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: 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

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) activation in experimental and human pulmonary arterial hypertension (PAH). a) Representative immunofluorescence micrograph of human lung sections from control and idiopathic PAH (IPAH) patients. Staining was undertaken for Pin1 (green) and vessel identity was visualised using α-smooth muscle actin (SMA) (red). Scale bar=50 µm. b, d) Protein expression of Pin1 in smooth muscle cells (control n=4, IPAH n=9) and endothelial cells (control n=4, IPAH n=5) isolated from pulmonary arteries of control and IPAH patients. Regulation at protein level was analysed using Western blot analysis followed by c, e) densitometric analysis. f–i) Correlation of Pin1 with clinical characteristics of IPAH patients, such as mean pulmonary arterial pressure (mPAP) (n=4, r=0.8177, p=0.0468), pulmonary capillary wedge pressure (n=6, r= −8825, p=0.1175), cardiac index (n=4, r= −0.8276, p=01724) and systolic pulmonary artery pressure (n=7, r= −0.6285, p=0.1306), respectively. j, l) Western blot analysis of Pin1 in lung homogenates exposed to Sugen5416/hypoxia (SuHx) (normoxia (NOX) n=4, SuHx n=4) and chronic hypoxia (HOX), respectively (NOX n=6, HOX n=7) followed by k, m) densitometric analysis. Pan-actin is taken as loading control. DAPI: 4′,6-diamidino-2-phenylindole; hPASMCs: human pulmonary artery smooth muscle cells; hPAECs: human pulmonary artery endothelial cells; ns : nonsignificant; A.U.: arbitrary unit. *: p<0.05, ***: p<0.001 (t-test).

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: Activation Assay, Immunofluorescence, Control, Staining, Expressing, Isolation, Western Blot

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) blockage results in the suppression of vascular cell proliferation in vitro . a) Human pulmonary artery smooth muscle cells (hPASMCs) from controls and idiopathic pulmonary arterial hypertension (IPAH) patients cultured in SmGM-2 were serum-starved and treated with Juglone or dimethyl sulfoxide (DMSO) (vehicle) in the presence of platelet-derived growth factor (PDGF)-BB for 24 h. b) Representative Western blots of Pin1 and proliferating cell nuclear antigen (PCNA) expression in control and IPAH hPASMCs followed by c) densitometric analysis 24 h after Pin1 mRNA knockdown. Immunofluorescence staining for Ki-67 + cells in d) Pin1-silenced (si) and f) Juglone-exposed hPASMCs. g) Human pulmonary artery endothelial cells (hPAECs) were serum-starved (0.2% fetal bovine serum (FBS) in M200) and stimulated with 10% FBS with or without Juglone for 24 h. Proliferation of Pin1-silenced e) hPASMCs and h) hPAECs of donor control and IPAH patients in presence or absence of e) PDGF-BB and h) 10% FBS determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. The rate of DNA synthesis for a, e, g and h) was examined by measuring of BrdU incorporation [ A 370 nm]. Scr: scrambled; ns : nonsignificant. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. **: p<0.01, ***: p<0.001, ****: p<0.0001 versus PDGF-BB or 10% FBS treated cells; # : p<0.05, ## : p<0.01, ### : p<0.001, #### : p<0.0001 versus si scrambled or dimethyl sulfoxide (DMSO)-treated cells; § : p<0.05, §§ : p<0.01, §§§ : p<0.001, §§§§ : p<0.0001 versus si scrambled treated or IPAH cells. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: In Vitro, Cell Culture, Derivative Assay, Western Blot, Expressing, Control, Knockdown, Immunofluorescence, Staining, BrdU Incorporation Assay, DNA Synthesis

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) blockage results in initiation of cell apoptosis in vitro . Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay after 24 h treatment with increasing concentration of Juglone of a) control and i) idiopathic pulmonary arterial hypertension (IPAH) human pulmonary artery smooth muscle cells (hPASMCs), and of e) control and o) IPAH human pulmonary artery endothelial cells (hPAECs). b, j, m) Representative Western blots and c, d, k, l, n) subsequent densitometric analysis of control and IPAH hPASMCs after Juglone treatment. f, p, s) Representative Western blots and g, h, q, r, t) subsequent densitometric analysis of control and IPAH hPAECs. PARP: poly (ADP-ribose) polymerase; PCNA: proliferating cell nuclear antigen. *: p<0.05; **: p<0.01; ***: p<0.001 versus dimethyl sulfoxide (DMSO)-treated control cells. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: In Vitro, TUNEL Assay, Concentration Assay, Control, Western Blot

