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: Genome biology

Article Title: Integrative analysis of vascular endothelial cell genomic features identifies AIDA as a coronary artery disease candidate gene.

doi: 10.1186/s13059-019-1749-5

Figure Lengend Snippet: Fig. 1 Transcriptomic and epigenomic profiling of teloHAEC. a RNAseq of teloHAEC non-treated (NT) or treated with TNFα identified 1316 differentially expressed (DE) genes (FDR < 0.1% and absolute log10-fold-change > 0.3) among three comparisons (NT vs. 4 h, NT vs. 24 h, 4 h vs. 24 h). Of these 1316 genes, 836 genes were DE in the NT vs. 4 h comparison. b Gene expression fold-change for DE genes are highly correlated between transformed teloHAEC and primary HCAEC. All three comparisons are highly significant (P < 2.2 × 10−16), but for simplicity we only show the NT vs. 4 h comparison. c ATACseq of teloHAEC NT or treated with TNFα identified 95,491 peaks, including 3138 differentially opened (or closed) (DO) peaks (FDR < 0.1% and absolute log10-fold-change > 0.3) among three comparisons (NT vs. 4 h, NT vs. 24 h, 4 h vs. 24 h). Of these 3138 peaks, 2654 peaks were DO in the NT vs. 4 h comparison. d Open chromatin regions (raw number of reads), identified by ATACseq, are highly correlated between teloHAEC and HCAEC. Results shown are for the 4 h TNFα treatment. Results are consistent for the NT and 24 h timepoints. The distribution falls under the diagonal because the coverage of the ATACseq teloHAEC libraries was higher than the coverage of the HCAEC libraries

Article Snippet: Primary human coronary artery endothelial cells (HCAEC) from a single male donor (ATCC, CC-2585) were grown in EGM2MV (Lonza, CC-3202) supplemented with 200 U/mL penicillin and 200 μg/mL of streptomycin.

Techniques: Comparison, Gene Expression, Transformation Assay

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

Journal: British Journal of Pharmacology

Article Title: Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

doi: 10.1111/bph.13371

Figure Lengend Snippet: cGMP accumulation in co‐cultures of human primary vascular smooth muscle cells following addition of serelaxin to endothelium. HUAEC , HUVEC or HCAEC were co‐cultured with (A) HUASMC or (B) HUVSMC (all n = 5), and the ECs were treated with serelaxin for 30 min. Serelaxin addition to HUAEC did not cause cGMP accumulation in HUAEC (▲) (C) HUASMC (□) or (D) HUVSMC (◯) co‐cultured with HUAEC, whereas direct stimulation of either (C) HUASMC (n = 5) or (D) HUVSMC with serelaxin caused a concentration‐dependent increase in cGMP accumulation (dashed lines). In contrast, serelaxin addition to HUVEC concentration‐dependently increased cGMP accumulation not only in HUVEC (■) but also in (E) HUASMC (□) or (F) HUVSMC (◯) co‐cultured with HUVEC with the responses in smooth muscle cells being greater or in the case of HUVSMC much greater than cGMP responses to direct stimulation of (E) HUASMC or (F) HUVSMC (dashed lines). A similar pattern of cGMP accumulation was observed with (G, H) HCAEC (●) and (G) HUASMC (□) or (H) HUVSMC (◯) co‐cultured with HCAEC.

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAEC), HUVEC, human coronary artery endothelial cells (HCAEC), human umbilical artery smooth muscle cells (HUASMC) and human umbilical vein smooth muscle cells (HUVSMC) were obtained from ScienCell Research Laboratories (San Diego, CA, USA ).

Techniques: Cell Culture, Concentration Assay

Journal: British Journal of Pharmacology

Article Title: Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

doi: 10.1111/bph.13371

Figure Lengend Snippet: Serelaxin‐mediated cGMP accumulation in monocultures of human primary vascular cells (all n = 5). Serelaxin (30 nM, 30 min) increased cGMP accumulation in (A) HUVEC, (B) HCAEC, (C) HUASMC and (D) HUVSMC. Pre‐incubation with l‐NOARG (30 μM, 30 min) or ODQ (1 μM, 30 min) almost abolished serelaxin‐mediated (30 nM, 30 min) cGMP accumulation in all cell types. Pre‐treatment with indomethacin (30 μM, 30 min) significantly inhibited serelaxin‐mediated (30 nM, 30 min) cGMP accumulation in (B) HCAEC but had no effect in (A) HUVEC, (C) HUASMC or (D) HUVSMC. *P < 0.05, **P < 0.02, ***P < 0.005; significantly different from serelaxin alone; one‐way anova with Dunnett's post hoc test.

