Journal: Cardiovascular Research

Article Title: Pannexin 1: a novel regulator of acute hypoxic pulmonary vasoconstriction

doi: 10.1093/cvr/cvab326

Figure Lengend Snippet: Smooth muscle Panx1 is required for the intact HPV response. Representative western blot (A) and quantitative densitometric analysis (B) show expression of Panx1 in human pulmonary artery smooth muscle cells (hPASMCs) and human pulmonary artery endothelial cells (hPAECs). GAPDH served as loading control (n = 3 per group). Panx1 expression on hPASMC and hPAEC were assessed on the same gel. Interspersed non-relevant lanes were removed and relevant sections were re-composed at the dotted line. Representative tracing (C) of pulmonary artery pressure (PAP) in isolated perfused mouse lungs and quantitative group data analysis (D) show attenuation of the vasoconstrictive response to hypoxia (1% O2)—measured as increase in PAP (ΔPAP)—by probenecid (50 mg/kg bw) (control n = 6, probenecid n = 3). (E) The pannexin 1 (Panx1) specific inhibitory peptide (10Panx1; 800 µMol/L) reduces the ΔPAP response to hypoxia by more than 50% as compared to scrambled peptide control (scrambled peptide n = 5, 10Panx1 n = 4). (F) Pulmonary vasoconstriction in response to KCl was attenuated by 10Panx1 (800 µMol/L) (KCl n = 4, KCl+10Panx1 n = 3). (G) Tamoxifen induced deletion of Panx1 in smooth muscle cells (SMMHC-CreERT2/Panx1fl/fl), yet not Panx1 deletion in endothelial cells (Cdh5-CreERT2/Panx1fl/fl) attenuates ΔPAP response to hypoxia in isolated perfused murine lungs (n = 4–5 per group). (I and J) Hypoxia did neither change Panx1 RNA levels (n = 4 per group) nor Panx1 protein expression (n = 3 per group). Data are mean ± SEM; Data were analysed using Mann–Whitney U-test (A–F, H–J) or Kruskal–Wallis test (Figure test); *P < 0.05; ns, not significant.

Article Snippet: 2.4 Cell culture Primary human PASMCs (hPASMCs) and primary human pulmonary artery endothelial cells (hPAECs) from five different male caucasian donors were purchased from PromoCell (Heidelberg, Germany) and cultured at 95% relative humidity, 37°C and 5% CO 2 using Smooth Muscle Cell Growth Medium (PromoCell; Heidelberg, Germany) and Endothelial Cell Growth Medium MV2 (PromoCell; Heidelberg, Germany), respectively, containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin.

Techniques: Western Blot, Expressing, Isolation, MANN-WHITNEY

Journal: Cardiovascular Research

Article Title: Pannexin 1: a novel regulator of acute hypoxic pulmonary vasoconstriction

doi: 10.1093/cvr/cvab326

Figure Lengend Snippet: Panx1 does not regulate HPV via ATP release and purinergic signalling. (A) Group data show ATP concentration in supernatant and cytosolic fraction of hPASMCs under normoxic (21% O2; white bars) and hypoxic (1% O2; grey bars) conditions after 5 min. Hypoxia decreased both extracellular and intracellular ATP levels (n = 3 per group). (B) Longitudinal measurements over 15 min of hypoxia show a corresponding decrease in extracellular ATP concentration. (C) In isolated perfused mouse lungs, pretreatment with the ATP degrading enzyme apyrase (8 U/mL and 16 U/mL) did not alter the vasoconstrictive response to hypoxia (1% O2), measured as increase in pulmonary artery pressure (ΔPAP) (control n = 5, apyrase 8 U/mL n = 3, apyrase 16 U/mL n = 3). (I) In isolated perfused mouse lungs, administration of an ATP bolus (0.9 µmol) induced pulmonary vasoconstriction (n = 6), measured as increase in pulmonary artery pressure (ΔPAP), which was reduced by apyrase (8 U/mL; n = 5). (J) In isolated perfused mouse lungs, pretreatment with the purinergic receptor blocker suramin (100 µMol/L) did not alter the vasoconstrictive response to hypoxia (control n = 5, suramin n = 3). (K) Suramin (100 µMol/L) attenuated the vasoconstrictive response to an ATP bolus (0.9 µmol) (ATP = 6, ATP + suramin n = 5). Data are mean ± SEM; data were analysed using Mann–Whitney U-test (A, D–F) or Kruskal–Wallis test (C); *P < 0.05; ns, not significant.

