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: 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: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Tree dendrogram depicting the relationship between different cell types. Whole-genome bisulfite sequencing (WGBS) data sets were included in the analysis. Average methylation was calculated for each sample within blocks of at least 3 CpGs and the top 30,000 blocks were selected that showed the highest variability across all samples. Unsupervised clustering of the reference WGBS samples was performed based on similarity in methylation status at these highly variable blocks. Samples from cell types with greater than n = 3 replicates were merged. ( B and C ) UMAP plot of human ( B ) and mouse ( C ) WGBS reference data sets. CAEC, coronary artery endothelial cell; CMEC, cardiac microvascular endothelial cell; CPEC, joint cardiopulmonary endothelial cell; HUVEV, human umbilical vein endothelial cell; LSEC, liver sinusoidal endothelial cell; MK, megakaryocyte; NK, natural killer cell; PAEC, pulmonary artery endothelial cell; PMEC, pulmonary microvascular endothelial cell.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Methylation Sequencing, Methylation

Journal: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Functions of genes adjacent to endothelium-specific methylation blocks (all P < 0.05). Blue color indicates nearby hypomethylated regulatory blocks. Yellow color indicates nearby hypermethylated regulatory blocks. ( B ) Example of the NOS3 locus specifically unmethylated in endothelial cells. This endothelium-specific, differentially methylated block (DMB; highlighted in light blue) is 157 bp long (7 CpGs), and is located within the NOS3 gene, an endothelium-specific gene (upregulated in paired RNA-seq data as well as in vascular endothelial cells, GTEx inset). The alignment from the UCSC genome browser (top) provides the genomic locus organization and is aligned with the average methylation (purple tracks) across cardiomyocyte, lung epithelial, liver sinusoidal endothelial cell (LSEC), cardiopulmonary endothelial cell (CPEC), hepatocyte, and immune (PBMC) samples ( n = 3/cell-type group). Results from RNA-seq generated from paired cell types are depicted (green tracks) as well as peak intensity from H3K27ac and H3K4me3 published ChIP-seq data generated in endothelial cells (blue tracks). ( C ) Expression levels of genes adjacent to tissue-specific endothelial methylation blocks. Expression data were generated from paired RNA-seq of the same CPEC and LSEC populations used to generate methylation reference data. Pan-endothelial genes upregulated in both populations (ALL) are identified as common endothelium-specific methylation blocks to both LSEC and CPEC tissue–specific endothelial populations. ( D ) Pathways related to the biological function of genes containing endothelium-specific methylation blocks (all Benjamini-Hochberg–corrected P < 0.05 by right-tailed Fisher’s exact test). Unique pathways to tissue-specific endothelial cells are highlighted in distinct colors. ( E ) Top 5 transcription factor binding sites enriched among endothelium-specific hypomethylated blocks, using HOMER de novo and known motif analysis (cumulative hypergeometric distribution statistic). The background for the HOMER analysis consisted of 3,589 non–endothelial cell-type–specific hypomethylated blocks. HUVEC, human umbilical vein endothelial cell.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Methylation, Blocking Assay, RNA Sequencing Assay, Generated, ChIP-sequencing, Expressing, Binding Assay

Journal: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Representative H&E staining of lung, heart, and liver tissues from mice treated with 3 Gy or 8 Gy radiation compared to sham control. Scale bar: 200 μm. ( B ) qPCR analysis of Cdkn1a (p21) mRNA. The expression in each sample was normalized to Actb and is shown relative to the expression in the sham control. Mean ± SD; n = 3. Kruskal-Wallis test was used for comparisons among groups: lung tissue, P = 0.004; heart tissue, P = 0.025; liver tissue, P = 0.004. ( C ) Lung endothelial, cardiomyocyte, and hepatocyte methylated cfDNA in the circulation of mice treated with 3 Gy and 8 Gy radiation compared to sham control expressed in genome equivalents per mL serum (Geq/mL). cfDNA was extracted from 18 mice ( n = 6 in each group), with cfDNA from 2 mice pooled in each methylome preparation. Mean ± SD; n = 3 independent methylome preparations. Kruskal-Wallis test was used for comparisons among groups. NS, P ≥ 0.05; * P < 0.05: lung endothelial, P = 0.01; cardiomyocyte, P = 0.01; hepatocyte, P = 0.13.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Staining, Control, Expressing, Methylation

Journal: JCI Insight

Article Title: Circulating cell-free methylated DNA reveals tissue-specific, cellular damage from radiation treatment

doi: 10.1172/jci.insight.156529

Figure Lengend Snippet: ( A ) Representative 3D-CRT treatment planning for patients with left-sided (left) and right-sided (right) breast cancer, respectively. The color map represents different radiation dose levels or isodose lines (green: 95% of prescription dose; isodose lines: yellow = 90%, cyan = 80%, orange = 70%, brown = 50%). ( B , D , and E ) Cardiopulmonary endothelial cell (CPEC), cardiomyocyte, and lung epithelial cfDNA (in Geq/mL) in serum samples. Fragment-level deconvolution used CPEC- ( n = 99), cardiomyocyte- ( n = 374), and lung epithelial cell–specific methylation blocks ( n = 69), respectively. Friedman test compared paired results at baseline, end of treatment (EOT), and recovery time points. * P < 0.05; CPEC P = 0.03, cardiomyocyte P = 0.01, lung epithelial P = 0.99. Mean ± SEM fold change relative to baseline levels is shown in bold ( n = 15). ( C ) Correlation of total endothelial cfDNA with dosimetry data. cfDNA is from deconvolution of pan-endothelial methylation blocks ( n = 131), the mean volume of the lung receiving the 5-Gy dose is represented by Lung V5 Mean (%). Spearman’s correlation r was calculated, and considered significant when * P < 0.05. ( F and G ) Hepatocyte and liver sinusoidal endothelial cell (LSEC) cfDNA (in Geq/mL) in serum samples. Fragment-level deconvolution used hepatocyte ( n = 200) and LSEC methylation blocks ( n = 61). Mean ± SEM fold change relative to baseline levels is shown in bold ( n = 8 right-sided, n = 7 left-sided breast cancer). Wilcoxon’s matched-pairs signed-rank test was used for comparison among groups. * P < 0.05; hepatocyte right-sided P = 0.02, hepatocyte left-sided P = 0.81, LSEC right-sided P = 0.02, and LSEC left-sided P = 0.93.

Article Snippet: Cryopreserved passage 2 human coronary artery endothelial cells (catalog C-14022), cardiac microvascular cells (catalog C-14029), and pulmonary artery endothelial cells (catalog C-14024) were isolated from single donor healthy human tissues purchased from PromoCell.

Techniques: Methylation, Comparison

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