Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial p53 Deletion Improves Angiogenesis and Prevents Cardiac Fibrosis and Heart Failure Induced by Pressure Overload in Mice

doi: 10.1161/JAHA.115.001770

Figure Lengend Snippet: Effect of p53 modulation on the angiogenic properties of endothelial cells in vitro. A, Representative western blot membranes showing the expression of p53 or its downstream effector p21 after incubation of human cardiac microvascular endothelial cells (HCMECs) with either PBS (1), vehicle (DMSO; 2), nutlin‐3a (10 μmol/L; 3) or pifithrin‐α (20 μmol/L; 4). Gapdh was used as internal control for equal protein loading. B, Representative pictures of HCMECs treated with either DMSO, nutlin‐3a or pifithrin‐α, followed by immunodetection of p53. C, Representative pictures of HCMEC spheroids after treatment with either DMSO, nutlin‐3a or pifithrin‐α. Magnification, ×200. D and E, Summary of the quantitative and statistical analysis. The number of sprouts (D) or the total sprout length (E) per spheroid were calculated using ImagePro Plus image analysis software. * P <0.05 and ** P <0.01 vs DMSO; ### P <0.001 vs nutlin‐3a, as determined using ANOVA. ANOVA indicates analysis of variance; DMSO, dimethyl sulfoxide.

Article Snippet: Human Cardiac Microvascular Endothelial Cells (HCMECs; PromoCell) were cultured at 37°C under 5% CO 2 in Endothelial Cell Growth Medium (EGM; PromoCell).

Techniques: In Vitro, Western Blot, Expressing, Incubation, Immunodetection, Software

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial p53 Deletion Improves Angiogenesis and Prevents Cardiac Fibrosis and Heart Failure Induced by Pressure Overload in Mice

doi: 10.1161/JAHA.115.001770

Figure Lengend Snippet: Effect of endothelial p53 deletion on transcription factors involved in mesenchymal differentiation. A through C, qPCR analysis of whole mouse hearts (n=9 per group) from End.p53‐KO (white bars) and End.p53‐WT mice (grey bars) 8 weeks after TAC for Snail (A), Slug (B) and Twist (C) mRNA. *** P <0.001 vs sham. D through F, HCMECs were stable transfected with lentiviral p53‐shRNA or negative control (scr) shRNA vector, treated with PBS or TGFβ1 (10 ng/mL) for 6 or 12 days and the expression of transcription factors regulating mesenchymal differentiation examined by qPCR analysis. * P <0.05, ** P <0.01 and *** P <0.001 vs PBS‐treated cells (n=3 to 6 separate experiments). Significant differences between p53 shRNA and scr shRNA transfected cells are indicated within the graphs. HCMEC indicates human cardiac microvascular endothelial cells; PCR, polymerase chain reaction; TAC, transverse aortic constriction; TGFβ, transforming growth factor‐beta.

Article Snippet: Human Cardiac Microvascular Endothelial Cells (HCMECs; PromoCell) were cultured at 37°C under 5% CO 2 in Endothelial Cell Growth Medium (EGM; PromoCell).

Techniques: Transfection, shRNA, Negative Control, Plasmid Preparation, Expressing, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Endothelial p53 Deletion Improves Angiogenesis and Prevents Cardiac Fibrosis and Heart Failure Induced by Pressure Overload in Mice

doi: 10.1161/JAHA.115.001770

Figure Lengend Snippet: Effect of p53 activation or inhibition on the TGFβ‐induced expression of the transcription factors Snail, Slug, and Twist. A through C, Quantitative real‐time PCR analysis of the transcription factors and EndMT surrogate markers Snail (A), Slug (B) and Twist (C) mRNA expression in human cardiac microvascular endothelial cells (HCMECs) after treatment with TGFβ1, alone or in combination with nutlin‐3a (to stabilize p53) or pifithrin‐α (to inhibit p53 activity) for 6 days. Similar findings were observed after treatment for 12 days (not shown). * P <0.05, ** P <0.01 and *** P <0.001 vs untreated cells; # P <0.05, ## P <0.01 and ## P <0.001 vs TGFβ1‐treated cells. PCR indicates polymerase chain reaction; TGFβ, transforming growth factor‐beta.

Article Snippet: Human Cardiac Microvascular Endothelial Cells (HCMECs; PromoCell) were cultured at 37°C under 5% CO 2 in Endothelial Cell Growth Medium (EGM; PromoCell).