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAEC), HUVEC, human coronary artery endothelial cells (HCAEC), human umbilical artery smooth muscle cells (HUASMC) and human umbilical vein smooth muscle cells (HUVSMC) were obtained from ScienCell Research Laboratories (San Diego, CA, USA ).

Techniques: Incubation

Journal: British Journal of Pharmacology

Article Title: Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

doi: 10.1111/bph.13371

Figure Lengend Snippet: Serelaxin‐mediated cGMP accumulation in human primary vascular smooth muscle cells co‐cultured with HUVEC or (A) HCAEC (all n = 6 except where otherwise indicated). Stimulation of HUVEC or HCAEC with serelaxin (30 nM, 30 min) increased cGMP accumulation not only in (B) HUVEC and (C) HCAEC but also in co‐cultures of (D, F) HUASMC or (E, G) HUVSMC. Pre‐incubation of HUVEC or HCAEC with l‐NOARG (30 μM, 30 min) before addition of serelaxin (30 nM, 30 min) significantly inhibited cGMP accumulation not only in HUVEC and (C) HCAEC but also in (D, F) HUASMC and (E, G) HUVSMC. Pre‐incubation of HUVEC with indomethacin (30 μM, 30 min) did not affect serelaxin‐mediated (30 nM, 30 min) cGMP accumulation in (B) HUVEC or in co‐incubated (D) HUASMC or (E) HUVSMC (n = 5). Pre‐incubation of HCAEC with indomethacin (30 μM, 30 min) had no significant effect on serelaxin‐mediated (30 nM, 30 min) cGMP accumulation in (C) HCAEC but produced marked and significant reductions in cGMP accumulation in co‐incubated (F) HUASMC or (G) HUVSMC (n = 5). Pre‐treatment of HUASMC or HUVSMC with ODQ (1 μM, 30 min) had no significant effect on serelaxin‐mediated (30 nM, 30 min) cGMP accumulation in (I) HUVEC or (J) HCAEC but reduced or abolished cGMP accumulation in (K, M) HUASMC or (L, N) HUVSMC (n = 5). *P < 0.05, **P < 0.02, ***P < 0.005 significantly different from serelaxin alone; one‐way anova with Dunnett's post hoc test.

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAEC), HUVEC, human coronary artery endothelial cells (HCAEC), human umbilical artery smooth muscle cells (HUASMC) and human umbilical vein smooth muscle cells (HUVSMC) were obtained from ScienCell Research Laboratories (San Diego, CA, USA ).

Techniques: Cell Culture, Incubation, Produced

Journal: British Journal of Pharmacology

Article Title: Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

doi: 10.1111/bph.13371

Figure Lengend Snippet: cAMP accumulation in co‐cultures of human primary vascular smooth muscle cells following addition of serelaxin to endothelium (all n = 5). HUAEC, HUVEC or HCAEC were co‐cultured with (A) HUASMC or (B) HUVSMC, and the endothelial cells were treated with serelaxin for 30 min. Serelaxin added to HUAEC did not cause cAMP accumulation either in (C, D) HUAEC (▲), (C) HUASMC (□) or (D) HUVSMC (◯), whereas direct stimulation of (C) HUASMC or (D) HUVSMC with serelaxin caused a concentration‐dependent increase in cAMP accumulation (dashed lines). Although direct addition of serelaxin to HUVEC concentration‐dependently increased cAMP accumulation in (E, F) HUVEC (■), there was no significant effect on cAMP accumulation in (E) HUASMC (□) or (F) HUVSMC (◯). Direct addition of serelaxin to (E) HUASMC or (F) HUVSMC stimulated cAMP accumulation (dashed lines). Serelaxin concentration‐dependently increased cAMP accumulation in (G, H) HCAEC (●) but also caused a robust concentration‐dependent increase in cAMP accumulation in both (G) HUASMC (□) and (H) HUVSMC (◯).

