Journal: Journal of Cell Science

Article Title: Contribution of protein–protein interactions to the endothelial-barrier-stabilizing function of KRIT1

doi: 10.1242/jcs.258816

Figure Lengend Snippet: Membrane-targeted KRIT1-RE does not rescue endothelial barrier function. (A) Permeability of transduced monolayers to 40 kDa FITC-dextran. Data shown are mean permeability±s.e.m., normalized to scramble shRNA alone (scr), from n =5 independent experiments. (B) TEER of confluent HPAEC monolayers. Resistance reading of an empty FN-coated Transwell was subtracted from resistance values of Transwells containing HPAEC, then multiplied by the growth area of the wells, yielding Ω*cm 2 values. Data shown are mean Ω*cm 2 values±s.e.m., normalized to scramble shRNA alone (negative control). * P <0.05 by Tukey post-hoc testing vs scramble shRNA alone. # P <0.05 by Tukey post-hoc testing vs shKRIT1 alone. P <0.0001 by one-way ANOVA.

Article Snippet: Human pulmonary artery endothelial cells (HPAEC; Cell Applications, Inc., San Diego, CA, Lot #2228) were cultured in Dulbecco's Modified Eagle's Medium DMEM/F-12 (1:1 ratio), supplemented with 5% fetal bovine serum (FBS), 1% antibiotic-antimycotic solution (Gibco/Thermo Scientific, Waltham, MA), 1% endothelial cell growth supplement (ECGS; ScienCell, Carlsbad, CA), and 50 μM heparin (Calbiochem, La Jolla, CA).

Techniques: Membrane, Permeability, shRNA, Negative Control

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A, B) Lung sections of mice conditionally lacking Egfr in ECs were stained for CD68 (green), iNOS (white, A) and Arg1 (red, B). (A, B) The numbers of iNOS + (A) and Arg1 + (B) macrophages were quantified by confocal imaging. (C, D, E) Human pulmonary arterial endothelial cells (PAECs) were transfected with either scrambled siRNA (si CTL ) and siRNA against AREG (si AREG ) and placed in a transwell chamber. They were then cultured in hypoxic conditions with or without leukocytes for 24 h. (C) AREG expression was assessed in leukocytes and HPAECs by qPCR. (D) Apoptosis of normoxic and hypoxic HPAECs was quantified by flow cytometry and shown as fold change compared with the level of apoptosis in si CTL PAECs. (E) HPAECs were plated on Matrigel, and tube formation was measured. (F) Human PAECs were treated with increasing concentrations (10–100 ng/ml) of recombinant amphiregulin or vehicle and placed under normoxic conditions. PAECs apoptosis was assessed by measuring caspase 3 + cells and caspase 3 MFI by flow cytometry. Isotype control was used to determine caspase 3 positivity. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Staining, Imaging, Transfection, Cell Culture, Expressing, Flow Cytometry, Recombinant

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: , (A) Schematic representing AREG and its upstream genes including BRCA1 , HLX , NCOA6 , PHB2 , RRP1B , TAF4 , TP63 , and VAV2 was generated using the Ingenuity Pathway Analysis Software. Each arrow represents the activation of AREG by each gene. (B) Schematic depicting HIF-1⍺–binding sites in PHB2 , RRP1B , and NCOA6 gene promoter regions. This schematic was designed using UCSC Genome Browser website ( https://genome.ucsc.edu ) and Snapgene software ( https://www.snapgene.com ). (C, D, E) Pulmonary arterial endothelial cells (PAECs) were transfected with scrambled siRNA (si CTL ) or siRNA against NCOA6 ( siNCOA6 ), PHB2 (si PHB2 ) or RRP1B (si RRP1B ) and placed in normoxia for 24 h. (C) Apoptotic PAECs were quantified by measuring caspase 3 + cells and caspase 3 MFI by flow cytometry. (D) PAECs were plated on Matrigel, and tube formation was assessed. (E) AREG expression was assessed by qPCR. (F) Human PAECs were transfected with either scrambled siRNA (si CTL ), or siRNA against HIF1A and placed in normoxic or hypoxic conditions for 24 h. AREG expression was quantified by qPCR. (G) Human PAECs were transfected with a lentivirus overexpressing HIF1-A for 48 h. The cells were transfected with siRNA against NCOA6 , PHB2 , or RRP1B and cultured in normoxic conditions for 48 h. AREG expression was assessed by qPCR. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01, *** P < 0.005.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Generated, Software, Activation Assay, Binding Assay, Transfection, Flow Cytometry, Expressing, Cell Culture

