pulmonary arterial ecs  (PromoCell)

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits EC migration but not proliferation. ( A ) Representative images from a wound healing scratch assay using control, wild-type TSP1, and phosphomutant TSP1-transfected BPAECs. Scratches were made in a confluent EC monolayer 24 h post-transfection. Images were captured at 0, 8, and 24 h post-scratch. Scale bar = 100 µm. ( B ) Wound closure was evaluated by measuring the open area at each time point, normalized to the 0 h image (**** p < 0.0001). Statistical analysis was completed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as mean ± SD ( n = 5). ( C ) Representative measurement of an in vitro wound healing assay performed using ECIS. Wounding was applied at 1 h. Each line represents the mean of three replicates ± SD. ( D ) Statistical analysis of EC migration rate was performed using one-way ANOVA with Tukey’s test (**** p < 0.0001). Data are represented as mean ± S.D ( n = 8). ( E ) BPAEC were either untransfected (ctr) or transfected with TSP1 WT -, TSP1 S93A -, or TSP1 S93D -expressing constructs. Cell proliferation was measured by an MTT assay. Absorbance values were normalized to 0 h. No significant differences in proliferation were observed between groups. Data represent SD ( n = 6).

Article Snippet: Bovine pulmonary artery endothelial cells (BPAECs) (culture line CCL 209) were obtained from the American Type Tissue Culture Collection (ATCC), Rockville, MD, USA) and subsequently used at passages 17–20, as described in [ ].

Techniques: Migration, Wound Healing Assay, Control, Transfection, In Vitro, Expressing, Construct, MTT Assay

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D inhibits FAK signaling and downstream targets during cell migration. ( A ) Control or TSP1-transfected confluent monolayers of BPAECs were scratched 24 h post-transfection. Samples were collected at the indicated time points (0, 4, and 8 h). Overexpression of TSP1 and the levels of signaling protein were analyzed by Western blot. ( B ) Quantitative analysis was performed by densitometry of the Western blot bands. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test ( n = 3–5) (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Article Snippet: Bovine pulmonary artery endothelial cells (BPAECs) (culture line CCL 209) were obtained from the American Type Tissue Culture Collection (ATCC), Rockville, MD, USA) and subsequently used at passages 17–20, as described in [ ].

Techniques: Migration, Control, Transfection, Over Expression, Western Blot

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D shows enhanced binding to ITGB1. ( A ) Bacterially expressed GST and GST-TSP1 1–221 WT, GST-TSP1 1–221 S93A, and GST-TSP1 1–221 S93D recombinant proteins immobilized on glutathione Sepharose beads were incubated with BPAEC lysate for pull-down assays. EC lysates and the eluted proteins were analyzed by Western blot using ITGB1- and TSP1-specific antibodies. ( B ) Quantitative analysis of pull-down samples. Statistical analysis was performed using one-way ANOVA ( n = 4) (** p < 0.01). ( C ) Control and TSP1-transfected BPAECs were subjected to immunoprecipitation using c-myc antibody to purify recombinant TSP1 proteins. Total cell lysates and immunocomplexes were tested for c-myc and ITGB1 by Western blot. ( D ) Quantitative analysis of IP. Statistical analysis was performed using one-way ANOVA ( n = 4) (*** p < 0.001).

Article Snippet: Bovine pulmonary artery endothelial cells (BPAECs) (culture line CCL 209) were obtained from the American Type Tissue Culture Collection (ATCC), Rockville, MD, USA) and subsequently used at passages 17–20, as described in [ ].

Techniques: Binding Assay, Recombinant, Incubation, Western Blot, Control, Transfection, Immunoprecipitation