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAEC), HUVEC, human coronary artery endothelial cells (HCAEC), human umbilical artery smooth muscle cells (HUASMC) and human umbilical vein smooth muscle cells (HUVSMC) were obtained from ScienCell Research Laboratories (San Diego, CA, USA ).

Techniques: Cell Culture, Concentration Assay

Journal: British Journal of Pharmacology

Article Title: Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

doi: 10.1111/bph.13371

Figure Lengend Snippet: Serelaxin‐mediated cAMP accumulation in monocultures of human primary vascular cells (all n = 5). Serelaxin (30 nM, 30 min) increased cAMP accumulation in (A) HUVEC, (B) HCAEC, (C) HUASMC and (D) HUVSMC that was not significantly altered by pre‐incubation with l‐NOARG (30 μM, 30 min) or ODQ (1 μM, 30 min). Pre‐treatment with indomethacin (30 μM, 30 min) significantly inhibited serelaxin‐mediated (30 nM, 30 min) cAMP accumulation in (B) HCAEC but not in (A) HUVEC, (C) HUASMC or (D) HUVSMC. *P < 0.05; significantly different from serelaxin alone; one‐way anova with Dunnett's post hoc test.

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAEC), HUVEC, human coronary artery endothelial cells (HCAEC), human umbilical artery smooth muscle cells (HUASMC) and human umbilical vein smooth muscle cells (HUVSMC) were obtained from ScienCell Research Laboratories (San Diego, CA, USA ).

Techniques: Incubation

Journal: British Journal of Pharmacology

Article Title: Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

doi: 10.1111/bph.13371

Figure Lengend Snippet: Serelaxin‐mediated cAMP accumulation in human primary vascular smooth muscle cells co‐cultured with HCAEC (A, E; all n = 5). Stimulation of HCAEC with serelaxin (30 nM, 30 min) increased cAMP accumulation not only in (B) HCAEC but also in co‐cultures of (C) HUASMC or (D) HUVSMC. Pre‐incubation of HCAEC with l‐NOARG (30 μM, 30 min) before addition of serelaxin (30 nM, 30 min) had no significant effect on cAMP accumulation in (B) HCAEC, (C) HUASMC or (D) HUVSMC. However, pre‐incubation of HCAEC with indomethacin (30 μM, 30 min) significantly inhibited serelaxin‐mediated (30 nM, 30 min) cAMP accumulation in (B) HCAEC and abolished cAMP accumulation in (C) HUASMC or (D) HUVSMC. Pre‐treatment of HUASMC or HUVSMC with ODQ (1 μM, 30 min) had no significant effect on serelaxin‐mediated (30 nM, 30 min) cAMP accumulation in (F) HCAEC, (G) HUASMC or (H) HUVSMC. *P < 0.05; significantly different from serelaxin alone; one‐way anova with Dunnett's post hoc test.

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAEC), HUVEC, human coronary artery endothelial cells (HCAEC), human umbilical artery smooth muscle cells (HUASMC) and human umbilical vein smooth muscle cells (HUVSMC) were obtained from ScienCell Research Laboratories (San Diego, CA, USA ).

Techniques: Cell Culture, Incubation

Journal: British Journal of Pharmacology

Article Title: Enhanced serelaxin signalling in co‐cultures of human primary endothelial and smooth muscle cells

doi: 10.1111/bph.13371

Figure Lengend Snippet: Signal transduction mechanisms activated by serelaxin in co‐cultures of human primary vascular cells. Activation of RXFP1 by serelaxin in HUVEC and HCAEC stimulates NO production and activates sGC and AC to produce cGMP and cAMP respectively. Endothelial NO also diffuses from the endothelial cells across the ThinCert membranes and activates sGC in both the arterial and venous smooth muscle cells. Additionally in HCAEC (blue lines) but not HUVEC, serelaxin stimulates prostanoid production that produces cAMP accumulation in both arterial and smooth muscle cells.

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAEC), HUVEC, human coronary artery endothelial cells (HCAEC), human umbilical artery smooth muscle cells (HUASMC) and human umbilical vein smooth muscle cells (HUVSMC) were obtained from ScienCell Research Laboratories (San Diego, CA, USA ).

Techniques: Transduction, Activation Assay