Journal: Diabetes & Vascular Disease Research

Article Title: Mitochondrial mitophagy protection combining rivaroxaban and aspirin in high glucose-exposed human coronary artery endothelial cell. An in vitro study

doi: 10.1177/14791641221129877

Figure Lengend Snippet: Panel a.- Representative Western-blot of the Factor Xa (FXA) expression in human coronary artery endothelial cells (HCAEC) incubated with normal D-glucose concentration (5mmol/L, control) and in the presence of 30 mmol/L D-Glucose (+Glucose). Panel b.- Representative Western-blot of tissue factor (TF) expression in HCAEC incubated with normal D-glucose concentration (5 mmol/L, control) or with high glucose, 30 mmol/L D-Glucose (+Glucose). Experiments adding 50 nmol/L Rivaroxaban, 0.33 mmol/L ASA and 12.5 nmol/L Rivaroxaban +0.33 mmol/L ASA to high D-glucose-incubated HCAEC are also represented. Panel c.- Representative dot-blots to measure mitochondrial content of Pink-1 and Parkin proteins HCAEC incubated with 5 mmol/L D- (control) or 30 mmol/L D-Glucose (+Glucose). Experiments adding 50 nmol/L Rivaroxaban, 0.33 mmol/L ASA and 12.5 nmol/L Rivaroxaban +0.33 mmol/L ASA to high D-glucose-incubated HCAEC are also represented. Panel d.- Reactive oxygen species (ROS) production and changes in mitochondria membrane potential (ΔΨm) in HCAEC incubated with 5 mmol/L D- (control) or 30 mmol/L D-Glucose (+Glucose). Experiments adding 50 nmol/L Rivaroxaban, 0.33 mmol/L ASA and 12.5 nmol/L Rivaroxaban +0.33 mmol/L ASA to high D-glucose-incubated HCAEC are also represented. All experiments were also performed in presence of submaximal thrombin concentration (0.025 U/mL). Bar graphs of the four panels show the densitometric analysis represented in arbitrary units (A.U.). Results are represented as mean ± SEM of six different experiments. * p < 0.05 compared to the experiments performed normal glucose (5 mmol/L, control). # p < 0.05 compared to the experiments performed with 30 mmol/L glucose (+Glucose).

Article Snippet: The human coronary artery endothelial cell (HCAEC) line, (Ref. 350–05a, Merck KGaA, Germany) was incubated under the following experimental conditions: HCAEC incubated with physiologic D-glucose concentration (5 mmol/L, control group), HCAEC incubated with 30 mmol/L D-Glucose to mimic an hyperglycemic condition (+Glucose group), HCAEC incubated with 30 mmol/L D-Glucose+50 nmol/L Rivaroxaban (Bay 59–7939, Rivaroxaban group), HCAEC incubated with 30 mmol/L D-Glucose+0.33 mmol/L acetylsalicylic acid (ASA group) and 30 mmol/L D-glucose incubated HCAEC with Rivaroxaban (12.5 nmol/L) +ASA (0.33 mmol/L) (Riva+ASA group).

Techniques: Western Blot, Expressing, Incubation, Concentration Assay

Journal: Biochemical and biophysical research communications

Article Title: Tanshinone IIA protects human coronary artery endothelial cells from ferroptosis by activating the NRF2 pathway.

doi: 10.1016/j.bbrc.2021.08.067

Figure Lengend Snippet: Fig. 1. TSA inhibits Erastin/RSL3-induced cell death in HCAEC cells. A, Morphological observation of HCAECs. The HCAECs were treated with either TSA (50 nM), Era (10 mM), RSL3 (0.5 mM), or combination, as indicated, for 24 h. Cells were observed under light microscopy. B, HCAECs were treated as indicated, and cytotoxicity was determined by the LDH release assay. Treatments: TSA (50 nM), Era (10 mM), RSL3 (0.5 mM), Fer-1 (10 mM). C, HCAECs were treated with either TSA (50 nM), Era (10 mM), or both for 24 h; cell death rate was measured by PI staining. D, HCAECs were treated with either TSA (50 nM), RSL3 (0.5 mM), or both for 24 h; cell death rate was measured by PI staining. Data are shown as mean ± SD. ***p < 0.001, ANOVA with multiple comparisons. TSA, Tanshinone IIA; Era, Erastin.

Article Snippet: The human coronary artery endothelial cell line HCAEC (ATCC®PCS-100-020TM) was obtained from American Type Culture Collection (Rockville, USA).

Techniques: Light Microscopy, Lactate Dehydrogenase Assay, Staining

Journal: Biochemical and biophysical research communications

Article Title: Tanshinone IIA protects human coronary artery endothelial cells from ferroptosis by activating the NRF2 pathway.

doi: 10.1016/j.bbrc.2021.08.067

Figure Lengend Snippet: Fig. 2. TSA may attenuate ferroptosis by reducing ROS and iron accumulation in HCAEC cells. A, HCAECs were treated with either TSA (50 nM), Era (10 mM), or both for 24 h. Total cellular ROS was detected by DCFDA staining, and mean fluorescence intensity was measured by flow cytometry. B, HCAECs were treated with either TSA (50 nM), RSL3 (0.5 mM), or both for 24 h. Total cellular ROS was detected by DCFDA staining, and mean fluorescence intensity was measured by flow cytometry. C, HCAECs were treated as indicated for 24 h. Cellular lipid ROS was detected by C11-BODIPY staining, and mean fluorescence intensity was measured by flow cytometry. D, HCAECs were treated as indicated for 24 h. Cellular lipid ROS was detected by C11-BODIPY staining, and mean fluorescence intensity was measured by flow cytometry. E, HCAECs were treated with either TSA (50 nM), Era (10 mM), or both for 24 h. Cellular GSH level was measured. F&G, HCAECs were treated with either TSA (50 nM), Era (10 mM), or both for 24 h. FTH1 protein expression was detected by Western blot assay. H, HCAEC cells were treated as indicated. The FTH1 mRNA level was detected by qPCR. Data are shown as mean ± SD. ***p < 0.001, ANOVA with multiple comparisons. TSA, Tanshinone IIA; Era, Erastin.