Journal: Biomolecules

Article Title: Phosphomimetic Thrombospondin-1 Modulates Integrin β1-FAK Signaling and Vascular Cell Functions

doi: 10.3390/biom16010084

Figure Lengend Snippet: TSP1 S93D enhances SMC migration and proliferation and induces morphological changes. ( A ) MOVAS cells were seeded at 10% confluence and treated with conditioned media from non-transfected (ctr) or TSP1-transfected BPAECs. Proliferation was assessed using MTT, with absorbance measured at 540 nm at 24, 48, and 72 h. Data are shown as means ± SEM ( n = 12). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test (* p < 0.01). ( B ) Migration of treated MOVAS cells was measured using an ECIS-based wound healing assay. Data presented mean ± S.D. from three chambers per condition. ( C ) Statistical analysis of migration rates was performed using one-way ANOVA with Tukey’s post hoc test ( n = 5; means ± S.D.; **** p < 0.0001). ( D ) Representative images of control and TSP1-treated MOVAS cells analyzed by HCS. Actin filaments were stained with Texas Red phalloidin (red) and nuclei with DAPI. White arrows indicate filopodia of cells. Scale bars: 50 μm. ( E ) Morphological parameters of MOVAS cells were analyzed using Harmony software on the Opera Phenix HCS system. Data are presented as mean ± SD (1000–1600 cells per well, n = 4). Statistical analysis was performed using ANOVA (**** p < 0.0001).

Article Snippet: Bovine pulmonary artery endothelial cells (BPAECs) (culture line CCL 209) were obtained from the American Type Tissue Culture Collection (ATCC), Rockville, MD, USA) and subsequently used at passages 17–20, as described in [ ].

Techniques: Migration, Transfection, Wound Healing Assay, Control, Staining, Software

Journal: Nature Communications

Article Title: Endothelial cell responses in sepsis are attenuated by targeting truncated procalcitonin

doi: 10.1038/s41467-025-68199-x

Figure Lengend Snippet: a Endothelial cells were isolated from murine lungs and subjected to bulk RNA sequencing. Created in BioRender. Brabenec, L. ( https://BioRender.com/mpa96bi ) b MDA plot showing DEGs in color that were more than 2-fold differentially regulated ( p < 0.05) in mice after cecal ligation and puncture (CLP) vs. mice subjected to laparotomy only (sham). Limma uses an empirical Bayes shrinkage method to moderate the standard errors of the estimated log-fold changes, which includes t-tests foreach gene To control for multiple testing the Benjamini and Hochberg method was used. c Principal component analysis of normalized expression values for n = 6 mice/group 18 h after sepsis induction. d Volcano plot representing DEGs in color and labelling of the 10 most up- and down-regulated genes in murine pulmonary endothelium. Limma uses an empirical Bayes shrinkage method to moderate the standard errors of the estimated log-fold changes, which includes t-tests foreach gene To control for multiple testing the Benjamini and Hochberg method was used. e List of the 10 most up- and down-regulated genes including the procalcitonin encoding gene Calca . f Up- and ( g ) down-regulated pathways in murine endothelium in response to polymicrobial sepsis. Enrichment analysis, statistical significance was assessed using adjusted p values (FDR correction) to account for multiple testing. h VEGFa and VEGFc is downregulated in isolated murine pulmonary endothelial cells 18 h after injection of human procalcitonin in mice, n = 3. i Human pulmonary microvascular endothelial cells show increased Calca expression after treatment with sepsis patients serum. This could be abolished by the use of procalcitonin antibody, n = 4 (Healthy), n = 5 (Sepsis), p = 0.0427. j , k Calcrl and Ramp1 show no difference in gene expression, n = 4 (Healthy), n = 5 (Sepsis), p = 0.0035. l VEGFa was downregulated in human pulmonary microvascular endothelial cells when treated with serum of septic patients, n = 4 (Healthy), n = 5 (Sepsis), p = 0.0047. m Expression of genes from LPS-treated human cells in our CLP study. We then took the top 50 up-regulated DEGs from the 8 h LPS study and found that 36 of these were also annotated in our dataset. Mean expression values for these genes from our study were calculated per group and then presented as heatmap with values scaled by row. Cluster 1: All genes in this cluster were up-regulated upon CLP as for LPS 8 h, and almost all DEGs (except ICAM1 ) were reduced in expression after AB treatment. Cluster 2: Genes in this cluster were down-regulated in our dataset upon CPL, but not up-regulated as in the LPS study. Of note, these DEGs were stronger down-regulated after AB treatment. One LPS-DEG gene, VCAM1 , was also up-regulated after CLP, but expressed higher in AB-treated samples compared to non-AB-treated CLP controls. Unpaired t test, data presented as mean ± SEM. p values as indicated. Source data are provided as a Source Data file.