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A) BAD expression in pulmonary arterial endothelial cells (PAECs) was quantified after AREG silencing (left panel) and recombinant Amphiregulin treatment (right panel). (B, C, F) PAECs were transfected with either scrambled siRNA (si CTL ) or siRNA against BAD (si BAD ) and placed in hypoxia for 24 h. (B, C) PAECs apoptosis was assessed by measuring caspase 3 + and caspase 3 MFI cells by flow cytometry (B), and tube formation ability was determined by a Matrigel assay (C). (D, E) PAECs were transfected with either scrambled siRNA (si CTL ), siRNA against AREG (si AREG ) or siRNA against both AREG and BAD (si BAD/AREG ) and placed in normoxic conditions for 24 h. (D, E) Apoptosis (D) and tube formation (E) were examined. (F) IFNB , IL1B , IL6 , and TNFA expression was assessed by qRT-PCR. n = 5 replicates per condition. Data are shown as mean. * P < 0.05, *** P < 0.005, **** P < 0.001.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Expressing, Recombinant, Transfection, Flow Cytometry, Matrigel Assay, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: Loss of Amphiregulin drives inflammation and endothelial apoptosis in pulmonary hypertension

doi: 10.26508/lsa.202101264

Figure Lengend Snippet: (A) BAD expression and the frequency of BAD + cells were determined by flow cytometry after AREG silencing in normoxic and hypoxic pulmonary arterial endothelial cells (PAECs). (B) PAECs were treated with increasing concentrations (10–100 ng/ml) of recombinant amphiregulin or vehicle and placed under normoxic conditions. BAD expression was measured by RT-qPCR. (C, D) HPAECs were co-cultured in a transwell with leukocytes and then treated with either control or BAD siRNA. (C) Granulocytes, monocytes, and T cells were enumerated by flow cytometry. (D) Cytokine concentrations were assessed by ELISA. (E) Mechanisms of increased PAEC apoptosis and exaggerated inflammation in the absence of AREG and epidermal growth factor receptor (EGFR) in pulmonary hypertension (PH). Our data support a model whereby decreased amphiregulin and EGFR expression in PAECs promote PH. Specifically, in the steady state, amphiregulin binds to the EGFR, which decreases the expression of BCL2-associated agonist of Cell Death (BAD), resulting in PAEC survival and suppressed inflammation. In PH, HIF-1⍺ binds to the promoters of NCOA6 , PHB2 , and RRP1B and increases their expression. These genes down-regulate AREG , resulting in augmented BCL2 expression. This pro-apoptotic gene, in turn, incites apoptosis and chemokine production. Elevated levels of the chemokines recruit inflammatory myeloid cells in lung vasculature. Mechanisms that were not investigated in the present study are labeled with a dotted arrow. The cartoon was designed with the online Biorender software ( https://biorender.com ). n = 5 replicates per condition. Data are shown as mean. * P < 0.05, ** P < 0.01.

Article Snippet: Human PAECs were obtained from Promocell and grew in Endothelial Basal Medium-2 (Promocell).

Techniques: Expressing, Flow Cytometry, Recombinant, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling, Software

