Journal: The American journal of the medical sciences

Article Title: Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells

doi: 10.1016/j.amjms.2016.03.019

Figure Lengend Snippet: Time course of hypoxia-induced HIF-1α activation in HPASMCs. (A) HPASMC HIF-1α mRNA levels were measured with qRT-PCR following exposure to hypoxia (1% O2) for 2, 8, 24 or 72 hours. Data are expressed relative to ribosomal 9S and displayed as fold change versus (vs.) normoxic (21% O2) control ± SE at the same time point (n = 4−16). *P < 0.05 vs. normoxia and **P < 0.01 vs. normoxia. (B) Quantitative densitometric analysis of Western blots for HIF-1α in nuclear protein extracts of HPASMCs exposed to normoxia (N, 21% O2) or hypoxia (1% O2) for 4 and 8 hours. In separate experiments, cells were exposed to normoxia or hypoxia for 72 hours. Each bar represents mean ± SE HIF-1α nuclear protein relative to fibrillarin levels in the same sample expressed as fold change over normoxia (n = 3−7). *P < 0.05 vs. normoxia and **P < 0.01 vs. normoxia. SE, standard error.

Article Snippet: Cell Culture HPASMCs were purchased from Lonza (Walkersville, MD) and cell monolayers (passages 3–4) were grown at 37°C in a 5% CO 2 atmostphere in culture media (SmGM-2, Lonza) containing 2% fetal calf serum, growth factors and antibiotics as prevoiusly reported.

Techniques: Activation Assay, Quantitative RT-PCR, Control, Western Blot

Journal: The American journal of the medical sciences

Article Title: Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells

doi: 10.1016/j.amjms.2016.03.019

Figure Lengend Snippet: Rosiglitazone attenuates early HIF-1α expression in HPASMCs exposed to hypoxia. HPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) and simultaneously treated with DMSO (Veh) or rosiglitazone (RSG) (10 μM) for 2–4 hours. (A) HPASMC HIF-1α mRNA levels following treatment for 2 hours. Each bar graph represents mean ± SE HIF-1α mRNA normalized to GAPDH in the same sample expressed as fold change over normoxia (n = 3). SE, standard error. (B) Representative immunoblots and averaged desitometric analysis of Western blotting for HIF-1α levels in HPASMC nuclear protein extracts treated for 4 hours. Each bar represents mean ± SE nuclear HPASMC HIF-1α protein levels relative to fibrillarin in the same sample expressed as fold change over normoxia (n = 3). **P < 0.05 versus (vs.) Normoxia-Veh and ##P < 0.001 vs. Hypoxia-Veh.

Article Snippet: Cell Culture HPASMCs were purchased from Lonza (Walkersville, MD) and cell monolayers (passages 3–4) were grown at 37°C in a 5% CO 2 atmostphere in culture media (SmGM-2, Lonza) containing 2% fetal calf serum, growth factors and antibiotics as prevoiusly reported.

Techniques: Expressing, Western Blot

Journal: The American journal of the medical sciences

Article Title: Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells

doi: 10.1016/j.amjms.2016.03.019

Figure Lengend Snippet: Hypoxia-induced PDK-1 expression in HPASMCs is attenuated by rosiglitazone. (A) HPASMCs PDK-1 mRNA levels were measured with qRT-PCR following exposure to hypoxia (1% O2) for 0, 2, 8 or 24 hours. Each bar represents the mean ± SE PDK-1 mRNA relative to ribosomal 9S expressed as fold change versus (vs.) normoxic control (n = 4). HPASMCs were then exposed to normoxia (21% O2) or hypoxia (1% O2) for 8 or 24 hours and simultaneously treated with DMSO (Veh) or rosiglitazone (RSG, 10 μM). (B) HPASMC PDK-1 mRNA levels were determined with qRT-PCR following exposure to hypoxia for 8 hours. Each bar represents mean ± SE PDK-1 mRNA relative to ribosomal 9S expressed as fold change vs. normoxic control (n = 4). ***P < 0.001 vs. Normoxia-Veh and #P < 0.05 vs. Hypoxia-Veh. (C) PDK-1 protein levels were examined in HPASMC lysates by Western blot analysis following exposure to hypoxia for 24 hours. Each bar represents mean ± SE PDK-1 relative to CDK4 levels in the same sample expressed as fold change over normoxia (n = 7). *P < 0.05 vs. normoxia. ##P < 0.01 vs. Hypoxia-Veh. SE, standard error.

