Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMP9 and BMP10 selectively activate SMAD1/5/8 signaling and induce proliferation in pulmonary artery endothelial cells but not pulmonary artery smooth muscle cells. ( A ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PAECs treated with the indicated TGF-β superfamily ligands (0.8 nM) or untreated control (UT); β-actin serves as a loading control. ( B ) PAEC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. ( C ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PASMCs treated with the indicated ligands (0.8 nM); β-actin serves as a loading control. ( D ) PASMC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. Data are shown as mean ± SD ( n = 3 replicate wells). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, BrdU Incorporation Assay

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMPR2 dosage-dependent model for BMP9/10 signaling output in pulmonary artery endothelial cells. Schematic illustrating how BMPR2 abundance constrains BMP9/10 (ALK1-dependent) canonical signaling output and downstream cellular programs in PAECs. ( A ) BMPR2-sufficient (~100%) state: BMP9/10 predominantly signal through ALK1–BMPR2 complexes, generating pSMAD1/5/8 output consistent with a threshold-like requirement for proliferation; bimagrumab (BiMab) produces no effect detected under BMPR2-replete conditions. ( B ) BMPR2-limiting (~50%) state: Reduced BMPR2 attenuates BMP9/10-induced canonical output and is associated with reduced proliferation and increased caspase-3/7 activity consistent with stress/injury. Under BMPR2-limiting conditions, residual canonical output becomes bimagrumab-sensitive, consistent with context-dependent contribution of Activin type II receptors (predominantly ACVR2A in PAECs; see for BMP10 affinity comparisons) to the remaining pSMAD1/5/8 signal. A putative non-canonical stress-signaling arm is shown as a proposed intermediate. Solid arrows denote observed relationships; dashed arrows and dashed-outline boxes denote proposed steps. Node shading and output gauges depict relative canonical signaling output.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondria are efficiently internalized by HPMEC. (A) Representative live imaging of mitochondrial transplantation. Mitochondria isolated from MSC (MSC-mt) were prestained with MitoTracker Red and co-cultured with HPMEC prestained with MitoTracker Green for 24 h. Top panels: control. Middle panels: MSC-mt. Confocal imaging of internalized MSC-mt mitochondria (Mitotracker Red) co-localized to endogenous mitochondria network of non-stimulated HPMEC (MitoTracker Green). Lower panel: LPS + MSC-mt. MSC-mt internalized and co-localized to the endogenous mitochondrial network of HPMEC stimulated with LPS. The images were taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (B) Representative images of orthogonal views (Scale bar = 20 μm) and 3D reconstruction (Scale bar = 5 μm) on the LPS + MSC-mt group. (C) Analysis of region of interest (ROI) colocalization of control, positive control, and MSC-mt group. Data presented as mean ± SD.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Imaging, Transplantation Assay, Isolation, Cell Culture, Control, Microscopy, Positive Control

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: MSC-mt transplantation effects on mtDNA copies, viability, inflammatory activation, and mitochondrial respiration of recipient cells. (A) Quantification of mtDNA copy numbers in LPS-stimulated HPMEC after mitochondrial transplantation (24 h). (B) Effects on HPMEC viability measured by levels of LDH release. ( n = 3–4). (C) Levels of interleukin (IL)-8 secretion by HPMEC measured by ELISA ( n = 3–4). (D) Levels of TNF-α secreted by THP1 macrophages measured by ELISA ( n = 3–4). (E) Representation of Seahorse Mito Stress assay curve showing OCR in HPMEC. (F–H) Values for respiratory parameters: basal respiration (F), maximal respiration (G), and ATP production (H) ( n = 4–5). Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. Differences were assessed using Kruskal–Wallis with post-hoc Dunn’s test.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondrial transplantation alleviates mitochondrial dysfunction in HPMEC. (A) Representative images of JC-1 fluorescence in HPMEC. Green areas indicate depolarized mitochondrial membranes (JC-1 monomers), and red areas indicate polarized mitochondrial membranes (JC-1 aggregates). FCCP was used to induce mitochondria depolarization. Images taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (B) Representative images of HPMEC mitochondrial superoxide production detected with MitoSOX. Mitotempo (MT) was used as positive control for ROS quenching. Images taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (C) Quantification of red to green JC-1 fluorescence intensity ratio in HPMEC analyzed by ImageJ software. (D) Quantitative MitoSOX fluorescence intensity (MFI) analyzed by ImageJ software. Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. Differences were assessed using one-way ANOVA analysis with post hoc Bonferroni’s test.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Fluorescence, Microscopy, Positive Control, Software

