primary human lung microvascular endothelial cells hlmvecs  (PromoCell)

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: FIGURE 1 | 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: FIGURE 3 | 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/perimeter2)) 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: FIGURE 4 | 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: FIGURE 6 | 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

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: Cigarette smoke extract alters PP2A activity and distribution within endothelial cells. (A) Human microvascular lung endothelial cells (HLMVEC) were grown to 80% confluence in 6-well plates and treated with 5% CSE for 10 min, 30 min, 1, 6, 12 and 24 h. PP2A activity assays were conducted on lysate protein isolated from the cells at baseline and after 5% CSE treatment. (B) PP2A activity assays were conducted on cytosolic, nuclear, cytoskeletal and membrane protein fractions of HLMVECs treated with and without 5% CSE for 24 h. (C) Immunoblots for the catalytic subunit of PP2A (PP2AC) were conducted on membrane fractions of HLMVECs treated with control media or 5% CSE for 24 h. Actin immunoblots were conducted on membrane fractions as a loading control.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Activity Assay, Isolation, Membrane, Western Blot, Control

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: . PTP1B regulates PP2A activity in endothelial cells and cigarette smoke extract reduces PTP1B activity (A) PP2A activity assays were conducted on human lung microvascular endothelial cells grown to 70% confluence in 6-well plates and then treated with control, NSMase or PTP1B siRNA for 24 h. (B) PTP1B activity assays were conducted on human microvascular lung endothelial cells (HLMVEC) grown to 80% confluence in 6-well plates and treated with 5% CSE for 6–24 h.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Activity Assay, Control

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: Cigarette smoke extract decreases calcium efflux and regulates PP2A activity in a calcium-dependent manner . (A) Human microvascular lung endothelial cells (HLMVEC) were grown to 80% confluence in 24-well plates and treated with control media or media with 1, 2, 5 or 10% CSE for 10 min. A calcium release assay was conducted on media collected from these cells. (B) PP2A activity assays were conducted on human microvascular lung endothelial cells (HLMVEC) grown to 80% confluence in 6-well plates and were treated with low calcium media that was switched to high calcium media or high calcium media that was switched to low calcium media after 5% CSE treatment.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Activity Assay, Control, Release Assay

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: Cigarette smoke extract and PP2A inhibition alter endothelial cell permeability and occluding phosphorylation. (A) FITC permeability assays were conducted on human microvascular endothelial cells monolayers grown to 100% confluence on 3-µm pore collagen-coated PTFE membranes that were treated with 5% CSE for 2, 4, 6, 18 and 24 h. (B) FITC permeability assays were conducted on human microvascular endothelial cell monolayers grown to 100% confluence on 3-µm pore collagen-coated PTFE membranes that were treated with 1-µM Fostriecin for 24 h. (C) Occludin protein was immunoprecipitated from the endothelial cell lysates using a specific antibody. Immunoblots for p-threonine and total occludin were conducted on the immunoprecipitated protein.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Inhibition, Permeability, Phospho-proteomics, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Cigarette smoke alters calcium flux to induce PP2A membrane trafficking and endothelial cell permeability

doi: 10.1038/s41598-024-77776-x

Figure Lengend Snippet: PP2A C silencing increases neutrophil adhesion in lung endothelial cells. Human lung microvascular endothelial cells were grown to 70% confluence in 6-well plates and then treated with control or PP2A C siRNA for 24 h. The wells were incubated with Calcein-labeled neutrophils and the fluorescence intensity of each well was then measured.

Article Snippet: Primary human lung microvascular endothelial cells (PromoCell Gmbh, Heidelberg, Germany) were grown to 70–80% confluence in endothelial cell growth media in 6-well plates.

Techniques: Control, Incubation, Labeling, Fluorescence