Article Snippet: The human coronary artery endothelial cell line HCAEC (ATCC®PCS-100-020TM) was obtained from American Type Culture Collection (Rockville, USA).

Techniques: Staining, Cytometry, Expressing, Western Blot

Journal: Biochemical and biophysical research communications

Article Title: Tanshinone IIA protects human coronary artery endothelial cells from ferroptosis by activating the NRF2 pathway.

doi: 10.1016/j.bbrc.2021.08.067

Figure Lengend Snippet: Fig. 3. TSA combined with Erastin activates the NRF2 pathway in HCAEC cells. A-E, HCAECs were treated with either TSA (50 nM), Era (10 mM), or both for 24 h. The indicated proteins were detected by Western blot assay. The intensity of protein bands was quantified using ImageJ software. FeI, HCAECs were treated with either TSA (50 nM), Era (10 mM), or both for 24 h. The mRNA levels of the indicated genes were detected by qPCR. Data are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA with multiple comparisons. TSA, Tanshinone IIA; Era, Erastin.

Article Snippet: The human coronary artery endothelial cell line HCAEC (ATCC®PCS-100-020TM) was obtained from American Type Culture Collection (Rockville, USA).

Techniques: Western Blot, Software

Journal: Biochemical and biophysical research communications

Article Title: Tanshinone IIA protects human coronary artery endothelial cells from ferroptosis by activating the NRF2 pathway.

doi: 10.1016/j.bbrc.2021.08.067

Figure Lengend Snippet: Fig. 4. NRF2 inhibition abolishes the protective effect of TSA on HCAEC cells. A, TSA promotes the nuclear translocation of NRF2. HCAECs were treated with either TSA, Era, or both for 24 h. NRF2 staining is indicated as green, and the cell nucleus was stained with DAPI (blue). B, HCAECs were treated as indicated for 24 h. The cytoplasmic and nuclear proteins were extracted, and NRF2 protein levels were detected by Western blot assay. The intensity of the protein bands was quantified. C, NRF2 inhibition by ML385 abolished the protective effect of TSA on HCAECs. HCAECs were treated as indicated, and cell death rate was determined by PI staining. Treatments: TSA (50 nM), Era (10 mM), ML385 (a NRF2 inhibitor, 10 mM). D, HCAECs were treated as indicated, and cellular lipid ROS was detected by C11-BODIPY staining. Mean fluorescence intensity was measured by flow cytometry. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001, ANOVA with multiple comparisons. TSA (50 nM), Era (10 mM), ML385 (a NRF2 inhibitor, 10 mM). TSA, Tanshinone IIA; Era, Erastin; H3, histone H3. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The human coronary artery endothelial cell line HCAEC (ATCC®PCS-100-020TM) was obtained from American Type Culture Collection (Rockville, USA).

Techniques: Inhibition, Translocation Assay, Staining, Western Blot, Cytometry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice

doi: 10.1161/atvbaha.118.311903

Figure Lengend Snippet: Figure 5. FURIN inhibition reduces neointimal plaque formation and inflammation in a wire injury model of atherosclerosis. Male Apoe−/− mice were fed a high-fat diet, treated with vehicle (dimethyl sulfoxide [DMSO]) or FURIN Inhibitor α-1-PDX and were subjected to wire injury of the common carotid artery. A, Representative photomicrographs of Pentachrome-stained sections 2 wk after injury, (B) significantly lower plaque area, (C) significantly lower neointima area, and (D) unchanged media area in FURIN inhibitor–treated mice. E, Significantly decreased vascular inflammatory cytokine TNF (tumor necrosis factor)-α lev- els (stained in green) and (F) unchanged endothelial adhesion molecule ICAM-1 (intercellular adhesion molecule 1) levels (stained in red) in FURIN inhibitor– treated mice. Groups are abbreviated as Apoe−/− mice (Control); Apoe−/− mice administered the FURIN inhibitor α-1-PDX (FURIN inhibitor). n=6 per group. Values represent mean±SEM. Data in A–E are normally distributed, and P values were assessed using Student t tests. Data in F was not normally distributed, and Mann-Whitney test was used.

Article Snippet: Primary human coronary artery endothelial cell line from ATCC (Manassas, VA) was cultured in EndoGRO-VEGF Complete Culture Media (Merck, Kenilworth, NJ) supplemented with 20% FBS, heparin, endothelial cell growth factor, nonessential amino acids, and antibiotics.

Techniques: Inhibition, Staining, Control, MANN-WHITNEY

Journal: Cell

Article Title: A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression

doi: 10.1016/j.cell.2017.06.049

Figure Lengend Snippet: Key Resources Table

Article Snippet: Human Coronary Artery Endothelial Cell Line , Lifeline Cell Technology , FC-0032.

Techniques: Virus, Clinical Proteomics, Recombinant, Knock-Out, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Reporter Assay, RNA Sequencing, Control, Plasmid Preparation, Software