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

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 is expressed in human cardiac microvascular (CM), coronary artery (CA), dermal microvascular (DM) and pulmonary microvascular (PM) endothelial cells. ( b , c ) The vasoconstrictor response of Gata5 -null mice mesenteric arteries to norepinephrine is unaltered ( n =7 per group), while the vasodilatory response to acetylcholine is decreased ( n =10–11 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (comparison of best-fit values—effector concentration for half-maximum response (EC 50 ) and Hill slope—using an F-test). ( d – f ) Deletion of Gata5 in endothelial (e Gata5 -null mice; n =4–5 per group) but not smooth muscle cells (sm Gata5 -null mice; n =5 per group) decreases mesenteric arteries sensitivity to acetylcholine and increases BP (e Gata5 -null mice n =6–10 per group; sm Gata5 -null mice n =6–9 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (two-factor ANOVA). ( g ) The vasodilatory response of Gata5 -null mice to diethylamine NONOate, an NO donor, is unaltered ( n =7 per group). The results are reported as mean±s.e.m. (comparison of best-fit values—EC 50 and Hill slope—using an F-test). ( h , i ) NOS3 and Akt phosphorylation are decreased in Gata5 -null mice mesenteric arteries. PTEN and PDK1 phosphorylation and expression are unaltered ( n =5–7 per group). Phosphorylated proteins are normalized to total proteins. Total proteins are normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice ( t -test). ( j ) Quantification of protein nitrotyrosination in mesenteric arteries of Gata5 -null mice and their controls as measured by ELISA. 3-Nitrotyrosine content is expressed as picomole of nitrotyrosine per milligram of protein ( n =5–7 per group). The results are reported as mean±s.e.m. ( t -test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 expression is significantly decreased in human dermal microvascular endothelial cells infected with a lentiviral vector containing an anti- GATA5 shRNA (HDMEC-GATA5-KD). Control cells were infected with a vector containing a control shRNA (targets no known mammalian gene) (HDMEC-pLKO-Ctrl, referred here as Ctrl). ( b ) Heatmap representation of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls as identified by transcriptomic analysis. Colour is function of Log2 RMA (Affymetrix microarray, n =3 per group). ( c ) Functional analysis of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls. Protein kinase A pathway is the most significantly enriched pathway. Fisher's exact test P value. ( d ) Validation by qPCR (upper panel) of genes predicted by microarray (lower panel) to be up- and downregulated in HDMEC-GATA5-KD endothelial cells. ( n =5 wells per condition). Downregulated genes: PRKACB codes for the PKA catalytic subunit β, PRKAR2B for the PKA regulator subunit 2β and PRKAA2 for the AMPK catalytic subunit α2. Upregulated genes: ICAM1 codes for the intercellular adhesion molecule 1, BMP4 for the bone morphogenetic protein 4 and IL6 for the interleukin 6. The results are reported as mean±s.e.m. ** P <0.01 versus Ctrl ( t -test). ( e ) Western blot representation of phospho-NOS3, NOS3 (Ser1177) and phospho-(Ser/Thr) PKA substrate motif in HDMEC-GATA5-KD cells and their controls. ( f ) Phosphorylation of NOS3 on Ser1177 is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-NOS3 is normalized to total NOS3. NOS3 is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( g ) Phosphorylation of (Ser/Thr) PKA substrate motif is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( h ) Western blot representation of phospho-(Ser/Thr) PKA substrate motif in mesenteric arteries of Gata5 -null mice and their controls. ( i ) In mesenteric arteries of Gata5 -null mice, there is a trend to decrease in the (Ser/Thr) PKA substrate motif phosphorylation ( n =4–5 per group). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. (Mann–Whitney test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Microarray, Functional Assay, Western Blot, MANN-WHITNEY

Journal:

Article Title: Bone Morphogenetic Protein-2 Induces Proinflammatory Endothelial Phenotype

doi: 10.2353/ajpath.2006.050284

Figure Lengend Snippet: A: Representative Western blot (left) and densitometric data (right) showing the effect of increasing concentrations of anti-p42/p44 siRNAs on the expression of p42/44 MAP kinase in primary human coronary arterial endothelial cells (HCAECs). B: Effect of pretreatment with anti-p42/p44 siRNAs on BMP-2- and BMP-4- (10 ng/ml, for 2 hours) induced adhesion of fluorescently labeled PMA-stimulated monocytes to HCAECs. PD98059 (30 minutes, 10 μmol/L) was used to pharmacologically inhibit MAP kinase activity. TNF-α (10 ng/ml) was used as positive control. Data are mean ± SEM. *P < 0.05 versus control, #P < 0.05 versus BMP-2/4 treatment. C–F: Representative Western blots (C, E) and densitometric data (D, F) showing the time course of p42/44 MAP kinase phosphorylation in BMP-2 (10 ng/ml)-treated HCAECs (C, D) and rat carotid arterial segments (E, F). G: Representative Western blot (top) and densitometric data (bottom) showing BMP-2 (10 ng/ml, 10 minutes)-induced phosphorylation of p42/44 MAP kinase in HCAECs pretreated with DPI, chelerythrine, and PD98059.

Article Snippet: Studies on Endothelial Cell Cultures Primary rat coronary arterial endothelial cells (CAECs; Celprogen, San Pedro, CA), rat aortic vascular smooth muscle cells (VSMCs; Cell Applications Inc., San Diego, CA), and SV-40-immortalized rat aortic smooth muscle cells (SV40-SMC, no. CRL-2018; American Type Culture Collection, Manassas, VA) were maintained in culture as described.

Techniques: Western Blot, Expressing, Labeling, Activity Assay, Positive Control