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: BMC Complementary Medicine and Therapies

Article Title: Ficus deltoidea suppresses endothelial activation, inflammation, monocytes adhesion and oxidative stress via NF-κB and eNOS pathways in stimulated human coronary artery endothelial cells

doi: 10.1186/s12906-020-2844-6

Figure Lengend Snippet: Cytotoxic effects of FDT, FDK, FDD and FDI on of HCAEC and RAW 264.7 cells. The viability of ( a ) HCAEC is good until FD concentration of 40 μg/ml; whilst ( b ) RAW 264.7 remains good at 500 μg/ml. Results are presented as percentage (%) of controls (untreated cells). Data are expressed as mean ± SD ( n = 3). Abbreviation: FDT (FD var. trengganuensis ), FDK (FD var. kunstleri ), FDD (FD var. deltoidea ), FDI (FD var. intermedia )

Article Snippet: Human coronary artery endothelial cells (HCAEC) provided by Lonza, Allendale, USA were cultured in endothelial growth medium (EGM) until confluent, at 37 °C in a humidified incubator set at 5% carbon dioxide (CO 2 ).

Techniques: Concentration Assay

Journal: BMC Complementary Medicine and Therapies

Article Title: Ficus deltoidea suppresses endothelial activation, inflammation, monocytes adhesion and oxidative stress via NF-κB and eNOS pathways in stimulated human coronary artery endothelial cells

doi: 10.1186/s12906-020-2844-6

Figure Lengend Snippet: Effects of FD extracts on a ) VCAM-1, b ) ICAM-1, c) E-selectin, d ) IL-6 protein expression in LPS-stimulated HCAEC. Before incubation, protein expression in the media was measured by ELISA. Data are expressed as mean ± SD (n = 3). Statistical analysis: ANOVA, post-hoc with Bonferroni correction; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to HCAEC incubated with LPS alone. Abbreviation: UNT (untreated), LPS (lipopolysaccharide), FDT (FD var. trengganuensis ), FDK (FD var. kunstleri ), FDD (FD var. deltoidea ), FDI (FD var. intermedia )

Article Snippet: Human coronary artery endothelial cells (HCAEC) provided by Lonza, Allendale, USA were cultured in endothelial growth medium (EGM) until confluent, at 37 °C in a humidified incubator set at 5% carbon dioxide (CO 2 ).

Techniques: Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: BMC Complementary Medicine and Therapies

Article Title: Ficus deltoidea suppresses endothelial activation, inflammation, monocytes adhesion and oxidative stress via NF-κB and eNOS pathways in stimulated human coronary artery endothelial cells

doi: 10.1186/s12906-020-2844-6

Figure Lengend Snippet: Effects of FD on a ) VCAM-1, b ) ICAM-1, c ) E-selectin, d ) IL-6 gene expression in LPS-stimulated HCAEC cell pellets. Data are expressed as mean ± SD (n = 3). Statistical analysis: ANOVA, post-hoc with Bonferroni correction; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to HCAEC incubated with LPS alone. Abbreviation: LPS (lipopolysaccharide), FDT (FD var. trengganuensis ), FDK (FD var. kunstleri ), FDD (FD var. deltoidea ), FDI (FD var. intermedia )

Article Snippet: Human coronary artery endothelial cells (HCAEC) provided by Lonza, Allendale, USA were cultured in endothelial growth medium (EGM) until confluent, at 37 °C in a humidified incubator set at 5% carbon dioxide (CO 2 ).

Techniques: Gene Expression, Incubation

Journal: BMC Complementary Medicine and Therapies

Article Title: Ficus deltoidea suppresses endothelial activation, inflammation, monocytes adhesion and oxidative stress via NF-κB and eNOS pathways in stimulated human coronary artery endothelial cells

doi: 10.1186/s12906-020-2844-6

Figure Lengend Snippet: Effects of FD on a ) NF-κB p50 in nuclear lysate protein expression, b ) NF-κB p50 in cytoplasmic lysate protein expression, c ) NF-κB p65 in nuclear lysate protein expression, d ) NF-κB p65 in cytoplasmic lysate protein expression, e ) NF-κB p50 gene expression in cell pellets, f ) NF-κB p65 gene expression in cell pellets, g ) eNOS protein expression in cell lysate, and h ) eNOS gene expression in cell pellets in LPS-stimulated HCAEC. Data are expressed as mean ± SD (n = 3). Statistical analysis: ANOVA, post-hoc with Bonferroni correction; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to HCAEC incubated with LPS alone. Abbreviation: UNT (untreated), LPS (lipopolysaccharide), FDT (FD var. trengganuensis ), FDK (FD var. kunstleri ), FDD (FD var. deltoidea ), FDI (FD var. intermedia )

Article Snippet: Human coronary artery endothelial cells (HCAEC) provided by Lonza, Allendale, USA were cultured in endothelial growth medium (EGM) until confluent, at 37 °C in a humidified incubator set at 5% carbon dioxide (CO 2 ).

