primary human pulmonary microvascular endothelial cells hpmec  (PromoCell)

Journal: Redox Biology

Article Title: TRPV2 channels facilitate pulmonary endothelial barrier recovery after ROS-induced permeability

doi: 10.1016/j.redox.2025.103720

Figure Lengend Snippet: H 2 O 2 induces HPMEC barrier instability and ADAM10-dependent VE-cadherin cleavage. Changes in HPMEC electrical resistance (normalized to baseline levels) were recorded with an ECIS device at 4000 Hz upon application of H 2 O 2 (75 μM, 300 μM) ( A ). The normalized resistance values 15 and 90 min after exposure were quantified ( B ). Representative Western blot (from one donor, 3 technical replicates) of the full length (FL) and C-terminal fragment (CTF) levels of VE-cadherin protein in HPMECs 2 h after H 2 O 2 exposure (300 μM) in the presence and absence of the ADAM10 inhibitor GI254023X (GI254, 3 μM) ( C ). β-actin was probed as a loading control. Normalized levels of VE-Cadherin CTF from these Western blots were quantified ( D ). Data reflect the mean ( A , B , D ) + SD ( B , D ) from 3 independent donors ( n = 3). ( E ) Mean ΔF/F 0 traces of HPMEC monolayer Ca 2+ influx following H 2 O 2 exposure (300 μM) in the presence and absence of the TRPM2 and TRPV2 inhibitors, econazole (10 μM) and tranilast (50 μM). Data represent the mean ± SD from one experiment, 35–50 cells/treatment group. This experiment was performed three times in HPMECs from a single donor at different passage numbers ( n = 3), and the area under the curve (AUC) of each mean ΔF/F 0 Ca 2+ trace was quantified ( E ), with bars reflecting the mean + SEM. Normality of data was confirmed using the Shapiro-Wilk test, and significance between means was analyzed using two- or one-way ANOVA and Tukey post hoc tests ( B , D , E ); ∗ p < 0.1, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMECs) [ ] from healthy donors were obtained from Promocell (Heidelberg, Germany, #C-12281) and cultured in endothelial cell growth medium MV (Promocell, #C-22020) at 37 °C and 5 % CO 2 , and were kept until passage 12.

Techniques: Western Blot, Control

Journal: Redox Biology

Article Title: TRPV2 channels facilitate pulmonary endothelial barrier recovery after ROS-induced permeability

doi: 10.1016/j.redox.2025.103720

Figure Lengend Snippet: TRPV2 and TRPM2 mediate VE-cadherin cleavage in HPMECs. Representative Western blot of FL and CTF VE-cadherin protein levels in HPMECs upon TRPV2 inhibition (50 μM tranilast) and 2 h exposure to H 2 O 2 (300 μM; A , quantified in B ). ( C ) Representative Western blot of FL and CTF VE-cadherin protein levels in HPMECs upon ADAM10 inhibition (3 μM GI254023X, GI254) and 2 h exposure to cannabidiol (CBD, 50 μM), quantified in ( D ). Representative Western blot of FL and CTF VE-cadherin protein levels in HPMECs upon TRPM2 inhibition (10 μM econazole) and 2 h exposure to H 2 O 2 (300 μM; E , quantified in F ). Representative Western blot of FL and CTF VE-cadherin protein levels after H 2 O 2 exposure (2 h, 300 μM) upon co-inhibition of TRPM2 and ADAM10 (10 μM econazole, 3 μM GI254023X, ( G , quantified in H )). For all Western blots, β-actin was probed for as a loading control; samples shown are from a single donor, 3 technical replicates. Quantified data reflect the mean + SD from 3 independent donors ( B , D , F ) or 3 consecutive passages from one donor ( H ); ( n = 3). Normality of data was confirmed using the Shapiro-Wilk test, and significance between means was analyzed using two-way ANOVA, with Tukey post hoc tests; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMECs) [ ] from healthy donors were obtained from Promocell (Heidelberg, Germany, #C-12281) and cultured in endothelial cell growth medium MV (Promocell, #C-22020) at 37 °C and 5 % CO 2 , and were kept until passage 12.

Techniques: Western Blot, Inhibition, Control

Journal: Redox Biology

Article Title: TRPV2 channels facilitate pulmonary endothelial barrier recovery after ROS-induced permeability

doi: 10.1016/j.redox.2025.103720

Figure Lengend Snippet: TRPM2 and TRPV2 facilitate HPMEC barrier recovery following H 2 O 2 exposure. Changes in barrier resistance (normalized to baseline) were measured in HPMECs which were preincubated with DMSO or econazole (econ, 10 μM, 1 h) and subsequently exposed to 75 μM H 2 O 2 ( A ). HPMEC resistance values (presented as % of control values) were quantified 15 and 90 min after H 2 O 2 application ( B ). Similar experiments were conducted with the TRPV2 inhibitor tranilast (tran, 50 μM, 1 h preincubation, ( C , D )) and the ADAM10 inhibitor GI254023X (GI254, 3 μM, 1 h preincubation, ( E , F )). Data represent the mean ( A – F ) + SD ( B , D , F ) of results from 3 independent donors ( n = 3). Normality of data was confirmed using the Shapiro-Wilk test, and significance between means was analyzed with two-way ANOVA and Tukey post hoc tests; ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMECs) [ ] from healthy donors were obtained from Promocell (Heidelberg, Germany, #C-12281) and cultured in endothelial cell growth medium MV (Promocell, #C-22020) at 37 °C and 5 % CO 2 , and were kept until passage 12.

Techniques: Control

Journal: Redox Biology

Article Title: TRPV2 channels facilitate pulmonary endothelial barrier recovery after ROS-induced permeability

doi: 10.1016/j.redox.2025.103720

Figure Lengend Snippet: TRPV2 and ADAM10 are necessary for altered localization of N- and VE-cadherin following H 2 O 2 exposure. ( A ) HPMEC immunofluorescence staining of VE-cadherin (red) and N-cadherin (green) over a timecourse of H 2 O 2 exposure (75 μM; 0 min, 15 min, 90 min) in the presence and absence of TRPM2, TRPV2 or ADAM10 inhibitors (10 μM econazole, 50 μM tranilast, 3 μM GI254023X, respectively). Nuclei were stained with DAPI (blue), scale bars: 100 μm. Signal intensities of VE-cadherin ( B ) and N-cadherin ( C ) at cell-cell junctions were quantified from stainings performed in HPMECs from one donor at 4 consecutive passages ( n = 4, 30 regions per n ). Colocalization analyses for N- and VE-cadherin were conducted for the same regions in three experiments ( n = 3, 30 regions per n ), and mean weighted colocalization coefficients for VE-cadherin – N-cadherin were plotted ( D ). Normality of data was confirmed using the Shapiro-Wilk test, and significance between means ( B – D ) were analyzed with two-way ANOVA and Tukey post hoc tests; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMECs) [ ] from healthy donors were obtained from Promocell (Heidelberg, Germany, #C-12281) and cultured in endothelial cell growth medium MV (Promocell, #C-22020) at 37 °C and 5 % CO 2 , and were kept until passage 12.

Techniques: Immunofluorescence, Staining