Article Snippet: Cell Culture HPASMCs were purchased from Lonza (Walkersville, MD) and cell monolayers (passages 3–4) were grown at 37°C in a 5% CO 2 atmostphere in culture media (SmGM-2, Lonza) containing 2% fetal calf serum, growth factors and antibiotics as prevoiusly reported.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot

Journal: The American journal of the medical sciences

Article Title: Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells

doi: 10.1016/j.amjms.2016.03.019

Figure Lengend Snippet: Rosiglitazone fails to attenuate chronic hypoxia-induced PDK-1 and GLUT1 protein expression in HPASMC. HPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) for 72 hours. During the final 24 hours of exposure, HPASMCs were treated with DMSO (Veh) or rosiglitazone (RSG, 10 μM). Western blotting was performed to determine PDK-1 (A), GLUT1 (B) or HIF-2α (C) levels in HPASMC. Each bar represents the mean ± SE PDK-1, GLUT1, or HIF-2α levels relative to β-actin in the same sample expressed as fold change over normoxia (n = 6). *P < 0.05 versus Normoxia. SE, standard error.

Article Snippet: Cell Culture HPASMCs were purchased from Lonza (Walkersville, MD) and cell monolayers (passages 3–4) were grown at 37°C in a 5% CO 2 atmostphere in culture media (SmGM-2, Lonza) containing 2% fetal calf serum, growth factors and antibiotics as prevoiusly reported.

Techniques: Expressing, Western Blot

Journal: The American journal of the medical sciences

Article Title: Time-dependent PPARγ Modulation of HIF-1α Signaling in Hypoxic Pulmonary Artery Smooth Muscle Cells

doi: 10.1016/j.amjms.2016.03.019

Figure Lengend Snippet: Depletion of HIF-1α attenuates chronic hypoxia-induced PDK-1 expression in HPASMC. HPASMCs were transfected with control siRNA or HIF-1α siRNA (to deplete HIF-1α) and then exposed to normoxia or hypoxia for 72 hours. Western blotting was performed to determine PDK-1 levels. Depletion of HIF-1α was verified by Western blotting using an antibody against HIF-1α. Each bar represents mean ± SE PDK-1 level relative to β-actin in the same sample expressed as fold change over normoxia (n = 3). **P < 0.01 versus (vs.) Normoxia; ##P < 0.01 vs. hypoxia. SE, standard error.

Article Snippet: Cell Culture HPASMCs were purchased from Lonza (Walkersville, MD) and cell monolayers (passages 3–4) were grown at 37°C in a 5% CO 2 atmostphere in culture media (SmGM-2, Lonza) containing 2% fetal calf serum, growth factors and antibiotics as prevoiusly reported.

Techniques: Expressing, Transfection, Control, Western Blot

Journal: Molecular Medicine Reports

Article Title: Iptakalim influences the proliferation and apoptosis of human pulmonary artery smooth muscle cells

doi: 10.3892/mmr.2016.5333

Figure Lengend Snippet: GLI reverses the effect on ET-1 induced change of apoptotic protein expression in HPASMCs by IPT (western blot, n=3). * P<0.05 vs. control group. # P<0.05 vs. ET-1 group. & P<0.05 vs. ET-1+IPT group. GLI, glibenclamide; HPASMC, human pulmonary artery smooth muscle cells; IPT, iptakalim.