Techniques: Expressing, Gene Expression, Incubation

Journal: BMC Complementary Medicine and Therapies

Article Title: Ficus deltoidea suppresses endothelial activation, inflammation, monocytes adhesion and oxidative stress via NF-κB and eNOS pathways in stimulated human coronary artery endothelial cells

doi: 10.1186/s12906-020-2844-6

Figure Lengend Snippet: Effects of FD extracts (5–40 μg/ml) on the monocyte-endothelial cell binding assay. Data expressed as mean ± SD. Statistical analysis: ANOVA, post-hoc with Bonferroni correction; * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to HCAEC incubated with LPS alone. Abbreviation: UNT (untreated), LPS (lipopolysaccharide), FDT (FD var. trengganuensis ), FDK (FD var. kunstleri ), FDD (FD var. deltoidea ), FDI (FD var. intermedia )

Article Snippet: Human coronary artery endothelial cells (HCAEC) provided by Lonza, Allendale, USA were cultured in endothelial growth medium (EGM) until confluent, at 37 °C in a humidified incubator set at 5% carbon dioxide (CO 2 ).

Techniques: Cell Binding Assay, Incubation

Journal: BMC Complementary Medicine and Therapies

Article Title: Ficus deltoidea suppresses endothelial activation, inflammation, monocytes adhesion and oxidative stress via NF-κB and eNOS pathways in stimulated human coronary artery endothelial cells

doi: 10.1186/s12906-020-2844-6

Figure Lengend Snippet: Effects of different concentrations of FD varieties (15.625–500 μg/ml) on the percentage increase of DCF fluorescence in RAW 264.7 cells stimulated with LPS and IFN-γ. Results are expressed as a percentage (%) of fluorescence intensity. Data are expressed as mean ± SD ( n = 3). Statistical analysis: ANOVA, post-hoc with Bonferroni correction; ** p < 0.01 and *** p < 0.001 compared to HCAEC incubated with LPS alone. Abbreviation: UNT (untreated), LPS (lipopolysaccharide), IFN-γ (interferon gamma), Q (Quercetin), FDT (FD var. trengganuensis ), FDK (FD var. kunstleri ), FDD (FD var. deltoidea ), FDI (FD var. intermedia )

Article Snippet: Human coronary artery endothelial cells (HCAEC) provided by Lonza, Allendale, USA were cultured in endothelial growth medium (EGM) until confluent, at 37 °C in a humidified incubator set at 5% carbon dioxide (CO 2 ).

Techniques: Fluorescence, Incubation

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: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Liver-Specific Knockdown of IGF-1 Decreases Vascular Oxidative Stress Resistance by Impairing the Nrf2-Dependent Antioxidant Response: A Novel Model of Vascular Aging

doi: 10.1093/gerona/glr164

Figure Lengend Snippet: A: Reporter gene assay showing the effects of recombinant insulin-like growth factor (IGF)-1 (200 ng/mL) on Nrf2/ARE reporter activity in cultured primary human coronary arterial endothelial cells (CAECs). Cells were transiently cotransfected with ARE-driven firefly luciferase and CMV-driven renilla luciferase constructs followed by IGF-1 treatment. Cells were then lysed and subjected to luciferase activity assay. After normalization relative luciferase activity was obtained from four to six independent transfections. Data are mean ± SEM. *p < .05. B–D: Effect of IGF-1 on messenger RNA expression of Nqo1, Hmox1, and Gclc in cultured primary CAECs. Data are mean ± SEM (n = 5 in each group). The effects of IGF-1 were significant (p < .05) for each target. E: Effect of IGF-1 on Nrf2/ARE reporter activity in CAECs transfected with plasmids expressing the wild-type human Akt1 (pMEV2HA-AKT1-WT) or a dominant negative mutant form of Akt1 (pMEV2HA-AKT1-K179A, pMEV2HA-AKT1-AA). Data are mean ± SEM (n = 6–8 in each group). *p < .05 versus untreated control, #p < .05 versus respective wild type.

