Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Upregulation of TMEM16A in human PAECs. ( a ) Immunofluorescence staining of TMEM16A in PAECs infected with Ctrl Ad or Ano1 Ad (BP = antibody blocking peptide; scale bar = 20 µm). ( b ) Representative whole-cell I ClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with Ctrl Ad . ( c ) Representative whole-cell I ClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with Ano1 Ad and overexpressing TMEM16A. ( d ) Consecutive, calculated Bbr-sensitive current comparing primary PAECs infected with Ctrl Ad or Ano1 Ad . Figures were generated with 8–13 cells from N = 2 healthy donors, data are presented as mean ± s.e.m.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Immunofluorescence, Staining, Infection, Blocking Assay, Transfection, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: TMEM16A-mediated membrane depolarization disrupts Ca 2+ dynamics of human PAECs. ( a ) Fluorometric measurements indicating shift in relative resting membrane potential (E m ) of donor PAECs infected with Ctrl Ad or Ano1 Ad using DiBAC 4 (3) dye. ( b ) Representative traces depict changes in intracellular Ca 2+ detected in PAECs transfected with Ctrl Ad or Ano1 Ad . ( c – e ) The effect of TMEM16A overexpression on cytosolic baseline Ca 2+ concentration ([Ca 2+ ] i ), store depletion and Ca 2+ influx using Fura-2 in donor PAECs infected with Ctrl Ad or Ano1 Ad (BHQ = butylhydroquinone). Figures were generated with 80-116 cells from N = 3 healthy donors. * p < 0.05, *** p < 0.001, paired ( a ) and unpaired t-tests ( c – e ).

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Infection, Transfection, Over Expression, Concentration Assay, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Enhanced TMEM16A mediates metabolic changes of PAECs. ( a – b ) Seahorse mitochondrial stress test profiles of TMEM16A-overexpressing primary PAECs showing the ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) OCR/ECAR. ( c ) Proliferation of human PAECs overexpressing TMEM16A measured with thymidine incorporation ( n = 5). ( d , e ) Cas3/Cas7 apoptosis assay and cell-cycle analysis of human PAECs overexpressing TMEM16A. (STS = staurosporin). ( f ) Western blots of PAECs infected with TMEM16A-overexpressing Ano1 Ad or control Ctrl Ad with quantifications of PCNA, cleaved PARP/PARP and Cyclin D1. Figures were generated with 13 separate sets of experiments. * p < 0.05, ratio-paired ( a ) or paired t-test.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Apoptosis Assay, Cell Cycle Assay, Western Blot, Infection, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Elevated TMEM16A activity disturbs eNOS activation: ( a ) Noninduced nitric oxide levels and ACh-induced nitric oxide production of control (Ctrl Ad ) and TMEM16A-overexpressing (Ano1 Ad ) human PAECs. Figures were generated with 8 sets of experiments with quadruplicate in each group and normalized to protein content. ( b ) Western blots showing ACh-induced changes in eNOS phosphorylation of Ctrl Ad and Ano1 Ad -infected donor PAECs with quantification following the eNOS phosphorylation pattern at activatory Ser1177 and inhibitory Thr495 sites after 5, 15 and 30 min of ACh stimulation. ( c ) Quantification of basal, noninduced level of eNOS phosphorylation at Ser1177 and Thr495 as well as phosphorylation of Ser1177 15 min after ACh stimulation. Figures were generated with 6 samples. * p < 0.05, *** p < 0.001, ratio-paired t-test.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Activity Assay, Activation Assay, Generated, Western Blot, Infection

Journal: Journal of Cellular and Molecular Medicine

Article Title: High-mobility group box-1 induces vascular remodelling processes via c-Jun activation

doi: 10.1111/jcmm.12519

Figure Lengend Snippet: Human pulmonary arterial endothelial cells and smooth muscle cells express the HMGB1 receptors TLR4 and RAGE. Relative TLR4 and RAGE mRNA expression (A and B) and protein (C) expression in human pulmonary arterial endothelial cells (PAEC) and human pulmonary arterial smooth muscle cells (PASMC); D1–D3 represent samples from three different donors for PASMC or lots for PAEC.

Article Snippet: Primary human arterial endothelial cells (PAEC) were purchased from Lonza (Basel, Switzerland) ( n = 6), cultured in VascuLife® VEGF Medium (LifeLine Technology, Frederick, MD, USA) and used for experiments until passage 8.

Techniques: Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: High-mobility group box-1 induces vascular remodelling processes via c-Jun activation

doi: 10.1111/jcmm.12519

Figure Lengend Snippet: HMGB1-induced proliferation of pulmonary arterial endothelial cells and smooth muscle cells. Effect of HMGB1 treatment on human pulmonary arterial smooth muscle cells (PASMC, A–D) and human pulmonary arterial endothelial cells (PAEC, E–H) on (A and E) proliferation n = 5–6, (B and F) apoptosis n = 5, (C and G) attachment n = 5 and (D and H) migration n = 4–6. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Primary human arterial endothelial cells (PAEC) were purchased from Lonza (Basel, Switzerland) ( n = 6), cultured in VascuLife® VEGF Medium (LifeLine Technology, Frederick, MD, USA) and used for experiments until passage 8.

Techniques: Migration

Journal: Journal of Cellular and Molecular Medicine

Article Title: High-mobility group box-1 induces vascular remodelling processes via c-Jun activation

doi: 10.1111/jcmm.12519

Figure Lengend Snippet: HMGB1 activates MAPK intracellular signalling pathways. Western blot analysis for MAPK and downstream factor activation in (A) human pulmonary arterial smooth muscle cells (PASMC) and (B) human pulmonary arterial endothelial cells (PAEC) following stimulation with 1 or 100 ng/ml HMGB1 for the indicated time-points. Blots are representative of a minimum three independent experiments.

Article Snippet: Primary human arterial endothelial cells (PAEC) were purchased from Lonza (Basel, Switzerland) ( n = 6), cultured in VascuLife® VEGF Medium (LifeLine Technology, Frederick, MD, USA) and used for experiments until passage 8.

Techniques: Western Blot, Activation Assay