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: DMOG suppresses endothelial cell proliferation, migration, and tube formation (A) Representative bright field images of formazan crystal formed after 3 h incubation of MTT with vehicle or DMOG-treated (1 mM) HPAEC. Right graph shows relative HPAEC proliferation assessed by MTT assay. Scale bar, 100μm. (B) Representative images of BrdU immunostaining. Right graph shows semi-quantitative analysis of BrdU positive cells/hpf. Scale bar, 50μm. (C) Representative histograms of cell cycle analysis for control and DMOG-treated cells. Right side graph demonstrates relative percentages of cell populations in G0/G1, S, and G2/M cell cycle phases. (D) Representative images of 2D scratch wound assay of control and DMOG-treated cells and semi-quantitative analysis of healed area after 24 h. Scale bar, 200μm. (E) Representative images of tubes formed at indicated time points in control and DMOG-treated cells and semi-quantitative analysis of different parameters at 20 h time point. Scale bar, 200μm. Data are pooled from 3 independent experiments and represented as mean ± SEM. Statistics were determined by two-tailed t-test. ∗∗, p < 0.01; ∗∗∗∗, p < 0.0001; ns, not statistically significant. See also Figure S1 .

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Migration, Incubation, MTT Assay, Immunostaining, Cell Cycle Assay, Control, Scratch Wound Assay Assay, Two Tailed Test

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: DMOG alters the endothelial cell metabolome (A) Shown are the top 25 downregulated (upper graph) and upregulated (lower graph) metabolic pathways detected by metabolites set enrichment analysis in DMOG-treated cells compared to control. Scaled intensity values indicating relative levels of metabolites related to glycolysis (B) and TCA cycle (C). (D) NAD + /NADH ratio in cells treated with vehicle or DMOG. Scaled intensity values indicating relative levels of lipid metabolites (E), nucleotides (F), and amino acids (G). n = 5 independent samples per condition. All statistical data are represented as mean ± SEM and statistics were determined by a Welch’s two sample t-test. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant. G6P, glucose-6-phosphate; FBP, fructose 1,6 bisphosphate; DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; AKG, alpha-ketoglutarate; DPA, docosapentaenoate; DHLA, dihomolinolenate; ALC, acetylcarnitine; CHOP, choline phosphate; GPC, glycerophosphorylcholine; PEA, phosphoethanolamine; GPEA, glycerylphosphorylethanolamine; G3P, glycerol 3-phosphate; 5′-AMP, adenosine-5′-monophosphate; 5′-ADP, adenosine-5′-diphopshate; 5′-CMP, cytidine 5′-monophosphate; CDP, cytidine diphosphate; 2′,3′-cCMP, cytidine 2′,3′-cyclic monophosphate; 5′-UDP, uridine-5-diphosphate; UTP, uridine 5′-triphosphate. See also Figure S4 and Table S1 .

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Control

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: Citrate supplementation partially rescues the DMOG-induced defects in endothelial migration and tube formation capacity (A) Representative bright field images of formazan crystal formed after MTT incubation with control, DMOG (1mM), DMOG + citrate and citrate (0.5mM)-treated HPAEC. Right graph shows relative HPAEC proliferation calculated by MTT assay. Scale bar, 100μm. (B) Representative images of BrdU immunostaining under the conditions indicated in A. Right graph shows semi-quantitative analysis of BrdU positive cells per hpf. Scale bar, 50μm. (C) Quantitative analysis of cell cycle showing relative percentage of cell population in G0/G1, S, and G2/M cell cycle phase. (D) Representative images of 2D scratch wound assay of control or DMOG-treated cells and semi-quantitative analysis of healed area after 24 h. Scale bar, 200μm. (E) Representative images of tubes formed at the indicated time points in control, DMOG, DMOG + citrate, and citrate-treated cells and semi-quantitative analysis of different parameters at 20 h time point. Scale bar, 200μm. Data are pooled from 3 independent experiments and represented as mean ± SEM. Statistics were determined by one-way ANOVA with Sidak correction for multiple comparisons. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant. Asterisks above bars indicate significant difference between control and treated group, whereas asterisks above lines indicate significant difference between DMOG and DMOG + citrate treated groups. See also Figures S5–S7 .

