Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients

doi: 10.1155/2018/2639081

Figure Lengend Snippet: (a-b) Real-time assessment of intracellular ROS production and collagen I synthesis in HPASMCs exposed to sera of IPF patients. (a) Before stimulation, subconfluent human pulmonary artery smooth muscle cells (HPASMCs) were loaded with 10 μ M of H 2 -DCFDA and then cultured in basal medium containing 10% ( v / v ) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD). Variations in intracellular ROS levels were kinetically determined in a 5-hour time-course (a randomly selected representative experiment is reported) and values at 2 hours (steady state) were used in the future comparisons. Fluorescence data were normalized for protein content and expressed as relative fluorescence units (RFUs). (b) Before stimulation, subconfluent HPASMCs were transduced with lentiviral particles obtained from the COL1A1-LV-tGFP and EF1 α -LV-FP602 lentivectors and then cultured in basal medium containing 10% ( v / v ) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD). Variations of COL1 promoter activation were kinetically followed for 10 hours (a randomly selected representative experiment is reported) and values at 8 hours (steady state) were used in the future comparison. Data are normalized for transduction efficiency by reporting the ratio of COL1A1-LV-tGFP to EF1 α -LV-FP602 relative fluorescence units (RFUs).

Article Snippet: In this study, pulmonary artery smooth muscle cells (HPASMCs) isolated from human pulmonary arteries of healthy donors were used (Innoprot, Spain).

Techniques: Cell Culture, Fluorescence, Transduction, Activation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients

doi: 10.1155/2018/2639081

Figure Lengend Snippet: (a-b) Effects of IPF sera on HPASMC intracellular ROS levels. Before stimulation, subconfluent human pulmonary artery smooth muscle cells (HPASMCs) were loaded with 10 μ M of H 2 -DCFDA, then cultured in basal medium containing 10% ( v / v ) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD). (b) In selected experiments, cells were pretreated for 60 min with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) before exposure to the sera. (a-b) Data represent the variations of intracellular ROS levels after 2 hours of sera stimulation. Fluorescence data were normalized for protein content and expressed as relative fluorescence units (RFUs). P value indicating that the significance is reported in the figure.

Article Snippet: In this study, pulmonary artery smooth muscle cells (HPASMCs) isolated from human pulmonary arteries of healthy donors were used (Innoprot, Spain).

Techniques: Cell Culture, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients

doi: 10.1155/2018/2639081

Figure Lengend Snippet: (a-b) Effects of IPF sera on HPASMC collagen I production. (a) Before stimulation, subconfluent HPASMCs were transduced with lentiviral particles obtained from the COL1A1-LV-tGFP and EF1 α -LV-FP602 lentivectors and then cultured in basal medium containing 10% ( v / v ) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD). Data represent the collagen I promoter activity after 8 hours of sera stimulation. Data are normalized for transduction efficiency by reporting the ratio of COL1A1-LV-tGFP to EF1 α -LV-FP602 relative fluorescence units (RFUs). (b) Subconfluent HPASMCs were stimulated for 48 hrs with basal medium containing 10% ( v / v ) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD) and processed for collagen I quantification as reported in . In selected experiments, cells were pretreated for 60 min with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) before exposure to the sera. Data are expressed as ng/ml collagen protein. P value indicating that the significance is reported in the figure.

Article Snippet: In this study, pulmonary artery smooth muscle cells (HPASMCs) isolated from human pulmonary arteries of healthy donors were used (Innoprot, Spain).

Techniques: Transduction, Cell Culture, Activity Assay, Fluorescence

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients

doi: 10.1155/2018/2639081

Figure Lengend Snippet: (a-b) Effects of IPF sera on HPASMC proliferation. Subconfluent HPASMCs were cultured for 48 hours in basal medium containing 10% ( v / v ) of sera from idiopathic pulmonary fibrosis (IPF), sera from idiopathic pulmonary fibrosis patients treated for 24 weeks with Pirfenidone (IPF + D), and healthy donors (HD). In selected experiments, cells were pretreated for 60 min with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) before exposure to the sera. Data are expressed as ng/ml collagen protein. (a-b) Data are expressed as relative light units/sec (RLU/s). P value indicating that the significance is reported in the figure.

Article Snippet: In this study, pulmonary artery smooth muscle cells (HPASMCs) isolated from human pulmonary arteries of healthy donors were used (Innoprot, Spain).

Techniques: Cell Culture

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition

doi: 10.1152/ajplung.00091.2017

Figure Lengend Snippet: Expression of photorelaxation proteins in rat pulmonary arteries (PAs), rat pulmonary arterial smooth muscle cells (PASMCs), and human PASMCs. A: Opsin 3 (Opn3) and Opsin 4 (Opn4) were both detected in PAs, but Opsin 5 (Opn5) was not. B: G protein-coupled receptor kinase 2 (GRK2) was detected in rat PA (n = 5). Expression of Opn3, Opn4, and GRK2 in isolated rPASMCs via qRT-PCR (C) and Western blot (D) (n = 5) is shown. E: immunofluorescence images of hPASMCs stained for Opn3, Opn4, or GRK2 (n = 5). F and G: RT-PCR and qRT-PCR of hPASMC mRNA showing expression of Opn3, Opn4, and GRK2 but not Opn5 (n = 4–5). H: Western blot of hPASMC lysates tested for Opn3, Opn4, and GRK2 (n = 5) (+, with reverse transcriptase, RT; -, no RT). ***P < 0.001.

