Journal: Cellular signalling

Article Title: PAI-1 contributes to homocysteine-induced cellular senescence

doi: 10.1016/j.cellsig.2019.109394

Figure Lengend Snippet: Cultures of EA.hy926 endothelial cells (A) and HCAEC (B, C) were pretreated with TM5441 (10 μM) (A, B) or TM5A15 (10 μM) (C) in triplicate followed by Homocysteine (Hcy) treatment for 4–5 days. Whole cell extracts were prepared and equal amount of pooled proteins from three wells were subjected to Western blot analysis for senescence markers and regulators using specific antibodies as indicated (A–C). Bar represents mean ± sem. Quantitative data are shown on the right (A’-C’). The levels of at least 2–3 senescence markers were determined in repeat experiments. D, E. Whole cell extracts (HCAEC) were prepared from two separate experiments and equal amount of pooled proteins from three wells were subjected to Western blot analysis for senescence markers and regulators p53 and pERK1/2 (D), integrin β3 and PAI-1 (E) using specific antibodies. Quantitative data in the lower panel showing the levels of each regulator relative to loading control Actin (D’, E’).

Article Snippet: Endothelial cell culture: treatment with Hcy and small molecule inhibitors of PAI-1 Primary cultures of Human Coronary Artery Endothelial Cells (HCAEC) (Cell Applications; Cat # 300–05a) and EA.hy926 (ATCC cat #CRL-2922) were grown in MesoEndo Cell Growth Media and Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin and streptomycin respectively and maintained at 37 °C in a 5% CO 2 incubator.

Techniques: Western Blot, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation

doi: 10.3389/fcvm.2021.651230

Figure Lengend Snippet: Overexpression of A20 in human coronary artery endothelial cells (HCAEC) increases endothelial nitric oxide synthase (eNOS) expression and prevents its downregulation by TNF, while A20 knockdown decreases eNOS levels. (A) Relative eNOS mRNA (qPCR) normalized by mRNA levels of the housekeeping (HKG) gene 28S and expressed as fold change of non-treated (NT) Ctrl HCAEC, and (B) eNOS protein (WB) levels in non-transduced HCAEC (Ctrl) and in HCAEC transduced with rAd.A20 or control rAd.βgal (100 MOI), before and 24 h after treatment with TNF. β-Actin was used to correct for loading. (C) Relative eNOS and A20 mRNA levels (qPCR) in HCAEC non-transfected (Ctrl) and transfected with A20 siRNA or with AllStars negative control siRNA (Ctrl siRNA) for 24 h, normalized by the mRNA levels of the HKG gene cyclophilin A (CycA), and expressed as fold change of non-transfected Ctrl HCAEC. All data in (A–C) are presented as mean ± SEM of three to five independent experiments. Significance between groups was determined by one- or two-way ANOVA followed by the Tukey or Bonferroni multiple comparison post hoc test, respectively; * p < 0.05, ** p < 0.01, *** p < 0.001. Significance between NT and TNF-treated HCAEC was determined by an unpaired t test and depicted as hashtag in lieu of asterisk symbols in order to better differentiate between the two statistical methods used to analyze the data. # p < 0.05, ## p < 0.01, ### p < 0.001. (D) Relative eNOS and A20 mRNA levels in the aortae of 3–4-week-old A20 knockout (KO) and heterozygous (HT) mice, as well as wild-type (WT) littermates. Data are presented as mean ± SEM of 5–11 animals/group. Significance between groups was determined by one-way ANOVA followed by the Tukey multiple comparison post hoc test; * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Representative eNOS immunohistochemistry (brown) in the brain of A20 wild-type (WT), heterozygous (HT), and knockout (KO) mice. Photomicrographs are representative of three animals per genotype, magnification = ×400.

Article Snippet: Human coronary artery endothelial cells (HCAEC) derived from six different donors from both genders were purchased from Lonza, Allendale, NJ, and cultured in EGM-2MV BulletKit medium.

