Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress activates PI3K/Akt in human PMVECs. Left: Representative immunoblots showing protein levels of phosphorylated Akt (p‐Akt), total Akt and β‐tubulin in whole cell lysates of human PMVECs exposed to different shear stress protocols. Right: Densitometry analysis showing corresponding ratio of p‐Akt to Akt. (a) Cells were pretreated with vehicle (0.1% DMSO) or PI3K inhibitor (LY294002; 10 μM) for 30 min and then exposed to physiological shear stress (12 dyn/cm 2 ) for 1 h, with static cells as controls. (b) After adapting to 0 or 12 dyn/cm 2 of shear stress for 24 h, human PMVECs were treated with vehicle (0.1% DMSO) or LY294002 (10 μM) while maintaining at respective static or shear stress conditions for another 24 h. (c) Human PMVECs were first adapted to shear stress (12 dyn/cm 2 ) for 24 h and then exposed to 30 min of reduced (3 or 0 dyn/cm 2 ) shear stress, with cells maintained at 12 dyn/cm 2 as control. For all graphs, symbols represent different results from different donors ( n = 4; open circles represent cells from male and closed circles represent cells from female). Bar graphs show mean ± SD values. Data were analyzed by one‐way ANOVA with Dunnett's multiple comparisons test (panel (a) interaction p = 0.0003 and panel (c) interaction p = 0.0005) or two‐way ANOVA with Tukey's multiple comparisons test (panel (b) interaction p = 0.1159).

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Western Blot, Control

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress reduces apoptosis in human PMVECs. After adapting PMVECs to 0 or 12 dyn/cm 2 of shear stress for 24 h, cells were treated with vehicle (0.1% DMSO) or LY294002 (10 μM) while maintaining at respective static conditions or shear stress for another 24 h. Apoptosis was evaluated by (a) Hoechst staining (Left: Representative images, white scale bar is 100 μm, red arrows denote condensed chromatin; Right: Summary data); (b) caspase 3/7 activity and (c) DNA fragmentation (image representative of 3 separate experiments). Bar graphs show mean ± SD values; symbols represent results in cells from different donors ( n = 4–5; open circles represent cells from male and closed circles represent cells from female). Data were analyzed by three‐way ANOVA (Factors are shear stress, STS and LY294002; panel (a) interaction p = 0.0451; panel (b) interaction p = 0.0393) followed by Sidak's multiple comparisons test. Shear stress groups are the same as respective panels in Figure .

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Staining, Activity Assay

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress attenuates STS‐induced apoptosis in human PMVECs. After culture under static conditions or adaptation to 12 dyn/cm 2 for 24 h, human PMVECs were treated with either vehicle (0.01% DMSO) or staurosporine (STS; 20 nM) and maintained at their respective static or shear stress conditions for another 24 h. Apoptosis was evaluated by (a) Hoechst staining (Left: Representative images, white scale bar is 100 μm; Right: Summary data); (b) caspase 3/7 activity and (c) DNA fragmentation (image representative of 3 separate experiments). Bar graphs show mean ± SD values; symbols represent results in cells from different donors ( n = 5; open circles represent cells from male and closed circles represent cells from female). Data were analyzed by two‐way ANOVA (panel (a) interaction p = 0.0830; panel (b) interaction p = 0.6835) followed by Tukey's multiple comparisons test.

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Staining, Activity Assay

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress‐induced increases in PI3K activity are not inhibited by STS. After adaptation to 0 or 12 dyn/cm 2 for 24 h, human PMVECs were treated with either vehicle (0.01% DMSO) or staurosporine (STS; 20 nM) while maintaining at their respective shear conditions (0 or 12 dyn/cm 2 ) for another 24 h. Immunoblot was performed in whole cell lysates to probe for p‐Akt, Akt and β‐tubulin. (a) Representative blot and (b) Densitometry analysis for p‐Akt to Akt ratio. Symbols represent different results from different donors ( n = 5; open circles represent cells from male and closed circles represent cells from female). Bar graphs show mean ± SD values; Data were analyzed by two‐way ANOVA (Interaction p = 0.0005) followed by Tukey's multiple comparisons test.

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Activity Assay, Western Blot

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: PI3K/Akt contributes to shear stress‐dependent apoptotic resistance to STS. After adaption to 0 or 12 dyn/cm of shear stress for 24 h, human PMVECs were treated with vehicle (0.1% DMSO) or LY294002 (10 μM) in combination with vehicle (0.01% DMSO) or staurosporine (STS; 20 nM) while maintaining at respective static or shear conditions for another 24 h. Apoptosis was evaluated by (a) Hoechst staining (Left: Representative images, white scale bar is 100 μm; Right: Summary data), (b) caspase 3/7 activity, and (c) DNA fragmentation (representative of three experiments). Symbols represent different results from different donors ( n = 4–5; open circles represent cells from male and closed circles represent cells from female). Bar graphs show mean ± SD values; Data were analyzed by three‐way ANOVA (Factors are shear stress, STS and LY294002; panel (a) interaction p = 0.0451; panel (b) interaction p = 0.0393) with Sidak's multiple comparisons test. Vehicle and vehicle LY294002 groups are the same as respective panels in Figure .

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Staining, Activity Assay

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Schematic summary of major findings. In human PMVECs, physiological shear stress rapidly and sustainably activates PI3K/Akt signaling, which contributes to apoptotic resistance upon staurosporine challenge. Apoptotic resistance conveyed by shear stress is not disrupted by PI3K inhibition, suggesting the existence of additional pro‐survival pathways.

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Inhibition