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: bioRxiv

Article Title: Dynamic Culture Improves the Predictive Power of Bronchial and Alveolar Airway Models of SARS-CoV-2 Infection

doi: 10.1101/2025.07.21.665885

Figure Lengend Snippet: (A) Schematic of the PhysioMimix® lung MPS co-culture system. Epithelial cells were seeded on the apical side of the Transwell® insert and human pulmonary microvascular endothelial cells (HPMVECs) on the basolateral side, under air–liquid interface (ALI) and dynamic flow conditions. (B) Representative H&E and Alcian blue-stained histological section of bronchial MPS co-culture after 14 days. Mucus is stained blue; endothelial cells are indicated by white arrows. Scale bar, 50 µm. (C) TEER measurements comparing epithelial monoculture, endothelial monoculture, and epithelial–endothelial co-culture over 14 days under ALI conditions. (D) TEER comparison of bronchial and alveolar co-cultures grown under static or dynamic flow MPS conditions over the 14-day ALI differentiation period. (E) Gene expression of Club cell (SCGB1A1) and Goblet cell (MUC5AC) markers in NHBE cells before culture (NHBE pellet) and after 14 days of differentiation in MPS co- culture. (F) Gene expression of alveolar markers - AT1 (AQP5) and AT2 (SFTPB) - in SAEC cells before culture (SAEC pellet) and after 14 days of MPS co-culture. (G) Immunofluorescence staining of bronchial MPS co-culture tissue for acetylated α-tubulin (yellow), mucus (MUC5AC, green), actin (phalloidin, red), and nuclei (Hoechst 33342, blue). Top row shows epithelial layer; bottom row shows endothelial layer. Scale bar, 100 µm. (H) Immunofluorescence staining of alveolar MPS co-culture tissue after 14 days of differentiation, showing surfactant (SFTPB, green), actin (phalloidin, magenta), and nuclei (Hoechst 33342, blue). Endothelial cells are marked with white arrows. Scale bar, 20 µm.

Article Snippet: Airway models were comprised of human pulmonary microvascular endothelial cells (HPMEC) (PromoCell, C-12281, lot.489Z024.1) and small airway epithelial cells (SAEC) (Lonza, CC-2547, lot.21TL214918) or normal human bronchial epithelial cells (NHBE) (Lonza, CC-2540, lot.21TL035497) for the alveolar and bronchial airway model, respectively.

Techniques: Co-Culture Assay, Staining, Comparison, Gene Expression, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Revealing the impact of Pseudomonas aeruginosa quorum sensing molecule 2’-aminoacetophenone on the human bronchial-airway epithelium and pulmonary endothelium using a human airway-on-a-chip

doi: 10.3389/fimmu.2025.1592597

Figure Lengend Snippet: Airway-on-a-chip RNA-sequencing analysis of 2-AA-treated endothelial and epithelial cells reveals multiple pathways to be differentially regulated by this quorum sensing molecule (A) Overview of the timeline of preparation and treatment of 2-AA of human pulmonary microvascular endothelial (HPMEC) and normal bronchial epithelial (NHBE) cells in a lung-on-chip setting. Volcano plot of the differentially expressed genes (DEGs) in HPMEC (B) and NHBE (C) cells after 20 hrs of continuous flow of 20 µM 2-AA. The blue dots represent genes that are upregulated (log 2 fold change>0.5), red dots for downregulated (log 2 fold change <-0.5), and grey dots for genes that are not significant. The two vertical dashed lines indicate log2-fold change > 0.5 and log2-fold change < -0.5. The horizontal dashed line indicates the significance of -log 10 (p-value) =1.3. (D-G) Horizontal lollipop plots showing Reactome pathway enrichment analysis of upregulated and downregulated pathways from the differentially regulated DEGs in human HPMEC and NHBE cells and compared to untreated cells with -log 10 (p-value) =1.3 on the x-axis. The numbers in the circles represent the count of genes for each pathway. DESeq2 determined the log2 fold change values used in volcano plots. The -log 10 (p-value) of 1.3 corresponds to a p-value of less than 0.05, as determined by Deseq2. The Reactome pathway enrichment analysis was done using the DAVID web server. The RNA sequencing analysis was performed in two independent experiments (n = 2).

Article Snippet: The HPMEC cells were cultured in a T-75 flask using Endothelial Cell Growth Medium MV 2 (Promocell, C-22221 and Growth Medium MV 2 Supplement Pack C39221 ), supplemented with 1X penicillin/streptomycin (Gibco).

Techniques: RNA Sequencing

Journal: Frontiers in Immunology

Article Title: Revealing the impact of Pseudomonas aeruginosa quorum sensing molecule 2’-aminoacetophenone on the human bronchial-airway epithelium and pulmonary endothelium using a human airway-on-a-chip

doi: 10.3389/fimmu.2025.1592597

Figure Lengend Snippet: 2-AA upregulates MMP-9 and VCAM-1 levels and enhances THP-1 monocyte adhesion in HPMECs. (A) Table depicting the differentially expressed genes and their associated pathways in HPMEC and NHBE cells following 2-AA treatment in the airway-on-a-chip. Genes and pathways upregulated in HPMEC cells and downregulated in NHBE cells (orange). Genes and pathways upregulated in NHBE cells and downregulated in HPMEC (purple). Significance is denoted by p<0.05 and FDR<0.05. (B) Representative confocal images of MMP-9 (red) in 2-AA-treated and untreated HPMEC cells. Nuclei were counterstained with DAPI. (C) Relative expression of MMP-9 fluorescence intensity normalized to DAPI. (D) Representative immunofluorescence images of VCAM-1 (red) and adherent THP-1 monocytes (green) in 2-AA-treated and untreated HPMEC cells. (E) Quantification of VCAM-1 and THP-1 fluorescence intensity normalized to DAPI. Composite images show all channels merged. The scale bar represents 50 μM. The images are representative of n=3 independent experiments, with six images analyzed per group. Each dot represents six different biological replicates (n = 6), error bars denote mean ± standard deviation. P-values were calculated using a two-tailed unpaired t-test, depicted on the graphs. The overall p-value summary is p < 0.0001, indicating statistical significance.

Article Snippet: The HPMEC cells were cultured in a T-75 flask using Endothelial Cell Growth Medium MV 2 (Promocell, C-22221 and Growth Medium MV 2 Supplement Pack C39221 ), supplemented with 1X penicillin/streptomycin (Gibco).

Techniques: Expressing, Fluorescence, Immunofluorescence, Standard Deviation, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Revealing the impact of Pseudomonas aeruginosa quorum sensing molecule 2’-aminoacetophenone on the human bronchial-airway epithelium and pulmonary endothelium using a human airway-on-a-chip

doi: 10.3389/fimmu.2025.1592597

Figure Lengend Snippet: Common genes and associated pathways regulated by 2-AA in HPMEC and NHBE cells. (A) A Venn diagram and table depict gene expression data. The Venn diagram shows 4,499 genes up- or down-regulated in HPMEC cells, 623 in NHBE cells, and 271 that are overlapping. (B) Upregulated (blue) and downregulated (red) genes and pathways, common in NHBE and HPMEC cells with a p-value < 0.05 and a False Discovery Rate (FDR) < 0.05. The DAVID web server was used for the Reactome pathway enrichment analysis.

Article Snippet: The HPMEC cells were cultured in a T-75 flask using Endothelial Cell Growth Medium MV 2 (Promocell, C-22221 and Growth Medium MV 2 Supplement Pack C39221 ), supplemented with 1X penicillin/streptomycin (Gibco).

Techniques: Gene Expression