Journal: The European Respiratory Journal

Article Title: Targeting peptidyl-prolyl isomerase 1 in experimental pulmonary arterial hypertension

doi: 10.1183/13993003.01698-2021

Figure Lengend Snippet: Peptidyl-prolyl cis/trans isomerase, NIMA interacting 1 (Pin1) controls the activity of multitude of transcription factors. a) Control and idiopathic pulmonary arterial hypertension (IPAH) human pulmonary artery smooth muscle cells (hPASMCs) after 24 h of serum starvation were subjected to platelet-derived growth factor (PDGF)-BB (50 ng·mL −1 ), epidermal growth factor (EGF) (5 ng·mL −1 ) and growth medium (GM) with 5% fetal bovine serum (FBS). Intracellular Pin1 levels were monitored by ELISA. *: p<0.05, ****: p<0.0001 versus control PASMCs; ## : p<0.01, #### : p<0.0001 versus IPAH hPASMCs; §§ : p<0.01 IPAH hPASMCs versus control hPASMCs. Statistical analysis was performed using one-way ANOVA with Newman–Keuls post hoc test for multiple comparisons. Data from three independent experiments are presented as mean± sem . b) Pin1-silenced and Juglone-treated hPASMCs were stimulated with GM for 24 h and nuclear protein extracts were used for transcription factor activation profile array, presented as log-transformed signals in a volcano plot. c) Log-transformed scatter plot of combined transcription factor activation/inactivation in Pin1-silenced and Juglone-treated hPASMCs. Data from two independent experiments are presented. d, f) Western blots and e, g) subsequent densitometry analyses of hypoxia-inducible factor (HIF)-1α and C/EBPα transcription factors in Pin1-silenced control and IPAH hPASMCs subjected to hypoxia for 24 h. h) Hypoxia-responsive element (HRE) luciferase activity in Pin1-silenced hPASMCs after 24 h of hypoxia. Scr: scrambled; ns : nonsignificant. *: p<0.05; ****: p<0.0001 for normoxia (NOX) si Scr versus hypoxia (HOX) si Scr; § : p<0.05; §§§§ : p<0.0001 for HOX si Scr versus HOX si Pin1. Data from three independent experiments are presented as mean± sem .

Article Snippet: Human pulmonary artery smooth muscle cells (hPASMCs) were either obtained from the Universities of Giessen and Marburg Lung Center Giessen Biobank, member of DZL Platform Biobanking or purchased from Lonza (Basel, Switzerland).

Techniques: Activity Assay, Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Transformation Assay, Western Blot, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: Urolithin A Protects against Hypoxia-Induced Pulmonary Hypertension by Inhibiting Pulmonary Arterial Smooth Muscle Cell Pyroptosis via AMPK/NF-κB/NLRP3 Signaling

doi: 10.3390/ijms25158246

Figure Lengend Snippet: UA alleviated the proliferation and migration of hPASMCs. ( a ) Cell viability of hPASMCs exposed to hypoxia for varying durations ( n = 6 per group). * p < 0.05 compared to the 0 h group, ** p < 0.01 compared to the 0 h group, *** p < 0.001 compared to the 0 h group. ( b ) Cell viability of hPASMCs exposed to hypoxia for 48 h with different concentrations of UA ( n = 6 per group). * p < 0.05 compared to the 0 μM UA group, ** p < 0.01 compared to the 0 μM UA group. ( c ) Representative images and ( d ) qualification analysis of wound confluency of hPASMCs ( n = 3 per group). ** p < 0.01 compared to the control group, *** p < 0.001 compared to the control group, # p < 0.05 compared to the hypoxia group, ### p < 0.001 compared to the hypoxia group. ( e ) Representative images and ( f ) qualification analysis of cell counts of hPASMCs using Transwell assay ( n = 3 per group). Scale bars: 100 μm. ** p < 0.01 compared to the control group, # p < 0.05 compared to the hypoxia group.

Article Snippet: The human pulmonary artery smooth muscle cells (hPASMCs) used in the cell experiments were purchased from Procell (CP-H007, Wuhan, China).

Techniques: Migration, Control, Transwell Assay

Journal: International Journal of Molecular Sciences

Article Title: Urolithin A Protects against Hypoxia-Induced Pulmonary Hypertension by Inhibiting Pulmonary Arterial Smooth Muscle Cell Pyroptosis via AMPK/NF-κB/NLRP3 Signaling

doi: 10.3390/ijms25158246

Figure Lengend Snippet: UA attenuated hypoxia-induced pyroptosis in hPASMCs. ( a , b ) Western blotting analysis for the protein expression of NLRP3, GSDMD, N-GSDMD, IL-1β, and Caspase-1 relative to β-actin in hPASMCs exposed to hypoxia with or without UA treatment ( n = 6 per group). ( c ) Representative immunofluorescence staining for α-SMA (greens), Caspase-1 (red) and DAPI (blue) in hPASMCs exposed to hypoxia with or without UA treatment ( n = 3 per group). Scale bars: 50 μm. ( d ) Qualification analysis of the NLRP3 + or Caspase-1 + areas. ** p < 0.01 compared to the control group, *** p < 0.001 compared to the control group, # p < 0.05 compared to the hypoxia group, ## p < 0.01 compared to the hypoxia group.