Article Snippet: Assessment of the Effects of IGF-1 on the Transcriptional Activity of Nrf2 in Cultured Human Coronary Artery Endothelial Cells In order to assess the direct effects of IGF-1 on endothelial Nrf2 signaling, human coronary artery endothelial cells (CAEC; Cell Applications, Inc., San Diego, CA; after Passage 4; age of the donors is unknown) were cultured in 96-well plates as described ( 28 ).

Techniques: Reporter Gene Assay, Recombinant, Activity Assay, Cell Culture, Luciferase, Construct, Transfection, RNA Expression, Expressing, Dominant Negative Mutation, Control

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Liver-Specific Knockdown of IGF-1 Decreases Vascular Oxidative Stress Resistance by Impairing the Nrf2-Dependent Antioxidant Response: A Novel Model of Vascular Aging

doi: 10.1093/gerona/glr164

Figure Lengend Snippet: A: Reporter gene assay showing the effects of oxLDL (40 μg/mL) on Nrf2/ARE reporter activity in primary human coronary arterial endothelial cells (CAECs) cultured in the presence of sera derived from Igf1f/f + MUP-iCre-AAV8 or control mice (n = 18–20 in each group). Data are mean ± SEM. *p < .05. B–C: Effect of oxLDL on messenger RNA expression of Nqo1 (B) and Hmox1 (C) in primary CAECs cultured in the presence of sera derived from Igf1f/f + MUP-iCre-AAV8 or control mice. Data are mean ± SEM. The differences between the groups are statistically not significant. D: Relative oxLDL-induced increases in O2·− production in primary CAECs cultured in the presence of sera derived from Igf1f/f + MUP-iCre-AAV8 or control mice. Cellular O2·− levels were assessed by flow cytometry using the redox-sensitive fluorescent dyes dihydroethidium. Data are mean ± SD. The difference between the groups is statistically not significant.

Article Snippet: Assessment of the Effects of IGF-1 on the Transcriptional Activity of Nrf2 in Cultured Human Coronary Artery Endothelial Cells In order to assess the direct effects of IGF-1 on endothelial Nrf2 signaling, human coronary artery endothelial cells (CAEC; Cell Applications, Inc., San Diego, CA; after Passage 4; age of the donors is unknown) were cultured in 96-well plates as described ( 28 ).

Techniques: Reporter Gene Assay, Activity Assay, Cell Culture, Derivative Assay, Control, RNA Expression, Flow Cytometry

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Liver-Specific Knockdown of IGF-1 Decreases Vascular Oxidative Stress Resistance by Impairing the Nrf2-Dependent Antioxidant Response: A Novel Model of Vascular Aging

doi: 10.1093/gerona/glr164

Figure Lengend Snippet: A–B: H2O2- (10−4 mol/L, for 24 hours) and oxLDL (40 μg/mL, for 24 hours)-induced increases in caspase 3/7 activity (A) and cytoplasmic histone-associated DNA fragments (B) in aorta segments isolated from Igf1f/f + MUP-iCre-AAV8 and control (Igf-1f/f + MUP-eGFP-AAV8) mice, indicating an increased rate of oxidative stress–induced apoptosis in insulin-like growth factor (IGF)-1 deficiency. Data are mean ± SEM (n = 8–10 in each group). *p < .05 versus Igf-1f/f + MUP-eGFP-AAV8. C: Representative TUNEL staining of aortas from IGF-1–deficient and control mice, treated with or without oxLDL. Nuclei from apoptotic endothelial and smooth muscle cells exhibit intense green fluorescence. Autofluorescence of elastic laminae (faint green) and nuclear counterstaining (propidium iodide, red) are shown for orientation purposes (original magnification: 20×). D: Apoptotic index (% of TUNEL positive cell nuclei) was significantly increased in the aortas of IGF-1–deficient mice after oxLDL treatment. *p < .05 versus Igf-1f/f + MUP-eGFP-AAV8. Data are mean ± SEM. Ten images per aorta were analyzed.

Article Snippet: Assessment of the Effects of IGF-1 on the Transcriptional Activity of Nrf2 in Cultured Human Coronary Artery Endothelial Cells In order to assess the direct effects of IGF-1 on endothelial Nrf2 signaling, human coronary artery endothelial cells (CAEC; Cell Applications, Inc., San Diego, CA; after Passage 4; age of the donors is unknown) were cultured in 96-well plates as described ( 28 ).

Techniques: Activity Assay, Isolation, Control, TUNEL Assay, Staining, Fluorescence