Techniques: Over Expression, Expressing, Knockdown, Transduction, Control, Transfection, Negative Control, Comparison, Knock-Out, Immunohistochemistry

Journal: Frontiers in Cardiovascular Medicine

Article Title: A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation

doi: 10.3389/fcvm.2021.651230

Figure Lengend Snippet: A20 overexpression in HCAEC increases eNOS transcription in an ERK5-dependent manner. (A) mRNA levels of eNOS, KLF2, KLF4, and ERK5 were measured by qPCR in non-transduced HCAEC (Ctrl) and HCAEC transduced with rAd.A20 or control rAd.βgal at 100 MOI for 48 h. Data were normalized by the 28S HKG and expressed as mean ± SEM fold change of Ctrl ( n = 5–9). (B) eNOS, (D) KLF2, and (E) KLF4 mRNA and (C) eNOS protein levels were measured by qPCR, and WB in non-transduced HCAEC (Ctrl) and HCAEC transduced with rAd.A20 or control rAd.βgal at 100 MOI for 3 h prior to 24 h treatment with the ERK5 inhibitor, XMD8-92 (10 μM). Graphs in (B) , (D) , and (E) depict relative mRNA levels, normalized by the 28S HKG and expressed as mean ± SEM fold change of non-treated (NT) Ctrl ( n = 6–10). In (C) , β-actin was used to correct for loading. Densitometry results are presented as fold change of NT Ctrl cells ( n = 3). (F) KLF2 mRNA levels were measured by qPCR in non-transduced HCAEC (Ctrl) and in HCAEC transduced with rAd.A20 or control rAd.βgal at 100 MOI for 48 h prior to 24 h treatment with TNF (200 U/mL). Graphs depict relative KLF2 mRNA levels normalized by the 28S HKG and expressed as mean ± SEM fold change of non-treated (NT) Ctrl ( n = 3–5). * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by two-way ANOVA followed by the Bonferroni post hoc test. Significance between NT and TNF-treated HCAEC was determined by an unpaired t test and depicted as hashtag in lieu of asterisk symbols in order to better differentiate between the two statistical methods used to analyze the data: # p < 0.05, ### p < 0.001.

Article Snippet: Human coronary artery endothelial cells (HCAEC) derived from six different donors from both genders were purchased from Lonza, Allendale, NJ, and cultured in EGM-2MV BulletKit medium.

Techniques: Over Expression, Transduction, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation

doi: 10.3389/fcvm.2021.651230

Figure Lengend Snippet: A20 overexpression in HCAEC increases eNOS transcription in a KLF2-dependent manner. HCAEC were transduced with 100 MOI of shRNA-KLF2 or scramble shRNA-Ctrl for 24 h, then retransduced with rAd.A20 or control rAd.βgal at 200–250 MOI for 48 h or left non-transduced (Ctrl). Cell lysates were evaluated by qPCR for mRNA levels of (A) KLF2, (B) KLF4, and (C) eNOS. Graphs shown depict relative mRNA levels, normalized by the 28S HKG and expressed as mean ± SEM fold change of Ctrl shRNA-Ctrl cells ( n = 3–6) and by (D) WB for eNOS protein expression. GAPDH was used to correct for loading. Densitometry results are presented as fold change of Ctrl shRNA-Ctrl cells and expressed as mean ± SEM ( n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001, as determined by two-way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: Human coronary artery endothelial cells (HCAEC) derived from six different donors from both genders were purchased from Lonza, Allendale, NJ, and cultured in EGM-2MV BulletKit medium.

Techniques: Over Expression, Transduction, shRNA, Control, Expressing

Journal: Frontiers in Cardiovascular Medicine

Article Title: A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation

doi: 10.3389/fcvm.2021.651230

Figure Lengend Snippet: Overexpression of A20 in HCAEC modulates the expression levels of KLF2-dependent genes to increase EC homeostasis. mRNA levels of endothelin-1 (END1), chemokine C-C motif ligand 2 (CCL2)/monocyte chemoattractant protein-1 (MCP1), thrombomodulin (THBD), eNOS, KLF2, and A20 were measured by qPCR in non-transduced HCAEC (Ctrl) and HCAEC transduced with rAd.A20 or control rAd.βgal. Data were normalized by the 28S HKG and expressed as mean ± SEM fold change of Ctrl ( n = 8–10). * p < 0.05, ** p < 0.01, *** p <0.001, as determined by one-way ANOVA followed by Tukey post hoc test.