Article Snippet: Human PASMCs (hPASMCs; PromoCell, Heidelberg, Germany) were maintained in smooth muscle cell growth medium 2 (PromoCell) in a humidified incubator at 37°C and 5% CO 2 and were used for experiments between passages 3 and 7 .

Techniques: Expressing, Isolation, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Opsin 3 and 4 mediate light-induced pulmonary vasorelaxation that is potentiated by G protein-coupled receptor kinase 2 inhibition

doi: 10.1152/ajplung.00091.2017

Figure Lengend Snippet: G protein-coupled receptor kinase 2 (GRK2) desensitizes the photorelaxation response and interacts directly with Opn3 and Opn4. A: repeated blue light (455 nm) stimulation on rat pulmonary arteries (PAs) with or without GRK2 inhibitor (n = 5). Attenuation in photorelaxation was observed in vessels not treated with GRK2 inhibitor, but no attenuation was observed in vessels treated with GRK2 inhibitor. B: representative myograph tracing showing repetitive blue light (blue arrows) response in rat PAs in the absence of GRK2 inhibitor followed by light response in the presence of GRK2 inhibitor. C: proximity ligation assay (PLA) of human pulmonary arterial smooth muscle cells (hPASMCs) tested for GRK2 and Opn3 or Opn4 proximity. Control stain was performed with only the GRK2 antibody (n = 5). D: PLA of hPASMCs tested for phosphoserine and Opn3 or Opn4 proximity. Control stain was performed with only the phosphoserine antibody. (n = 5). ***P < 0.001.

Article Snippet: Human PASMCs (hPASMCs; PromoCell, Heidelberg, Germany) were maintained in smooth muscle cell growth medium 2 (PromoCell) in a humidified incubator at 37°C and 5% CO 2 and were used for experiments between passages 3 and 7 .

Techniques: Proximity Ligation Assay, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Bone morphogenetic protein (BMP)-4, transforming growth factor (TGF)-β1, serotonin (or 5-hydroxytryptamine; 5-HT), endothelin (ET)-1, and glycogen synthase kinase (GSK)-3β inhibitors increase pulmonary smooth muscle cell size and protein synthesis. A: change in forward scatter in human pulmonary artery smooth muscle cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, SB-216763, and EGF. B: overall protein synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]leucine incorporation (cpm/well). C: Overall DNA synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]thymidine incorporation (cpm/well); n = 3, means ± SE; *P < 0.05, ANOVA.

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: DNA Synthesis

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Phosphorylation of GSK-3β is required for BMP-4-, TGF-β1-, 5-HT-, and ET-1-induced hypertrophy. A: representative immunoblots for phospho-GSK-3β and total GSK-3β in human pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763. B: GSK-3β-A9 was expressed in A7R5 cells via retroviral gene transfer. Expression of GSK-3β-A9 acts as a “dominant-negative,” decreasing the binding of upstream kinases and scaffolding proteins to native GSK-3β. This leads to a relative reduction of phosphorylated, inactive GSK-3β, and an increase in GSK-3β activity. C: effect of GSK-3β-A9 overexpression on the size of cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763 (*different from MSCV-transduced cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Expressing, Dominant Negative Mutation, Binding Assay, Scaffolding, Activity Assay, Over Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Mechanism of GSK-3β-mediated cell hypertrophy. A: representative immunoblots for phospho- and total eIF2B in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, and GSK-3β inhibitors. B: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on serum response factor (SRF) reporter activity. A7R5 cells were transiently transfected with SV40 Renilla luciferase vector and SRF-luc. Forty-eight hours after treatment, cells were lysed and luciferase activity determined. Each stimulus increased SRF activity (n = 8, means ± SE; *different from control cells, P < 0.05, ANOVA). C: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on α-actin mRNA in human pulmonary artery cells. Cells were treated for 4 days and processed for qPCR analysis of α-actin mRNA levels relative to GAPDH mRNA. Each stimulus increased α-actin mRNA (n = 3, means ± SE, *different from control cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: BMP-4, TGF-β1, 5-HT, and ET-1 activate the p70S6K signaling pathway. A: representative immunoblots for phospho-p70S6K, total p70S6K (top), phospho-S6, and total S6 (bottom) in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, and ET-1. B: group mean data (n = 3, ± SE, *different from unstimulated cells, P < 0.05, ANOVA). C: specific siRNAs against p70S6K (top) and S6 (bottom) block phosphorylation of these proteins. D: group mean data (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Blocking Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Activation of the p70S6K pathway is required for cell hypertrophy. Pulmonary artery smooth muscle cells were transfected with either nontargeting siRNA, specific siRNA against p70S6K (A), or siRNA against S6 (B), and treated with BMP-4, TGF-β1, 5-HT, or ET-1. Cell size was measured by flow cytometry. C: representative immunoblots for α-actin and β-actin from cells transfected with either nontargeting siRNA, p70S6K siRNA, or S6 siRNA. D: group mean data for p70S6K siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA). E: group mean data for S6 siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Activation Assay, Transfection, Flow Cytometry, Western Blot