Techniques: Activation Assay, Inhibition, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: Arterial thrombosis in the context of HCV-associated vascular disease can be prevented by protein C

doi: 10.1038/cmi.2016.10

Figure Lengend Snippet: Effect of poly (I:C) on the endothelial expression of procoagulatory factors and clotting time. HMEC were stimulated with poly (I:C) (10 μg/ml) for 12 h and the expression of tissue factor (a) and PAI-1 (b) was analyzed by RT–PCR (n=4, *P<0.05. mean±s.e., statistics with t-test (sigma plot); rel. to ct, relative to control). Comparable results were obtained in two series of independent experiments. (c) HMEC were stimulated with poly (I:C) (10 μg/ml) or TNFα (5 ng/ml) as a positive control for 24 h and then lysed. Whole blood samples were stimulated with cell lysates and the clotting time was analyzed as described in the Materials and methods section (n=5–6, *P<0.05. mean±s.e., statistics with t-test (sigma plot); rel. to ct, relative to control). Comparable results were obtained in two series of independent experiments. HMEC, human microvascular endothelial cell; poly (I:C), polyriboinosinic:polyribocytidylic acid; RT–PCR, reverse transcription–PCR. **P<0.01.

Article Snippet: Human microvascular endothelial cells (HMEC) were provided by Ades et al. 14 and were cultured in M199 media supplement with 10% fetal calf serum, 10% endothelial growth media (PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin as described previously.

Techniques: Expressing, Coagulation, Reverse Transcription Polymerase Chain Reaction, Positive Control

Journal: Cellular and Molecular Immunology

Article Title: Arterial thrombosis in the context of HCV-associated vascular disease can be prevented by protein C

doi: 10.1038/cmi.2016.10

Figure Lengend Snippet: Poly (I:C) did not influence platelet aggregation and activation. Light transmission aggregometry (the method by Born)18 was performed in platelet-rich-plasma (PRP) from healthy human volunteers, as described in the Materials and methods section. The percent light transmission of platelet-rich plasma (PRP) was compared with platelet poor plasma (PPP) on stimulation with poly (I:C) (10 μg/ml) or ADP (10 μM) (a and b). PRP was incubated with poly (I:C) (10 μg/ml) for different time intervals (10, 20, 30, 45 min) and ADP-dependent (5 μM) platelet aggregation was analyzed (c). PRP was incubated with poly (I:C) (10 μg/ml) for 15 min in the presence of ADP at a low concentration (5 μM), ADP at a high concentration (10 μM), thrombin-receptor-activated peptide (TRAP) (20 μM) or collagen (10 μg/ml) and platelet aggregation was analyzed (n=4, P>0.05, mean±s.e., statistics with t-test (sigma plot)) Comparable results were obtained in two series of independent experiments. (d) Human platelets were isolated as described in the Materials and methods section. The platelets were stimulated with poly (I:C) (10 μg/ml) for different time intervals (10, 60 min) alone or in the presence of thrombin (2 U/ml) and FACS analysis with a monoclonal antibody against P-selectin (e and f) and GPIIbIIIa (g and h) was performed (n=3–4, P>0.05, mean±s.e., statistics with one-way ANOVA (sigma plot)). Comparable results were obtained in two series of independent experiments. ANOVA, analysis of variance; HMEC, human microvascular endothelial cell; poly (I:C), polyriboinosinic:polyribocytidylic acid.

Article Snippet: Human microvascular endothelial cells (HMEC) were provided by Ades et al. 14 and were cultured in M199 media supplement with 10% fetal calf serum, 10% endothelial growth media (PromoCell, Heidelberg, Germany) and 1% penicillin/streptomycin as described previously.