Article Snippet: Human coronary artery endothelial cells (HCAEC) derived from six different donors from both genders were purchased from Lonza, Allendale, NJ, and cultured in EGM-2MV BulletKit medium.

Techniques: Over Expression, Expressing, Transduction, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: A20/TNFAIP3 Increases ENOS Expression in an ERK5/KLF2-Dependent Manner to Support Endothelial Cell Health in the Face of Inflammation

doi: 10.3389/fcvm.2021.651230

Figure Lengend Snippet: A20 overexpression increases basal Ser-1177 eNOS phosphorylation in HCAEC. Representative WB of total eNOS and P-eNOS (Ser-1177) in non-transduced HCAEC (Ctrl) and HCAEC transduced with rAd.A20 or rAd.βgal at 100 MOI for 48 h; GAPDH was used to correct for loading. Densitometry results are presented as mean fold change of Ctrl ± SEM ( n = 4). * p < 0.05, as determined by one-way ANOVA followed by the Tukey post hoc test.

Article Snippet: Human coronary artery endothelial cells (HCAEC) derived from six different donors from both genders were purchased from Lonza, Allendale, NJ, and cultured in EGM-2MV BulletKit medium.

Techniques: Over Expression, Phospho-proteomics, Transduction

Journal: Cellular signalling

Article Title: Targeting acid sphingomyelinase with anti-angiogenic chemotherapy

doi: 10.1016/j.cellsig.2016.09.010

Figure Lengend Snippet: Chemotherapy-induced ASMase activation, ceramide generation and induction of apoptosis in cultured endothelium. (A) In a time-course experiment, ASMase activity was quantified in BAEC after treatment with 50 µM etoposide by radioenzymatic assay using [N-methyl-14C] sphingomyelin as substrate. (B) In parallel, a time-course of ceramide generation in response to etoposide was measured using the diacylglycerol kinase assay in BAEC. (C) Ceramide generation was measured using the diacylglycerol kinase assay at 10 and 30 min after treatment in HCAEC with 50 µM etoposide. (D) Ceramide generation was measured using the diacylglycerol kinase assay at 10 and 30 min after treatment in HCAEC with 100 nM paclitaxel. Data (mean±SD) represent triplicate points from two independent experiments.

Article Snippet: Human coronary artery endothelial cells (HCAEC) were obtained from CloneticsTM Coronary Artery Endothelial Cell Systems (Cambrex Bio Science Inc.).

Techniques: Activation Assay, Cell Culture, Activity Assay, Radioenzymatic Assay, Kinase Assay

Journal: Cellular signalling

Article Title: Targeting acid sphingomyelinase with anti-angiogenic chemotherapy

doi: 10.1016/j.cellsig.2016.09.010

Figure Lengend Snippet: CRM generation signals chemotherapy-induced apoptosis in endothelium. Cultured BAEC were treated with paclitaxel (100 nM) (A) or etoposide (50 µM) (B) and at the indicated times incidence of apoptosis was scored by bis-benzamide trihydrochloride staining. (C) Treatment of BAEC with cisplatin does not lead to endothelial cell dysfunction. BAEC were treated with increasing doses of cisplatin (0.1–50 µM) and apoptosis was detected by bis-benzamide trihydrochloride staining. Paclitaxel and etoposide induce apoptosis in HCAEC. HCAEC were treated with 100 nM paclitaxel (D) or 50 µM etoposide (E) and the incidence of apoptosis was scored at the time points indicated. (F) BAEC were pre-incubated for 30 min with bFGF (2 ng/mL), VEGF (2 ng/mL) or nystatin (30 µg/mL) prior to treatment with etoposide (50 µM), and apoptosis was evaluated after 8 hours. Each value (mean±SD) represents duplicate determinations from three independent experiments.

Article Snippet: Human coronary artery endothelial cells (HCAEC) were obtained from CloneticsTM Coronary Artery Endothelial Cell Systems (Cambrex Bio Science Inc.).

Techniques: Cell Culture, Staining, Incubation