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondrial transplantation restores HPMEC barrier integrity in the presence of LPS or ARDS plasma. (A) Real-time impedance analysis of HPMEC exposed to LPS and treated with MSC-mt and their respective cell impedance analysis of XCelligence RTCA measurements at a 24-h timepoint. Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. (B) Representative real-time impedance analysis of HPMEC exposed to hypoinflammatory ARDS plasma and their respective cell impedance analysis of XCelligence RTCA measurements at a 24 and 48-h timepoint. (C) Representative real-time impedance analysis of HPMEC exposed to hyperinflammatory ARDS plasma and their respective cell impedance analysis of XCelligence RTCA measurements at a 24 and 48-h timepoint. Data presented as mean ± SD. Differences were assessed using Kruskal–Wallis with post-hoc Dunn’s test (A) and one-way ANOVA analysis with post hoc Bonferroni’s test (B, C).

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Clinical Proteomics

Journal: Pharmaceutica analytica acta

Article Title: Omeprazole does not Potentiate Acute Oxygen Toxicity in Fetal Human Pulmonary Microvascular Endothelial Cells Exposed to Hyperoxia

doi: 10.4172/2153-2435.1000424

Figure Lengend Snippet: Omeprazole-treated HPMEC display increased CYP1A1 and NQO1 expression: Human pulmonary microvascular endothelial cells (HPMEC) were treated with dimethylsulfoxide (DMSO) or omeprazole (OM) at concentrations of 0.5 (OM 0.5), 5 (OM 5), 50 (OM 50) or 100 (OM 100) μM for up to 48 h, following which: RNA was extracted for CYP1A1 (A) and NQO1 (B) mRNA expression; and whole-cell protein was extracted for immunoblotting using anti-NQO1 or β-actin antibodies (C). Densitometric analyses wherein NQO1 band intensities were quantified and normalized to β-actin (D). Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). *, p<0.05 vs. DMSO-treated cells.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMEC), the primary microvascular endothelial cells derived from the lungs of human fetus were obtained from ScienCell research laboratories (San Diego, CA; 3000).

Techniques: Expressing, Western Blot

Journal: Pharmaceutica analytica acta

Article Title: Omeprazole does not Potentiate Acute Oxygen Toxicity in Fetal Human Pulmonary Microvascular Endothelial Cells Exposed to Hyperoxia

doi: 10.4172/2153-2435.1000424

Figure Lengend Snippet: Effects of OM on hyperoxia-induced cytotoxicity in HPMEC: Human pulmonary microvascular endothelial cells (HPMEC) treated with DMSO, OM 5 or OM 100 were exposed to air or hyperoxia for up to 48 h, following which: (A) Cell viability was assessed by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) reduction activities; and (B) Cell proliferation was determined based on the measurement of cellular DNA content via fluorescent dye binding using the CyQUANT NF cell proliferation assay. Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). Two-way ANOVA showed an effect of hyperoxia but not of OM treatment for the dependent variables, cell viability and proliferation, in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p<0.05.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMEC), the primary microvascular endothelial cells derived from the lungs of human fetus were obtained from ScienCell research laboratories (San Diego, CA; 3000).

Techniques: Binding Assay, CyQUANT Assay, Proliferation Assay

Journal: Pharmaceutica analytica acta

Article Title: Omeprazole does not Potentiate Acute Oxygen Toxicity in Fetal Human Pulmonary Microvascular Endothelial Cells Exposed to Hyperoxia

doi: 10.4172/2153-2435.1000424

Figure Lengend Snippet: Effects of OM on hyperoxia-induced H 2 O 2 generation in HPMEC: Human pulmonary microvascular endothelial cells (HPMEC) treated with DMSO, OM 5, or OM 100 were exposed to air or hyperoxia for up to 24 h, following which the H 2 O 2 levels was measured by ROS-Glo ™ H 2 O 2 assay. Data are representative of at least three independent experiments. Values are presented as means ± SEM (n=3). Two-way ANOVA showed an effect of hyperoxia and OM 100 and an interaction between them for the dependent variable, H 2 O 2 levels, in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p<0.05. Significant differences between hyperoxia-exposed DMSO-, OM 5-, and OM 100–treated cells are indicated by †, p<0.05. Significant differences between air-exposed DMSO-, OM 5-, and OM 100–treated cells are indicated by π, p<0.05.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMEC), the primary microvascular endothelial cells derived from the lungs of human fetus were obtained from ScienCell research laboratories (San Diego, CA; 3000).