Techniques: Activation Assay, Transmission Assay, Incubation, Concentration Assay, Isolation

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: To evaluate replication efficiency, lung microvascular endothelial cells (LMECs) were plated on the (A) apical or (B) basolateral side of a transwell filter. LMECs were inoculated with pH1N1, H1N1, or H3N2 virus at MOI 1 and at the indicated time points supernatants of the apical as well as basolateral side were harvested and virus titers were determined by endpoint titration. Infection efficiency was determined by immunofluorescence staining. LMECs plated on the (C) apical and (D) basolateral were inoculated with pH1N1, H1N1, or H3N2 virus. Cells were fixed 24 hours post inoculation and stained for the endothelial cell marker Vascular Endothelial-Cadherin (VE-CAD, magenta) and influenza A virus nucleoprotein (NP (green). Hoechst (blue) was used to visualize nuclei. (E) Percentage of infection determined by flow cytometry at 24 and 72 hours post inoculation. (F) Viral RNA genome copies were quantified by quantitative real time PCR at indicated time points. Data represent mean +/- standard deviation (SD) from at least three independent experiments performed in biological duplicates and flow cytometry was performed in biological triplicates. A one-way ANOVA multiple comparison test was used to compare groups (*< 0.05, **<0.01, ***<0.005). Scale bar: 20 μm.

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Titration, Infection, Immunofluorescence, Staining, Marker, Flow Cytometry, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: (A) Well-differentiated airway organoids at air-liquid interface (AO at ALI) in co-culture with lung microvascular endothelial cells (LMECs) were inoculated with pH1N1, H1N1 or H3N2 virus at MOI 1. At the indicated timepoints virus titers were determined in the supernatants of the apical and basolateral compartments. (B) Detection of influenza A virus (IAV) nucleoprotein (NP) by immunohistochemistry of the AO at ALI-LMEC co-cultures 24 hours post inoculation (C) Hematoxylin and eosin (H&E) staining of the co-cultures 72 hours post inoculation (scale bar 20 μm) (D) At 72 hours post inoculation well-differentiated AO at ALI were stained for IAV NP (green), the cilia marker acetylated-α-tubulin (cyan) and the tight-junction marker Zona occludin 1 (ZO-1, magenta) on the apical compartment of the transwell. The basolateral compartment containing the LMECs was stained for IAV NP (green) and the endothelial cell marker Vascular-Endothelial Cadherin (VE-CAD, magenta). In both cases the nuclei were visualized with Hoechst (scale bar 20 μm). (E) Epithelial cells (AO at ALI) or endothelial-epithelial co-cultures were inoculated with pH1N1, H1N1 or H3N2 virus at MOI 1. At 24 hours post-inoculation cytokines were measured in the apical compartment using the Legendplex assay. Data represented here show individual data points of cytokines derived from three independent experiments performed in biological duplicates and the mean +/- standard deviation (SD) is depicted. Mock of each condition was subtracted from the values of virus infected cells. Statistical significance was determined with Students-T-test (*<0.05, **<0.01, ***<0.005, ****<0.001).

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Co-Culture Assay, Immunohistochemistry, Staining, Marker, Derivative Assay, Standard Deviation, Infection

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: Lung microvascular endothelial cells (LMECs) or differentiated airway organoids at air-liquid interface in co-culture with LMECs were inoculated with pH1N1 virus at MOI 1. (A) percentage of infection and (B) viral genome copies in LMEC single cultures compared to co-cultures were determined by flow cytometry or qRT-PCR at 24 hours post-inoculation. Data represented here show pooled data of virus titers derived from three independent experiments performed in biological duplicates and the mean +/- standard deviation is depicted. A student T-test was used to compare groups (*<0.05, **<0.01, ***<0.005, ****<0.001).

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Co-Culture Assay, Infection, Flow Cytometry, Quantitative RT-PCR, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: The pro-inflammatory response to influenza A virus infection is fueled by endothelial cells

doi: 10.1101/2022.08.19.504520

Figure Lengend Snippet: (A) Endothelial cells (lung microvascular endothelial cells), epithelial cells (airway organoids at air-liquid interface) and endothelial-epithelial co-cultures were inoculated with pH1N1, H1N1 and H3N2 virus at MOI 1. At 24 hours post-inoculation cytokines were measured in the basolateral compartment using a Legendplex assay. Data represented here show individual data points of cytokines derived from three independent experiments performed in biological duplicates and the mean +/- standard deviation is depicted. Mock of each condition was subtracted from the values of virus inoculated cultures. Statistical significance was determined with One-Way Anova and each group was compared to each other (*<0.05, **<0.01, ***<0.005, ****<0.001).

Article Snippet: Primary human lung microvascular endothelial cells (LMEC) were purchased at passage 3 from PromoCell-PromoKine (#C-12285) and cultured in 1% gelatine coated cell cultures flasks with Endothelial Cell Growth Medium MV-2 kit according to the manufacturer’s protocol (#C-22121, PromoCell-PromoKine), LMECs were only used up to passage 10 to ensure organ specificity.