Article Snippet: HPASMCs were purchased from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: Expressing, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin inhibits mTORC1 and mTORC2. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-p70S6k, p70S6k, p-AKT (S473), p-AKT (T308), AKT. (B) immunoblotting analyses of p-p70S6k, p70S6k, p-AKT (S473), and AKT in hPASMCs, which were stimulated with 5 μg/ml insulin for 24h before treatment with 100 nM rapamycin. (C) hPASMCs were treated with 100 nM rapamycin for the indicated times, and then cell lysates were prepared for and immunoprecipitation (IP) with mTOR antibody. The elution from IP was analyzed by immunoblotting for the levels of mTOR and Rictor. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Western Blot, Immunoprecipitation, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin in hPASMCs. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-PDGFRα/β, PDGFRα, PDGFRβ. (B) Immunoblotting analyses of p-PDGFRα/β, PDGFRα, PDGFRβ, p-AKT (S473), p-AKT (T308), p-S6 and S6 in hPASMCs treated with vehicle (Control), 100 nM rapamycin (Rap), 5 uM imatinib (Ima) and 100 nM rapamycin + 5 uM imatinib (Rap + Ima) for 48 h. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on the viability, proliferation and migration of hPASMCs. (A) Cell viability was determined by measuring the absorbance at 0, 24, 48 and 72 h after different drug treatments. (B) A scratch was applied to cell monolayers, and migration of the cells towards the wound was recorded by photomicrographs at 0, 4, and 8h ( n = 3); summarized data showing percent wound closure [(0h wound area–4h or 8h wound area)/0h wound area] * 100%. (C) BrdU assay was performed to determine hPASMCs proliferation under normoxia and hypoxia (3% 0 2 ) for 24 and 48 h. (D) BrdU assay was performed to determine hPASMCs proliferation at 24 and 48 h after different drug treatments. Data are presented as the mean ± SE. Two-way ANOVA was used for statistical analysis. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Rap; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Ima.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Migration, BrdU Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by MCT. (A) H&E staining in lung tissue sections. Summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus MCT with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus MCT with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by Hypoxia/Sugen. (A) H&E staining of lung tissue sections and summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained with α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means no significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Hypoxia/Sugen with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Hypoxia/Sugen with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on mTOR and PDGFR signaling pathways in pulmonary artery. (A) Pulmonary artery vessels of were isolated for protein extraction, and the expression of mTORC 1, mTORC 2 and PDGFR signaling pathway related proteins were detected by immunoblotting. Data are presented as the mean ± SE. One-way ANOVA followed by Graphpad prism was used for statistical analysis. NS means no significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control. ### p < 0.001; ## p < 0.01; # p < 0.05 versus MCT with vehicle. (B) The schematic representation of the findings of this study: rapamycin chronic treatment in hPASMCs induced the highly expression of phosphorylation of PDGFRs. Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin. Abbreviations: GF, growth factors; RTK, receptor tyrosine kinase; PDGF, platelet derived growth factor; PDGFR, platelet derived growth factor receptor; PI3K, phosphoatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-bisphosphate; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Protein-Protein interactions, Isolation, Protein Extraction, Expressing, Western Blot, Control, Phospho-proteomics, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: Sildenafil attenuated the hypoxia-induced downregulation of PPARγ expression and inhibited the hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. (A) HPASMCs were identified by immunofluorescence staining for α-SMA in cells grown under normoxic conditions. (B) Western blot and (C) RT-qPCR analysis of the increase in PPARγ protein and mRNA expression in HPASMCs induced by different concentrations of sildenafil under hypoxic conditions. Each of the four groups were treated with 0, 1, 10 or 50 nM sildenafil. (D) Western blot and (E) RT-qPCR analysis of the sildenafil-mediated attenuation of hypoxia-induced downregulation of PPARγ expression and sildenafil-mediated inhibition of hypoxia-induced upregulation of TRPC and Ki67 expression in HPASMCs. There were three experimental groups: i) The normoxic control group (60 h), ii) the hypoxia group (60 h, 4% O 2 ), and iii) the hypoxia + sildenafil group (60 h, 4% O 2 , 50 nM sildenafil). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; SMA, smooth muscle actin; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Inhibition, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: A PPARγ inhibitor (GW9662) inhibited the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the inhibitory effects of different concentrations of a PPARγ inhibitor (GW9662) on PPARγ protein expression in HPASMCs under hypoxic conditions. Cells were treated with either 0, 1 or 10 nM GW9662. (C) Western blot and (D) RT-qPCR analysis of the inhibitory effect of a PPARγ inhibitor (GW9662) on the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + GW9662 group (60 h, 4% O 2 , 10 nM), and (iv) the hypoxia + sildenafil + GW9662 group (60 h, 4% O 2 , 10 nM). Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control, # P>0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; TRPC, transient receptor potential canonical; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Sildenafil protects against pulmonary hypertension induced by hypoxia in neonatal rats via activation of PPARγ-mediated downregulation of TRPC

doi: 10.3892/ijmm.2021.5074

Figure Lengend Snippet: si-PPARγ reversed the sildenafil-induced downregulation of TRPC and Ki67 expression under hypoxic conditions. (A) Western blot and (B) RT-qPCR analysis of the downregulation of PPARγ protein expression in HPASMCs transfected with si-PPARγ under normoxic conditions. There were four groups: si-NC, siR-PP1, siR-PP2 and siR-PP3. (C) Western blot and (D) RT-qPCR analysis of the reversal of the sildenafil-induced downregulation of TRPC and Ki67 protein expression in HPASMCs induced by PPARγ siRNA under hypoxic conditions. There were four groups: i) The hypoxic control group (60 h, 4% O 2 ), ii) the hypoxia + sildenafil group (60 h, 4% O 2 ), iii) the hypoxia + siR-PP1 group (60 h, 4% O 2 ), and iv) the hypoxia + sildenafil + siR-PP1 group. Data are presented as the mean ± standard deviation of three repeats. * P<0.05 vs. control. PPARγ, peroxisome proliferator-activated receptor γ; HPASMC, human pulmonary artery smooth muscle cell; siRNA, small interfering RNA; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: HPASMCs derived from ScienCell primary cells were used for the in vitro experiments.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Control, Standard Deviation, Small Interfering RNA, Negative Control, Reverse Transcription, Real-time Polymerase Chain Reaction