Article Snippet: Human pulmonary endothelial cells (PromoCell) were cultured in cell culture media and exposed to serum from human sepsis patients diluted 1:1 in serum-free media.

Techniques: Isolation, RNA Sequencing, Ligation, Control, Expressing, Injection, Gene Expression

Journal: Nature Communications

Article Title: Endothelial cell responses in sepsis are attenuated by targeting truncated procalcitonin

doi: 10.1038/s41467-025-68199-x

Figure Lengend Snippet: a , b Representative immunoblot and quantitative summary showing phosphorylated VE-cadherin at tyrosine residue 685 in murine endothelial cell lysates after stimulation with septic mice plasma for 15, 30 and 60 min, n = 4 (60), n = 5(ctrl,15,30) independent experiments/group, timesfold vs. control. Samples derive from the same experiment and blots were processed in parallel. Uncropped blots in Source Data. c Procalcitonin plasma concentrations during the course of sepsis in mice, n = 5 (CLP12h, 6 h), n = 12 (sham), n = −18 (CLP18h). d Schematic depicting the strategy of antibody generation targeting murine truncated procalcitonin. Created in BioRender. Brabenec, L. ( https://BioRender.com/7xr1vmf ) e Representative immunoblot showing phosphorylated VE-cadherin at tyrosine residue 685 in endothelial cell lysates 30 min after exposure to recombinant procalcitonin, n = 7, timesfold vs. control. Samples derive from the same experiment and blots were processed in parallel.Uncropped blots in Source Data. f Fluorescence-labeled macromolecule permeability of murine pulmonary endothelial cells exposed to septic mice’s plasma after pre-treatment with 1 µg of the antibody directed against truncated procalcitonin (PCT AB) and respective IgG control for two h, n = 48 (ctrl AB), n = 7 (ctrl IgG), n = 8 (Sepsis IgG, Sepsis AB). Data presented as mean ± SEM. One-way ANOVA/Bonferroni. p values as indicatedSource data are provided as a Source Data file.

Article Snippet: Human pulmonary endothelial cells (PromoCell) were cultured in cell culture media and exposed to serum from human sepsis patients diluted 1:1 in serum-free media.

Techniques: Western Blot, Residue, Clinical Proteomics, Control, Recombinant, Fluorescence, Labeling, Permeability

Journal: Nature Communications

Article Title: Endothelial cell responses in sepsis are attenuated by targeting truncated procalcitonin

doi: 10.1038/s41467-025-68199-x

Figure Lengend Snippet: a Summary of the number of up- (red) and down-(blue) differentially regulated genes (DEGs) in murine endothelial cells. Results are derived from relating sham AB to sham IgG [left column], CLP IgG to sham IgG, CLP AB to CLP IgG and CLP AB to sham AB, respectively. b VENN diagram deciphering the number of DEGs from the same contrasts. c Scatter plot of genes showing mean log 2 fold differences of CLP controls and CLP AB treated mice to the respective sham-treated controls. For this comparison, the up-regulated genes of the cytokine-mediated signaling pathway were used that were commonly up-regulated DEGs in CLP IgG and CLP AB treated versus the respective sham controls. d Heatmap of expression levels of DEGs from all contrasts. Values were scaled by row. red: up-regulated DEGs, blue: down-regulated DEGs. Each column represents one mouse. e Expression differences in Il17 pathway. Colors represent differences in normalized expression levels from the strongest CLP AB responder (sample CLP_PCT_AB_11_02) versus a strong CLP IgG responder (sample CLP_IgG_ctrl_16_07) projected on the mouse IL-17 signaling pathway (KEGG pathway mmu04657). f Inhibition of procalcitonin activation by DPP4 inhibitor sitagliptin and blocking procalcitonins receptor by the PCTR antagonist olcegepant, reduced IL-17 plasma levels in septic mice. n = 7 (Sham IgG, CLP PCTAB), n = 6 (CLP IgG), n = 5 (PCTS Antagonist, DPP4 Inhibitor), timesfold vs. control. g Heatmap showing cytokine and chemokine expression profiles in murine blood 18 h after sepsis induction by cecal ligation and puncture (CLP)/control (sham) surgery following injection of the antibody and respective control IgG 6 h after surgery, data shown as ng/mL, n = 7 mice/group. Data is presented as mean ± SEM. One- and Two-way ANOVA and Bonferroni-correction. p values as indicated.Source data are provided as a Source Data file.

