human pulmonary microvascular endothelial cells pmvecs  (PromoCell)

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

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: AMs aggravate LPS-induced injury in PMVECs via M1 polarization-mediated inflammatory response. (A) PMVECs were cultured in the presence of AMs or alone, followed by 100 ng/mL LPS treatment. Equivalent PBS was used as the control. After 24 h of culture, (B) PMVEC permeability was assessed using HRP leakage assays. (C) Western blot analysis revealed the relative expressions of both Claudin-5 and ZO-1 proteins, with GAPDH as the internal reference. (D) Annexin V-FITC/PI staining and (E) TUNEL assays demonstrated the apoptosis rate of PMVECs. (E) ELISA was used for inflammatory cytokines (F) IL-6 and (G) TNF-α concentration determination. (H) The western blot analysis for iNOS and Arg-1 represented the level of M1 polarization. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, *** p < 0.001. PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: Mouse pulmonary microvascular endothelial cells (PMVECs; CP-M001) were purchased from Procell Life Science (Wuhan, China) and maintained in endothelial cell growth medium (Procell) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (p/s).

Techniques: Cell Culture, Control, Permeability, Western Blot, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: MSC-exos attenuate LPS-induced PMVEC injury by promoting M2 polarization of AMs. MSC-exos were added to the LPS-treated co-culture system to evaluate their effects on PMVECs. (A) PMVEC permeability was assessed using HRP leakage assays. (B) Western blot analysis was performed to detect the expression levels of tight junction proteins Claudin-5 and ZO-1. (C) Annexin V-FITC/PI staining and (D) TUNEL assays demonstrated the apoptosis rate of PMVECs to determine the anti-apoptosis effect of MSC-exos. (E-F) ELISA for inflammatory cytokines IL-6 and TNF-α in cell supernatant. (G) Western blot analysis for M1 polarization markers assessed the relative levels of iNOS and Arg-1 proteins. (H) M2 polarization was determined via flow cytometry by calculate the percentage of CD206-positive cells. (I) Gas chromatography-mass spectrometry was used to measure ITA concentration. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, ** p < 0.01, and *** p < 0.001. MSC-exos: mesenchymal stem cell-derived exosome; PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: Mouse pulmonary microvascular endothelial cells (PMVECs; CP-M001) were purchased from Procell Life Science (Wuhan, China) and maintained in endothelial cell growth medium (Procell) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (p/s).

Techniques: Co-Culture Assay, Permeability, Western Blot, Expressing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Gas Chromatography, Mass Spectrometry, Concentration Assay, Derivative Assay