited at 355??55?nm [22,24]. Molecular oxygen quenches the phosphorescence signal, and the signal decay is inversely proportional to the concentration of O2. Phosphoresence intensity after excitation was measured after 30?s?(ambient O2 levels in the plate reader were fit by exponential analysis. 18?kPa O2: 20.7??1.7?nmol/mg.protein) (Fig. 2D). Intracellular GSH (Fig. 2E) and catalase (Fig. 2F) levels were significantly.