Article Snippet: Human pulmonary endothelial cells (PromoCell) were cultured in cell culture media and exposed to serum from human sepsis patients diluted 1:1 in serum-free media.

Techniques: Derivative Assay, Comparison, Expressing, Inhibition, Activation Assay, Blocking Assay, Clinical Proteomics, Control, Ligation, Injection

Journal: The FASEB Journal

Article Title: SARS ‐ CoV ‐2 Nucleocapsid Protein Does Not Induce Inflammation in Endothelial Cells or Monocytes

doi: 10.1096/fj.202501433R

Figure Lengend Snippet: Effects of recombinant N‐protein (endotoxin nondepleted, NPnon) on inflammatory marker mRNA levels in human microvascular endothelial cells. Cells were treated with vehicle, LPS (1 μg/mL), or NPnon (10 μg/mL, ThermoFisher) for 4 h. The total RNA was isolated, and qPCR analysis was performed to measure the mRNA levels of TNFα, IL‐6, E‐selectin, and ICAM‐1. Data are presented as mean ± SEM and analyzed using one‐way ANOVA followed by Bonferroni's multiple comparisons test. n = 4–5 independent cell cultures per group. **** p < 0.0001 vs. control.

Article Snippet: Human pulmonary artery microvascular endothelial cells (HMEC, American Type Culture Collection (ATCC)) were cultured in endothelial cell media as described above, in an incubator at 37°C with 5% CO 2 .

Techniques: Recombinant, Marker, Isolation, Control

Journal: The FASEB Journal

Article Title: SARS ‐ CoV ‐2 Nucleocapsid Protein Does Not Induce Inflammation in Endothelial Cells or Monocytes

doi: 10.1096/fj.202501433R

Figure Lengend Snippet: Effects of recombinant N‐protein (endotoxin nondepleted, NPnon) on inflammatory marker mRNA levels in mouse microvascular endothelial cells. Cells were treated with vehicle, LPS (1 μg/mL), NPnon (10 μg/mL, ThermoFisher), LPS + polymyxin B (PMB) (1.25 mg/mL), NPnon + PMB (250 μg/mL), or PMB (250 μg/mL) for 4 h. The total RNA was isolated and qPCR analysis was performed to measure the mRNA levels of TNFα, IL‐6, E‐selectin, and ICAM‐1. Data are presented as mean ± SEM and analyzed using one‐way ANOVA followed by Bonferroni's multiple comparisons test. n = 5 independent cell cultures per group. *** p < 0.001, ** p < 0.01 vs. control.

Article Snippet: Human pulmonary artery microvascular endothelial cells (HMEC, American Type Culture Collection (ATCC)) were cultured in endothelial cell media as described above, in an incubator at 37°C with 5% CO 2 .

Techniques: Recombinant, Marker, Isolation, Control

Journal: The FASEB Journal

Article Title: SARS ‐ CoV ‐2 Nucleocapsid Protein Does Not Induce Inflammation in Endothelial Cells or Monocytes

doi: 10.1096/fj.202501433R

Figure Lengend Snippet: Effects of endotoxin‐depleted N‐protein (NPd) on inflammatory marker mRNA levels in mouse microvascular endothelial cells. Cells were treated with vehicle, LPS (1 μg/mL), NPd (50 μg/mL), NPd + PMB (250 μg/mL), or PMB (250 μg/mL) alone (control) for 4 h. Total RNA was isolated and RT‐qPCR analysis was performed to measure mRNA levels of TNFα and E‐selectin. Data are presented as mean ± SEM and analyzed using one‐way ANOVA followed by Bonferroni's multiple comparisons test. n = 3 independent cell cultures per group. *** p < 0.001, ** p < 0.01 vs. control.

Article Snippet: Human pulmonary artery microvascular endothelial cells (HMEC, American Type Culture Collection (ATCC)) were cultured in endothelial cell media as described above, in an incubator at 37°C with 5% CO 2 .

Techniques: Marker, Control, Isolation, Quantitative RT-PCR