Article Snippet: The human pulmonary artery smooth muscle cells (hPASMCs) used in the cell experiments were purchased from Procell (CP-H007, Wuhan, China).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: Urolithin A Protects against Hypoxia-Induced Pulmonary Hypertension by Inhibiting Pulmonary Arterial Smooth Muscle Cell Pyroptosis via AMPK/NF-κB/NLRP3 Signaling

doi: 10.3390/ijms25158246

Figure Lengend Snippet: UA attenuated PASMC pyroptosis through inhibiting the NF-κB/NLRP3 signaling pathway. ( a , b ) Western blotting analysis for the protein expression of p-P65 relative to P65 and p-IκB-α relative to IκB-α in hPASMCs exposed to hypoxia with or without UA treatment ( n = 6 per group). ** p < 0.01 compared to the control group, # p < 0.05 compared to the hypoxia group, ## p < 0.01 compared to the hypoxia group. ( c , d ) Western blotting analysis for the protein expression of p-P65 relative to P65 and p-IκB-α relative to IκB-α from lungs of mice exposed to normoxia (NOR) or hypoxia with UA (HX + UA) or vehicle (HX) treatment ( n = 6 per group). ** p < 0.01 compared to the NOR group, # p < 0.05 compared to the HX group.

Article Snippet: The human pulmonary artery smooth muscle cells (hPASMCs) used in the cell experiments were purchased from Procell (CP-H007, Wuhan, China).

Techniques: Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Urolithin A Protects against Hypoxia-Induced Pulmonary Hypertension by Inhibiting Pulmonary Arterial Smooth Muscle Cell Pyroptosis via AMPK/NF-κB/NLRP3 Signaling

doi: 10.3390/ijms25158246

Figure Lengend Snippet: UA inhibited NF-κB/NLRP3 pathway via activating AMPK. ( a ) Molecular structure of UA. ( b , c ) The molecular docking models of UA with ( b ) AMPK-α1 and ( c ) AMPK-α2, respectively. The solid blue lines represent hydrogen bonds, the gray dotted lines represent hydrophobic effect, and the green dotted lines represent π-π stacking interaction. ( d , e ) Western blotting analysis for the protein expression of p-AMPK relative to β-actin in hPASMCs exposed to hypoxia with or without UA treatment ( n = 6 per group). ** p < 0.01 compared to the control group, # p < 0.05 compared to the hypoxia group. ( f , g ) Western blotting analysis for the protein expression of p-AMPK relative to β-actin from lungs of mice exposed to normoxia (NOR) or hypoxia with UA (HX + UA) or vehicle (HX) treatment ( n = 6 per group). ** p < 0.01 compared to the NOR group, # p < 0.05 compared to the HX group.

Article Snippet: The human pulmonary artery smooth muscle cells (hPASMCs) used in the cell experiments were purchased from Procell (CP-H007, Wuhan, China).

Techniques: Western Blot, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Urolithin A Protects against Hypoxia-Induced Pulmonary Hypertension by Inhibiting Pulmonary Arterial Smooth Muscle Cell Pyroptosis via AMPK/NF-κB/NLRP3 Signaling

doi: 10.3390/ijms25158246

Figure Lengend Snippet: The AMPK selective inhibitor Compound C hindered the protective effect of UA on hPASMCs. ( a , b ) Western blotting analysis for the protein expression of p-AMPK relative to β-actin in hPASMCs administered with different concentrations of Compound C ( n = 3 per group). * p < 0.05 compared to the 0μM Compound C group, *** p < 0.001 compared to the 0 μM Compound C group, ns means nonsignificant. ( c , d ) Western blotting analysis for the protein expression of p-IκB-α relative to IκB-α and p-P65 relative to P65 in hPASMCs exposed to hypoxia with or without UA or Compound C treatment ( n = 6 per group).( e , f ) Western blotting analysis for the protein expression of NLRP3, N-GSDMD, IL-1β, and Caspase-1 relative to β-actin in hPASMCs exposed to hypoxia with or without UA or Compound C treatment ( n = 6 per group). ** p < 0.01 compared to the control group, *** p < 0.001 compared to the control group, # p < 0.05 compared to the hypoxia group, ## p < 0.01 compared to the hypoxia group, ^ p < 0.05 compared to the hypoxia + UA group, ^^ p < 0.01 compared to the hypoxia + UA group.

Article Snippet: The human pulmonary artery smooth muscle cells (hPASMCs) used in the cell experiments were purchased from Procell (CP-H007, Wuhan, China).

Techniques: Western Blot, Expressing, Control