Techniques:

Journal: Pharmaceutica analytica acta

Article Title: Omeprazole does not Potentiate Acute Oxygen Toxicity in Fetal Human Pulmonary Microvascular Endothelial Cells Exposed to Hyperoxia

doi: 10.4172/2153-2435.1000424

Figure Lengend Snippet: Effects of OM on NQO1 protein expression in hyperoxia-exposed HPMEC: Human pulmonary microvascular endothelial cells (HPMEC) were treated with DMSO, OM 5, or OM 100 for up to 48 h, following which whole-cell protein was extracted for immunoblotting using anti-NQO1 or β-actin antibodies (A). Densitometric analyses wherein NQO1 band intensities were quantified and normalized to β-actin (B). Values are presented as means ± SEM (n=3). Two-way ANOVA showed an effect of hyperoxia and OM 100 and an interaction between them for the dependent variable, NQO1 protein levels, in this figure. Significant differences between air- and hyperoxia-exposed cells are indicated by *, p<0.05. Significant differences between hyperoxia-exposed DMSO-, OM 5-, and OM 100–treated cells are indicated by †, p<0.05. Significant differences between air-exposed DMSO-, OM 5-, and OM 100–treated cells are indicated by π, p<0.05.

Article Snippet: Human pulmonary microvascular endothelial cells (HPMEC), the primary microvascular endothelial cells derived from the lungs of human fetus were obtained from ScienCell research laboratories (San Diego, CA; 3000).

Techniques: Expressing, Western Blot

Journal: JCI Insight

Article Title: 25-Hydroxycholesterol exacerbates vascular leak during acute lung injury

doi: 10.1172/jci.insight.155448

Figure Lengend Snippet: ( A ) Serum angiopoietin-2 was measured in mice at the indicated time points relative to high-dose LPS aerosol inhalation ( n = 13–14 per genotype). ( B ) Lung tissue from Ch25h +/+ and Ch25h –/– mice was analyzed by quantitative PCR (qPCR) for the targets shown following LPS inhalation ( n = 4–5 per genotype). ( C ) Lung homogenates were evaluated by immunoblot for VE-cadherin and β-actin (loading control) in Ch25h +/+ and Ch25h –/– mice at baseline and 48 hours or 72 hours after high-dose LPS aerosol inhalation. Immunoblot results from independent mice are shown in the left panel, and densitometry for actin-normalized VE-cadherin signal in the right panel ( n = 3–8 per condition). The dashed line indicates juxtaposition of nonadjacent portions of the original gel. ( D ) Lung tissue from WT mice was analyzed by qPCR for Vcam1 following i.p. treatment with 25HC or vehicle ( n = 10 per treatment). ( E ) Mouse pulmonary microvascular endothelial cells were cultured for 24 hours in 50% medium/50% BALF (0 or 48 hours after inhaled LPS) collected from mice of the indicated genotype and then analyzed by qPCR for Vcam1 . * P < 0.05, ** P < 0.01 by unpaired 2-tailed t test. FC, fold change.

Article Snippet: Transendothelial electrical resistance was measured across confluent human pulmonary artery endothelial monolayers (passage 4–6; Lonza) in medium containing 2% FBS, using an electrical cell-substrate impedance sensing system (Applied Biophysics), as previously described ( ).