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: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Arterial stiffness induces remodeling phenotypes in pulmonary artery smooth muscle cells via YAP/TAZ-mediated repression of cyclooxygenase-2

doi: 10.1152/ajplung.00173.2017

Figure Lengend Snippet: Overexpression of active YAP and TAZ represses COX-2. Human PASMCs (Lonza) were stably transfected with FLAG-tagged, nuclear-localizing YAP (YAP5SA) or TAZ (TAZ4SA), similar constructs lacking TEAD-binding capability (YAP5SA S94A, TAZ4SA S51A), or control vector (pLVX-Puro). RNA was isolated and assessed for YAP (A), TAZ (B), and COX-2 (C) expression via qPCR. Kruskal-Wallis ANOVA testing was performed on ΔCT values, and Dunn’s posttest was performed. Relative expression is displayed ± SD; n = 2–4 independent experiments. D–H: protein was isolated and Western blot was performed for TAZ, YAP, ANKRD1, FLAG, and COX-2. Quantification represents 3 independent experiments; representative blots are shown. *P < 0.05 for YAP5SA compared with pLVX-Puro and TAZ4SA. **P < 0.05 for TAZ4SA compared with pLVX-Puro and YAP5SA. #P < 0.05 for TAZ4SA compared with pLVX-Puro. ¥P < 0.01 for pLVX-Puro compared with TAZ4SA and YAP5SA. I: levels of 6-keto-PGF1α were measured by ELISA. Statistical significance was determined by one-way ANOVA. *P = 0.02, **P = 0.009 compared with pLVX-Puro. #P = NS. n = 3 experiments. J: TAZ4SA-expressing cells and control (pLVX-Puro)-expressing cells were seeded onto discrete stiffness polyacrylamide gels with shear moduli of 0.4, 6.4, and 25.6 kDa. 6-keto-PGF1α concentrations were measured by ELISA and normalized to cell number. Statistical significance was determined by two-way ANOVA; n = 2 independent experiments.

Article Snippet: ELISA hPASMCs (Lonza) or stable YAP or TAZ overexpressing lines derived from hPASMCs (above) were seeded at a density of 100 cells/mm 2 in 24-well plates containing discrete stiffness polyacrylamide gels with stiffness of 0.4, 6.4, or 25.6 kPa or on TCP and cultured at 37°C and 5% CO 2 .

Techniques: Over Expression, Stable Transfection, Transfection, Construct, Binding Assay, Control, Plasmid Preparation, Isolation, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Shear

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

Article Title: Arterial stiffness induces remodeling phenotypes in pulmonary artery smooth muscle cells via YAP/TAZ-mediated repression of cyclooxygenase-2

doi: 10.1152/ajplung.00173.2017

Figure Lengend Snippet: YAP/TAZ knockdown enhances COX-2 expression and activity. Human PASMCs (Lonza) were grown on discrete stiffness polyacrylamide gels with shear moduli of 0.4, 6.4, and 25.6 kPa with or without siControl or siYAP/TAZ transfection. A–E: RNA was isolated 48 h after treatment, and qPCR was performed to assess COX-2 (A and B), COX-1 (C), PTGER2 (D), and PTGIR (E) expression. Kruskal-Wallis ANOVA testing was performed on ΔCT values, and Dunn’s posttest was performed. Relative expression is displayed ± SD; n = 6 independent experiments. No significant differences in COX-1, PTGER2, or PTGIR expression were observed. F and G: protein was isolated and Western blotting performed using anti-COX-2 (Santa Cruz) and anti-GAPDH (Santa Cruz) antibodies. n = 3 independent experiments, representative blots are shown. H: levels of 6-keto-PGF1α were measured in media by ELISA and normalized to cell number. Statistical significance was determined by two-way ANOVA; n = 3 independent experiments.