Techniques: Derivative Assay, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Modular Microphysiological System for Modeling of Biologic Barrier Function

doi: 10.3389/fbioe.2020.581163

Figure Lengend Snippet: Recapitulating alveolar microenvironment: (A) Cartoon of alveolar expansion during inspiration. Created with BioRender.com. (B) Calculated bi-axial strain in response to applied pressure in the microfluidic chip. (C) Application of a cyclic pressure in the apical channel using a sine wave to mimic breathing at approximately 20 breaths per minute. A constant pressure was applied to the basal channel to drive the flow of PBS. (D) Workflow for development of ALI co-culture. Endothelial cells (LMECs) were first seeded on the basal surface of a transwell culture insert, followed alveolar epithelial cells (AECs). Dexamethasone was added to the apical chamber on Day 3 to enhance the epithelial cell barrier. On Day 7, the ALI was induced by removing media from the apical chamber. On Day 10, the membrane was cut from the transwell support and bonded into the microfluidic chip and exposed to dynamic strain. (E) TEER measurements during liquid-liquid co-culture in the transwell with (Dex +) and without (Dex-) dexamethasone. Statistical analysis by unpaired t -test ( N = 3). (F) 3D visualization of fluorescent z -stack after culture for 24 h under cyclic pressure exposure in the MPS. Cytokeratin (green) staining shows the alveolar endothelial cells on the apical side of the membrane, while PECAM1 (red) staining indicates the lung microvascular endothelial cells on the basal side.

Article Snippet: Primary human glomerular microvascular endothelial cells (GMECs; Cell Systems) were culture per the manufacturer in complete endothelial cell growth media 2 (PromoCell).

Techniques: Co-Culture Assay, Staining

Journal: bioRxiv

Article Title: Aging-regulated TUG1 is dispensable for endothelial cell function

doi: 10.1101/2022.02.27.482212

Figure Lengend Snippet: (A) Top 10 expressed lncRNAs based on transcript counts from HUVEC bulk RNA sequencing data (n = 4). TUG1 is highlighted in green. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) and Kinase Insert Domain Receptor ( KDR ) were used as controls. (B) RNA expression levels of TUG1 in different human cell types of the cardiovascular system (n=3). Vascular ECs are highlighted by grey bars. AoEC: Aortic ECs, PAEC: Pulmonary Artery ECs, CAEC: Coronary Artery ECs, CMEC: Cardiac Microvascular ECs, DMEC: Dermal Microvascular ECs, PMVEC: Pulmonary Microvascular ECs, SaVEC: Saphenous Vein ECs, HUVEC: Human Umbilical Vein ECs, DLEC: Dermal Lymphatic ECs, MSC: Mesenchymal Stem Cells, AoAF: Aortic Arterial Fibroblasts, AoSMC: Aortic Smooth Muscle Cells, CM: Cardiomyocytes (C) TUG1 expression levels in low (P3) vs. high (P16) passage HUVECs as determined by RT-qPCR. Expression is relative to GAPDH (n = 5-6; SEM; Mann-Whitney-test). (D) Tug1 expression from bulk RNA-sequencing data of the intima of the carotid arteries of young (10 weeks) vs. aged mice (18 months) (n = 3; SEM; Mann-Whitney-test).. (E) Quantification of the expression levels of the lncRNAs Differentiation Antagonizing Non-Protein Coding RNA ( DANCR ), TUG1 and Metastasis Associated Lung Adenocarcinoma Transcript 1 ( MALAT1 ) in subcellular fractions of wild type HUVECs using RT-qPCR (n=3). Results are expressed as percentages of the subcellular fractions associated to cytoplasm, nucleoplasm and chromatin. Expression is normalized to GAPDH as determined by RT-qPCR.

Article Snippet: Alternatively, total RNA was isolated from cell pellets from cardiomyocytes, aortic fibroblasts, pericytes, aortic smooth muscle cells, mesenchymal stem cells, dermal lymphatic endothelial cells, umbilical vein endothelial cells, saphenous vein endothelial cells, pulmonary microvascular endothelial cells, dermal microvascular endothelial cells, cardiac microvascular endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells and aortic endothelial cells (all human; Promocell) with the miRNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions including DNase digest.

Techniques: RNA Sequencing Assay, RNA Expression, Expressing, Quantitative RT-PCR, MANN-WHITNEY