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Migration, Incubation, Control, MTT Assay, Immunostaining, Scratch Wound Assay Assay

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet: Nicotinamide Riboside supplementation partially rescues the DMOG-induced defects in endothelial migration and tube formation capacity (A) Representative bright field images of formazan crystal formed after incubation with MTT in control, DMOG (1mM), DMOG + NR and NR (200μM)-treated HPAEC. Right graph shows relative HPAEC proliferation assessed by MTT assay. Scale bar, 100μm. (B) Representative images of BrdU immunostaining. Right graph shows semi-quantitative analysis of BrdU positive cells/hpf. Scale bar, 50μm. (C) Representative images of 2D scratch wound assay and semi-quantitative analysis of healed area after 24 h. Scale bar, 200μm. (D) Representative images of tubes formed at different time points in control, DMOG, DMOG + NR and NR-treated ECs and semi-quantitative analysis of different parameters at 20 h time point. Scale bar, 200μm. Data are pooled from 3 independent experiments and represented as mean ± SEM. Statistics were determined by one-way ANOVA with Sidak correction for multiple comparisons. ∗∗, p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001; ns, not significant. Asterisks above bars indicate significant difference between control and treated group, whereas asterisks above lines indicate significant difference between DMOG and DMOG + NR-treated groups. NR, nicotinamide riboside.

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Migration, Incubation, Control, MTT Assay, Immunostaining, Scratch Wound Assay Assay

Journal: iScience

Article Title: Chemical inhibition of oxygen-sensing prolyl hydroxylases impairs angiogenic competence of human vascular endothelium through metabolic reprogramming

doi: 10.1016/j.isci.2022.105086

Figure Lengend Snippet:

Article Snippet: Primary Pulmonary Artery Endothelial Cells; Normal, Human (HPAEC) , ATCC , Cat# PCS-100-022.

Techniques: Purification, Recombinant, Lysis, Extraction, Protease Inhibitor, Angiogenesis Assay, In Vitro, Enzyme-linked Immunosorbent Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Software, Western Blot, Membrane

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Protective role of N-acetylcysteine and Sulodexide on endothelial cells exposed on patients’ serum after SARS-CoV-2 infection

doi: 10.3389/fcimb.2023.1268016

Figure Lengend Snippet: Intracellular generation of free radicals in CAEC exposed to culture medium (Medium), culture medium supplemented with 20% control serum (Control), 20% Post-COVID-19-serum (Post-COVID), 20% Post-COVID-19 serum supplemented with N-Acetylcysteine 1 mmol/L (Post-COVID+NAC), or Post-COVID-19 serum with Sulodexide 0.5 LRU/mL (Post-COVID+Sul).

Article Snippet: During the experiments, the primary cultures of human coronary artery endothelial cells (CAEC) obtained from Cell Applications, Inc. (San Diego, California, USA) were used.

Techniques: Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Protective role of N-acetylcysteine and Sulodexide on endothelial cells exposed on patients’ serum after SARS-CoV-2 infection

doi: 10.3389/fcimb.2023.1268016

Figure Lengend Snippet: Synthesis of IL-6 (A) and vWF (B) in CAEC exposed to culture medium (Medium), culture medium supplemented with 20% control serum (Control), 20% Post-COVID-19 serum (Post-COVID), 20% Post-COVID-19 serum supplemented with N-Acetylcysteine 1 mmol/L (Post-COVID+NAC), or Post-COVID-19 serum with Sulodexide 0.5 LRU/mL (Post-COVID+Sul).

Article Snippet: During the experiments, the primary cultures of human coronary artery endothelial cells (CAEC) obtained from Cell Applications, Inc. (San Diego, California, USA) were used.

Techniques: Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Protective role of N-acetylcysteine and Sulodexide on endothelial cells exposed on patients’ serum after SARS-CoV-2 infection

doi: 10.3389/fcimb.2023.1268016

Figure Lengend Snippet: Synthesis of tPA (A) and PAI-1 (B) in CAEC exposed to culture medium (Medium), culture medium supplemented with 20% control serum (Control), 20% Post-COVID-19 serum (Post-COVID), 20% Post-COVID-19 serum supplemented with N-Acetylcysteine 1 mmol/L (Post-COVID+NAC),or Post-COVID-19 serum with Sulodexide 0.5 LRU/mL (Post-COVID+Sul).

Article Snippet: During the experiments, the primary cultures of human coronary artery endothelial cells (CAEC) obtained from Cell Applications, Inc. (San Diego, California, USA) were used.

Techniques: Control