Article Snippet: ELISA hPASMCs (Lonza) or stable YAP or TAZ overexpressing lines derived from hPASMCs (above) were seeded at a density of 100 cells/mm 2 in 24-well plates containing discrete stiffness polyacrylamide gels with stiffness of 0.4, 6.4, or 25.6 kPa or on TCP and cultured at 37°C and 5% CO 2 .

Techniques: Knockdown, Expressing, Activity Assay, Shear, Transfection, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Expression Profiles of circRNAs and Identification of hsa_circ_0007608 and hsa_circ_0064656 as Potential Biomarkers for COPD-PH Patients

doi: 10.2147/COPD.S424712

Figure Lengend Snippet: qRT-PCR validation of circRNAs expression in serum and pulmonary vascular cell. ( A and B ) Two significantly up-regulated circRNAs in serum, n=12. ( C ) hsa_circ_0007608 was constantly up-regulated with the prolongation of hypoxia in HPAECs, n=3. ( D and E ) hsa_circ_0064656 decreased significantly in HPAECs, while increased in HPASMCs, n=3. (*P<0.05, **P<0.01, *** P<0.001).

Article Snippet: Pulmonary arterial smooth muscle cells (HPASMCs) were purchased from iCell Research Laboratory cultured in smooth muscle culture medium.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Expression Profiles of circRNAs and Identification of hsa_circ_0007608 and hsa_circ_0064656 as Potential Biomarkers for COPD-PH Patients

doi: 10.2147/COPD.S424712

Figure Lengend Snippet: Identification and validation of hub mRNAs. ( A ) Correlation heat map of 35 key mRNA. ( B ) The protein–protein interaction (PPI) of key mRNAs.( C – E ) VCAM1, VCAN and THBS1 were up-regulated in HPAECs after 48 hours of hypoxia treatment, n=3 (**P<0.01, *** P<0.001). ( F – H ) VCAM1, VCAN and THBS1 were up-regulated in HPASMCs after 48 hours of hypoxia treatment, n=3 (*** P<0.001). ( I and J ) Representative immunohistochemistry staining and quantitative analysis of VCAN in two groups of human lung tissue, ×200, scale bar: 100μm, n=3. (***P< 0.001, normal vs COPD-PH group, Student t test).

Article Snippet: Pulmonary arterial smooth muscle cells (HPASMCs) were purchased from iCell Research Laboratory cultured in smooth muscle culture medium.

Techniques: Biomarker Discovery, Immunohistochemistry, Staining

Journal: Biomedicines

Article Title: H 2 S Donor Therapy Reverses Established Pulmonary Arterial Hypertension and Pulmonary Vascular Structural Remodeling in Rats

doi: 10.3390/biomedicines14040760

Figure Lengend Snippet: H 2 S donors restored the expression of key H 2 S-producing enzymes and antagonized hypoxia-induced proliferation of hPASMCs. ( A ). Schematic diagram of cell grouping and treatment regimen. ( B ). Immunoblotting assays and quantitative analysis showing the effects of hypoxia exposure and NaHS treatment on the protein expression levels of CSE, CBS, and MPST in hPASMCs. ( C ). Immunoblotting assays and quantitative analysis showing the effects of hypoxia exposure and GYY4137 treatment on the protein expression levels of CSE, CBS, MPST, PCNA, and phospho-ERK1/2 in hPASMCs. All data are presented as mean ± SEM, n = 6. * p < 0.05, ** p < 0.01.

Article Snippet: The hPASMCs (Procell, Wuhan, China) were cultured in specialized medium at 37 °C and 5% CO 2 .

Techniques: Expressing, Western Blot

Journal: Biomedicines

Article Title: H 2 S Donor Therapy Reverses Established Pulmonary Arterial Hypertension and Pulmonary Vascular Structural Remodeling in Rats

doi: 10.3390/biomedicines14040760

Figure Lengend Snippet: H 2 S inhibited hPASMC proliferation via upregulation of ETAR persulfidation. ( A ). Biotin-switch assay (BSA) quantitation of ETAR persulfidation and total ETAR in rat lung tissues. ( B ). Schematic diagram of cell grouping and treatment regimen. ( C ). BSA quantification of ETAR persulfidation and total ETAR in cultured hPASMCs. ( D ). Western blot detection of ERK1/2 phosphorylation and PCNA in hPASMCs. All data are expressed as mean ± SEM, n = 6. * p < 0.05, ** p < 0.01, ns: not statistically significant.

Article Snippet: The hPASMCs (Procell, Wuhan, China) were cultured in specialized medium at 37 °C and 5% CO 2 .

Techniques: Biotin Switch Assay, Quantitation Assay, Cell Culture, Western Blot, Phospho-proteomics