Techniques: Aerosol, Real-time Polymerase Chain Reaction, Western Blot, Control, Cell Culture

Journal: JCI Insight

Article Title: 25-Hydroxycholesterol exacerbates vascular leak during acute lung injury

doi: 10.1172/jci.insight.155448

Figure Lengend Snippet: ( A – C ) Mice chimeric for Ch25h expression in hematopoietic and/or non-hematopoietic cells were produced by bone marrow transfer from Ch25h +/+ (WT) or Ch25h –/– (KO) mice into WT or KO recipients following sublethal irradiation (graphs show donor > recipient). Chimeras were then exposed to inhaled LPS. At 72 hours after exposure, protein ( A ) and albumin ( B ) were measured in BALF, and 25HC was measured in BALF, lung, or serum ( C ) ( n = 4–10 per condition, representative of 2 independent experiments). ( D ) Endothelial cells (CD45 – EpCAM – CD31 + ) were FACS-sorted from mouse lung 72 hours after saline or LPS inhalation. RNA was prepared and qPCR performed for Ch25h ( n = 2 saline, n = 6 LPS). In A – C , all chimeras with statistically significant differences in 25HC compared with WT>WT are indicated with asterisks. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 compared with WT>WT by 1-way ANOVA with Dunnett’s post hoc test. FC, fold change.

Article Snippet: Transendothelial electrical resistance was measured across confluent human pulmonary artery endothelial monolayers (passage 4–6; Lonza) in medium containing 2% FBS, using an electrical cell-substrate impedance sensing system (Applied Biophysics), as previously described ( ).

Techniques: Expressing, Produced, Irradiation, Saline

Journal: JCI Insight

Article Title: 25-Hydroxycholesterol exacerbates vascular leak during acute lung injury

doi: 10.1172/jci.insight.155448

Figure Lengend Snippet: ( A and B ) Human pulmonary artery endothelial cells (HPAECs) were cultured in medium with 2% FBS supplemented with vehicle or the indicated concentrations of 25HC ( A ) or cholesterol (CH; a negative control) ( B ), and transendothelial electrical resistance (TER) measurements were performed over a 70-hour time period. ( C ) HPAECs were incubated with vehicle or heat-killed S . aureus (HKSA; 2 × 10 8 particles/mL) for 30 minutes followed by treatment with vehicle or 25HC (20 μM) and TER monitored over 70 hours. ( D ) HPAECs were incubated with vehicle or LPS (100 ng/mL) for 30 minutes followed by treatment with vehicle or 25HC (20 μM) and TER monitored for 70 hours. Data are mean ± SEM and are representative of 2–3 independent experiments. The number of replicates per condition is given in the figure. Intercurve differences at 60 hours were analyzed by 1-way ANOVA with all pairwise post hoc comparisons and Tukey’s adjustment. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Transendothelial electrical resistance was measured across confluent human pulmonary artery endothelial monolayers (passage 4–6; Lonza) in medium containing 2% FBS, using an electrical cell-substrate impedance sensing system (Applied Biophysics), as previously described ( ).

Techniques: Cell Culture, Negative Control, Incubation

Journal: JCI Insight

Article Title: 25-Hydroxycholesterol exacerbates vascular leak during acute lung injury

doi: 10.1172/jci.insight.155448

Figure Lengend Snippet: ( A ) Lung tissue from Ch25h +/+ and Ch25h –/– mice was analyzed by qPCR for Chop 72 hours after LPS inhalation ( n = 10 per genotype). ( B ) Lung tissue from WT mice was analyzed by qPCR for the indicated targets after i.p. treatment with 25HC or vehicle ( n = 8–10 per condition). ( C ) Mouse pulmonary microvascular endothelial cells were cultured for 24 hours in 50% medium/50% BALF (0 or 48 hours after inhaled LPS) collected from mice of the indicated genotypes and then analyzed by qPCR for the indicated targets. ( D ) Mice of the indicated genotypes were treated with 100 mg/kg 4-phenylbutyric acid (PBA) or PBS vehicle (Veh.) i.p. at –1, +6, and +24 hours in relation to LPS inhalation and then had BALF IgM and albumin quantified at 72 hours after LPS ( n = 15–18 per condition). ( E ) Mice were treated as in D and then had the indicated cytokines quantified in BALF ( n = 15 per condition). Data are mean ± SEM and are representative of 2–3 independent experiments. Ψ P = 0.06, * P < 0.05, ** P < 0.01, *** P < 0.001 by unpaired 2-tailed t test. FC, fold change.

Article Snippet: Transendothelial electrical resistance was measured across confluent human pulmonary artery endothelial monolayers (passage 4–6; Lonza) in medium containing 2% FBS, using an electrical cell-substrate impedance sensing system (Applied Biophysics), as previously described ( ).

Techniques: Cell Culture