cell lines 369 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

Journal: Microcirculation (New York, N.y. : 1994)

Article Title: Oxidized Cell‐Free Hemoglobin Induces Mitochondrial Dysfunction by Activation of the Mitochondrial Permeability Transition Pore in the Pulmonary Microvasculature

doi: 10.1111/micc.70012

Figure Lengend Snippet: CFH3+ induces mitochondrial superoxide and cellular ROS formation. MitoSOX stained HLMVECs were treated for the indicated time points before being analyzed on a BD LSRFortessa in the PE channel. (A) Quantification by background adjusted MFI of MitoSOX staining of HLMVECs at the time points of 1, 6, and 24 h. CellROX stained HLMVECs were imaged on a BioTek Lionheart FX microscope using the Cy5 filter set (628/685). (B) Quantification of CellROX Deep Red staining at the time points of 1, 6, and 24 h. (C) Pre‐treatment with Hp for 1 h reduced CellROX staining from CFH3+ exposure at 24 h (Mann–Whitney test). (A,C) n = 6 from 3 donors (two male, one female), (B) n = 7–15 from 4 donors (two male, two female). * = p < 0.05, ** = p < 0.01 , and **** = p < 0.0001. Graphs show median with interquartile range.

Article Snippet: Primary human lung microvascular endothelial cells (HLMVECs) were purchased from PromoCell (C‐12281; Heidelberg, Germany) and cultured in Endothelial Cell Growth Media MV2 (C‐22121; PromoCell).

Techniques: Staining, Microscopy, MANN-WHITNEY

Journal: Microcirculation (New York, N.y. : 1994)

Article Title: Oxidized Cell‐Free Hemoglobin Induces Mitochondrial Dysfunction by Activation of the Mitochondrial Permeability Transition Pore in the Pulmonary Microvasculature

doi: 10.1111/micc.70012

Figure Lengend Snippet: CFH3+ induces mitochondrial morphological changes. (A) Quantification of the mitochondrial footprint per field of view using the Mitochondrial Network Analysis (MiNA) ImageJ plugin. Representative images of the MiNA output for (B) vehicle, (C) CFH2+, (D) CFH3+, and (E) Paraquat treated cells with nuclei highlighted (white arrows; scale bar = 10 um). Representative TEM micrographs for (F) vehicle, (G) CFH2+, and (H) CFH3+ treated HLMVECs with circularity demonstration (red circles) and mitochondria highlighted (black arrows; scale bar = 400 nm). Quantification of mitochondrial morphology parameters of (I) mean electron density, (J) circularity (4pi (area/perimeter 2 )) and (K) aspect ratio (major axis: Minor axis). (A–E) n = 5, (F, vehicle) n = 228, (G, CFH2+) n = 163, (H, CFH3+) n = 416. * = p < 0.05, ** = p < 0.01 , and **** = p < 0.0001.

Article Snippet: Primary human lung microvascular endothelial cells (HLMVECs) were purchased from PromoCell (C‐12281; Heidelberg, Germany) and cultured in Endothelial Cell Growth Media MV2 (C‐22121; PromoCell).

Techniques:

Journal: Microcirculation (New York, N.y. : 1994)

Article Title: Oxidized Cell‐Free Hemoglobin Induces Mitochondrial Dysfunction by Activation of the Mitochondrial Permeability Transition Pore in the Pulmonary Microvasculature

doi: 10.1111/micc.70012

Figure Lengend Snippet: CFH3+ decreases mitochondrial mass. (A) Quantification of MitoGreen by object MFI based on the DNA counterstain. Representative images (Blue = nuclei, Green = mitochondria; scale bar = 2000 um) from HLMVECs stained with MitoTracker Green and treated with (B) vehicle and CFH3+ at the (C) 1, (D) 3, and (E) 6 h time points. n = 6 from 3 donors (two male, one female). * = p < 0.05, *** = p < 0.001, **** = p < 0.0001.

Article Snippet: Primary human lung microvascular endothelial cells (HLMVECs) were purchased from PromoCell (C‐12281; Heidelberg, Germany) and cultured in Endothelial Cell Growth Media MV2 (C‐22121; PromoCell).

Techniques: Staining

Journal: Microcirculation (New York, N.y. : 1994)

Article Title: Oxidized Cell‐Free Hemoglobin Induces Mitochondrial Dysfunction by Activation of the Mitochondrial Permeability Transition Pore in the Pulmonary Microvasculature

doi: 10.1111/micc.70012

Figure Lengend Snippet: CFH3+ activates the mPTP and induces release of mtDNA. (A) Quantification of HLMVECs stained with calcein‐AM and cobalt chloride measured by MFI. (B) Representative stacked histogram of the FITC fluorescence shift from CFH3+ treatment. (C) Quantification of the change in mtDNA release in the media supernatant by qPCR. (D) Quantification of the circulating mtDNA copy number by ddPCR in patient plasma in association with CFH levels. Probes are specific to the mt‐ND4L gene. (A) n = 7 from 3 donors (two male, one female), (C) n = 14 from 5 donors (three male, two female), and (D) n = 36. * = p < 0.05.

Article Snippet: Primary human lung microvascular endothelial cells (HLMVECs) were purchased from PromoCell (C‐12281; Heidelberg, Germany) and cultured in Endothelial Cell Growth Media MV2 (C‐22121; PromoCell).

Techniques: Staining, Fluorescence, Clinical Proteomics