Journal: bioRxiv

Article Title: Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

doi: 10.1101/2021.02.02.429321

Figure Lengend Snippet: ( A ) Real-time qPCR analysis for CDKIs and SASP factors in pulmonary artery ECs (PAECs) transfected with either GFP (control cells) or TRF2DN (premature senescent cells) (n = 5-8 each). ( B ) Schemes for co-culture experiments of PAECs and pulmonary artery SMCs (PASMCs). ( C ) Immunocytochemistry for Ki-67 in PASMCs directly (n = 7 each) or indirectly (n = 5 each) co-cultured with control or premature senescent PAECs. ( D ) Migration capacity was assessed by a modified Boyden chamber assay in PASMCs directly or indirectly co-cultured with control or premature senescent PAECs (n = 3 each). ( E ) Immunoblotting for cleaved caspase-3, total caspase-3, and GAPDH in PASMCs directly or indirectly co-culture PASMCs with control or premature senescent PAECs. Apoptosis was induced by incubating with 500 nM hydrogen peroxide for 3 h (n = 3 each). Data are presented as mean ± SEM. Two-tailed student’s t -test was used for the analysis of the differences between two groups. Two-way ANOVA with Tukey’s post hoc test was used for the analysis of the differences between groups more than three. Scale bars: 50 μM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Human pulmonary arterial endothelial cells (PAECs) and smooth muscle cells (PASMCs) were purchased from PromoCell.

Techniques: Transfection, Co-Culture Assay, Immunocytochemistry, Cell Culture, Migration, Modification, Boyden Chamber Assay, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

doi: 10.1101/2021.02.02.429321

Figure Lengend Snippet: ( A ) Real-time qPCR analysis for differentiation markers in PASMCs directly (n = 4 each) or indirect (n=4 each) co-cultured with PAECs. ( B ) Phalloidin staining in PASMCs directly or indirectly co-cultured with PAECs. ( C ) Real-time qPCR analysis for Notch ligands in PAECs transfected with either GFP (n = 6 each) or TRF2DN (n = 6 each). Cells were treated with with either vehicle or 10 μM 5-azacytidine. Data are presented as mean ± SEM. Two-tailed student’s t -test was used for the analysis of the differences between two groups. Two-way ANOVA with Tukey’s post hoc test was used for the analysis of the differences between groups more than three. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Article Snippet: Human pulmonary arterial endothelial cells (PAECs) and smooth muscle cells (PASMCs) were purchased from PromoCell.

Techniques: Cell Culture, Staining, Transfection, Two Tailed Test

Journal: bioRxiv

Article Title: Endothelial cell senescence exacerbates pulmonary hypertension through Notch-mediated juxtacrine signaling

doi: 10.1101/2021.02.02.429321

Figure Lengend Snippet: ( A ) Real-time qPCR analysis for Notch ligands in PAECs transfected with either GFP (control cells) or TRF2DN (premature senescent cells) (n= 6-8 each). ( B ) Real-time qPCR for Notch target genes in PASMCs directly or indirectly co-cultured with control or premature senescent PAECs (n = 4-5 each). ( C ) Immunocytochemistry for Ki-67 in PASMCs directly co-cultured with control or premature senescent PAECs. Cells were treated with either vehicle or 10 μM DAPT (n = 7-8 each). ( D ) Migration capacity was assessed by a modified Boyden chamber assay in PASMCs directly co-cultured with control or premature senescent PAECs. Cells were treated with either vehicle or 10 μM DAPT (n= 3 each). ( E ) Real-time qPCR analysis for Notch ligands in ECs isolated from the lungs of WT (n = 8-11) and TRF2DN-Tg (n = 9-10) mice exposed to chronic hypoxia. ( F ) Real-time qPCR analysis for Notch target genes in the lungs of WT (n = 11-12) and TRF2DN-Tg (n = 9-10) mice exposed to chronic hypoxia. Data are presented as mean ± SEM. Two-tailed student’s t -test was used for the analysis of the differences between two groups. Two-way ANOVA with Tukey’s post hoc test was used for the analysis of the differences between groups more than three. Scale bars: 50 μM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 .

Article Snippet: Human pulmonary arterial endothelial cells (PAECs) and smooth muscle cells (PASMCs) were purchased from PromoCell.

Techniques: Transfection, Cell Culture, Immunocytochemistry, Migration, Modification, Boyden Chamber Assay, Isolation, Two Tailed Test