Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: Whole-cell currents in HUVEC adapted to 5 kPa or 18 kPa O 2 . ( A ) Raw current traces under indicated conditions with voltage protocol using the Nanion P-a-P. HUVEC exhibit inward- and outward rectifying K + currents showing differential responses under defined physiological O 2 levels and following incubation with an NO donor. ( B ) Steady-state current-voltage (IV) relationships differ in HUVEC under indicated conditions (left: F Treatment (17, 738) = 38.02, P < 0.0001, F Voltage (1, 738) = 50.83, P < 0.0001; middle: F Treatment (17, 558) = 29.60, P < 0.0001, F Voltage (1, 558) = 0.3886, P = 0.5333; right: F Treatment (17, 648) = 58.70, P < 0.0001, F Voltage (1, 648) = 32.51, P < 0.0001, two-way ANOVA). Notably, inward currents are smaller under 5 kPa compared to 18 kPa O 2 , whereas outward currents are larger. NO induced stronger relative potentiation of inward and outward currents under 18 kPa O 2 . ( C ) Direct comparisons of current amplitudes at −20 mV and +50 mV under indicated conditions (−20 mV, P = 0.0127, +50 mV, P = 0.012, one-way ANOVA). ( D ) Exponential fits to the rising phase of current activation. Schematic illustrates the best fit (green line) by a second order exponential with time constants of activation shown in graphs for currents at −120 mV and + 50 mV ( P = 0.0465, one-way ANOVA). Data denote mean ± S.E.M., n = 7–21 cells per condition.

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Incubation, Activation Assay

Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: NO application modulates channel activation and membrane potentials differently in HUVEC adapted to 5 kPa or 18 kPa O 2 . ( A, B ) Boltzmann fits to G/G max for indicated conditions. Data following NO treatments suggests a leftward shift of half activation voltages (V 1/2 ) at 18 kPa pericellular O 2 (5 kPa: 17.0 ± 1.2 mV, 5 kPa + NO: 13.5 ± 1.3 mV; 18 kPa: 16.1 ± 1.2 mV, 18 kPa + NO: 11.7 ± 1.9 mV, n = 9–23, one-way ANOVA, P = 0.0061, n = 7–21 cells per condition). ( C ) Membrane potentials were recorded in control HUVEC under indicated conditions. NO application induces a hyperpolarization in cells adapted to hyperoxic 18 kPa O 2 (one-way ANOVA, P = 0.0176, n = 5–8 cells per condition). Data denote mean ± S.E.M.

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Activation Assay, Membrane, Control

Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: Whole-cell currents in human brain microvascular endothelial cells adapted to 5 kPa or 18 kPa O 2 . ( A ) Raw current traces under indicated conditions with voltage protocol using the Nanion P-a-P. hCMEC/D3 cells exhibit outward rectifying K + currents showing differential responses under defined O 2 levels and following incubation with an NO donor. ( B ) Steady-state current-voltage (IV) relationships differ between hCMEC/D3 cells recorded under indicated conditions (left: F Treatment (8, 243) = 84.08, P < 0.0001, F Voltage (1, 243) = 23.33, P < 0.0001; middle: F Treatment (8, 252) = 119.3, P < 0.0001, F Voltage (1, 252) = 13.89, P = 0.0002; right: F Treatment (9, 268) = 58.59, P < 0.0001, F Voltage (1, 268) = 5.876, P = 0.0160, two-way ANOVA). Notably, outward currents are larger under 5 kPa compared to 18 kPa O 2 . NO induces a stronger potentiation of outward currents under 5 kPa O 2 . C , Direct comparisons of current amplitudes at −20mV and +60 mV under indicated conditions (−20 mV: ∗ P = 0.0426, ∗∗ P = 0.0087; +60 mV: 5 kPa versus 18 kPa: P = 0.0318, 5 kPa + NO versus 18 kPa + NO: P = 0.0309, one-way ANOVA). D , Exponential fits to the rising phase of current activation. Schematic illustrates the best fit (green line) by a second order exponential with time constants of activation shown in graphs for currents at +60 mV (∗∗ P = 0.0035, ∗∗∗∗ P < 0.0001, one-way ANOVA, n = 14–15 cells per condition). Data denote mean ± S.E.M.

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Incubation, Activation Assay

Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: K + channel contributions to whole-cell current vary between cells adapted to 5 kPa or 18 kPa O 2 . ( A) Raw current traces under indicated conditions recorded from HUVEC exhibit outward rectifying whole-cell currents. ( B) Comparisons of normalised current amplitudes (relative to current at +50 mV) under indicated conditions show current inhibition relative to basal control from cells cultured at 5 kPa or 18 kPa using 10 mM TEA, 5 μM TRAM-34, 100 nM apamin (5 kPa: Basal vs. TEA: P = 0.0381, Basal vs. TEA + TRAM-34: P = 0.0095, Basal vs. TEA + TRAM-34+apamin: P = 0.0022; 18 kPa: Basal vs. TEA: P = 0.03723, Basal vs. TEA + TRAM-34: P = 0.0076, Basal vs. TEA + TRAM-34+apamin: P = 0.0042; two-way ANOVA, data denote mean ± S.E.M.). ( C ) Steady-state current-voltage (IV) relationships showing differential effects of TEA, TRAM-34 and apamin in HUVEC adapted to either O 2 level (5 kPa O 2 : F Treatment (3, 90) = 49.88, P < 0.0001, F Voltage (12, 90) = 131.0, P < 0.0001; 18 kPa O 2 : F Treatment (3, 104) = 15.50, P < 0.0001, F Voltage (12, 104) = 55.47, P < 0.0001, n = 2–3 cells each, two-way ANOVA, data denote mean (+S.E.M.).

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Inhibition, Control, Cell Culture

Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: K + channel contributions to whole-cell current vary between cells adapted to 5 kPa or 18 kPa O 2 . ( A) Raw current traces under indicated conditions recorded from hCMEC/D3 cells exhibit outward rectifying whole-cell currents. ( B ) Comparisons of normalised current amplitudes (relative to current at +50 mV) under indicated conditions show current inhibition relative to basal control from cells cultured at 5 kPa or 18 kPa using 10 mM TEA, 5 μM TRAM-34, 100 nM apamin (5 kPa: Basal vs. TEA: P = 0.0375, Basal vs. TEA + TRAM-34: P = 0.0013, Basal vs. TEA + TRAM-34+apamin: P < 0.0001; 18 kPa: Basal vs. +TEA + TRAM-34+apamin: P = 0.0244, two-way ANOVA, data denote mean ± S.E.M.). ( C ) Steady-state current-voltage (IV) relationships showing differential effects of TEA, TRAM-34 and apamin in hCMEC/D3 cells adapted to either O 2 level (5 kPa O 2 : F Treatment (3, 102) = 173.0, P < 0.0001, F Voltage (12, 102) = 131.4, P < 0.0001; 18 kPa O 2 : F Treatment (3, 103) = 14.96, P < 0.0001, F Voltage (12, 103) = 38.59, P < 0.0001, n = 3 cells each, two-way ANOVA, data denote mean (+S.E.M.).

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Inhibition, Control, Cell Culture

Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: Expression of KCa2.3 in human umbilical vein and human brain microvascular endothelial cells adapted to 5 kPa versus 18 kPa O 2 . ( A, B ) Immunoblot analysis of KCa2.3 (SK3) channel expression in human umbilical vein (HUVEC) and brain microvascular (hCMEC/D3) endothelial cells adapted for 5 days to 5 kPa or 18 kPa O 2 reveals no difference (n = 3 independent cultures, Student's t -test, P > 0.05). Data denote mean ± S.E.M.

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Expressing, Western Blot

Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: Expression of KCa3.1 channels in human umbilical vein and human brain microvascular endothelial cells adapted to 5 kPa versus 18 kPa O 2 . Representative immunocytochemical images of HUVEC (top) and hCMEC/D3 (bottom) immunostained for KCa3.1 (SK4) channel protein with quantifications of fluorescence signals normalised to DAPI in HUVEC and hCMEC/D3. Data denote mean ± S.E.M., n = 6 independent cultures, Student's t -test, P > 0.05).

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Expressing, Fluorescence

Journal: Redox Biology

Article Title: Physiological oxygen levels reset K + channel activity in human vascular endothelial cells

doi: 10.1016/j.redox.2025.103981

Figure Lengend Snippet: Expression of KCa1.1 channels in human umbilical vein and human brain microvascular endothelial cells adapted to 5 kPa versus 18 kPa O 2 . Representative immunocytochemical images of HUVEC (top) and hCMEC/D3 (bottom) immunostained for KCa1.1 (BK) channel protein with quantifications of fluorescence signals normalised to DAPI in HUVEC and hCMEC/D3. Data denote mean ± S.E.M., n = 4 independent cultures, Student's t -test, P > 0.05).

Article Snippet: HUVEC and hCMEC/D3 cell monolayers were maintained for at least 5 days in an O 2 -controlled Scitive dual or InVivO 2 ®400 workstation (Baker, USA), gassed to 18 kPa or 5 kPa O 2 under 5 % CO 2 at 37 o C. We previously established that lowering ambient O 2 in the workstation from 18 kPa to 5 kPa rapidly upregulates HIF1-α mRNA expression with protein expression of HIF1-α and its targets only returning to baseline after 5 days adaptation [ ].

Techniques: Expressing, Fluorescence