Article Snippet: 2.4 Cell culture Primary human PASMCs (hPASMCs) and primary human pulmonary artery endothelial cells (hPAECs) from five different male caucasian donors were purchased from PromoCell (Heidelberg, Germany) and cultured at 95% relative humidity, 37°C and 5% CO 2 using Smooth Muscle Cell Growth Medium (PromoCell; Heidelberg, Germany) and Endothelial Cell Growth Medium MV2 (PromoCell; Heidelberg, Germany), respectively, containing 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin.

Techniques: Concentration Assay, Isolation, MANN-WHITNEY

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: PLoS ONE

Article Title: MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

doi: 10.1371/journal.pone.0153780

Figure Lengend Snippet: ( A ) miR-214 binding sites in the 3’-UTR of LMOD1 and MEF2C transcripts. Seed and target sequences are in blue, and base-pairing between miR-214 and target site marked by vertical lines. Mutations in the reporter plasmid construct are indicated in red letters. The double mutant (not shown) carries mutations to both sites in the 3’UTR construct vs. the single site mutants shown in 3A. ( B-E ) For luciferase assays, HEK293 cells (B, D) or hPASMCs (C, E) were transfected with miR-214 mimic or non-targeting mimic control and wildtype vs. mutant constructs for LMOD1 (B, C) or MEF2C (D, E). Relative luciferase activities were determined 48 hrs post transfection. ( n = 3–5 ), $ , p<0.05 WT scrmb vs. mimic; *, p<0.05 WT mimic vs. mutants mimic.

Article Snippet: We investigated miR-214 expression in cultured hPASMCs (Lonza) exposed to hypoxia (known to mimic clinical features of PAH). miR-214 was significantly upregulated in hPASMCs exposed hypoxia vs. normoxia ( ).

Techniques: Binding Assay, Plasmid Preparation, Construct, Mutagenesis, Luciferase, Transfection, Control

Journal: PLoS ONE

Article Title: MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

doi: 10.1371/journal.pone.0153780

Figure Lengend Snippet: HPASMCs were transfected with miR-214 (Mimic) or scrambled control (Scrmb) under normoxic conditions for 72 hrs and lysed for Western blot analysis. Overexpression of miR-214 using its mimic significantly decreased the expression of MEF2C ( A ), MYOCD ( B ), LMOD1 ( C ), MYH11 ( D ), smoothelin ( E ), CNN1 ( F ), ( n = 4–9 ). Graphs represent mean ± SEM (*, p<0.05, **, p<0.01, ***, p<0.001).

Article Snippet: We investigated miR-214 expression in cultured hPASMCs (Lonza) exposed to hypoxia (known to mimic clinical features of PAH). miR-214 was significantly upregulated in hPASMCs exposed hypoxia vs. normoxia ( ).

Techniques: Transfection, Control, Western Blot, Over Expression, Expressing

Journal: PLoS ONE

Article Title: MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

doi: 10.1371/journal.pone.0153780

Figure Lengend Snippet: HPASMCs transfected with anti-miR-214 or scrambled control (Scrmb) were exposed to normoxia (open bars) or hypoxia (filled bars) for 24 hrs and lysed for Western blot. Hypoxia significantly decreased the expression of contractile proteins, while transfection with anti-miR-214 significantly reversed the hypoxia-induced suppression of contractile proteins, MEF2C ( A ), MYOCD ( B ), LMOD1 ( C ), MYH11( D ), smoothelin ( E ) and CNN1 ( F ), ( n = 3–6 ). Graphs represent mean ± SEM (*, p<0.05, **, p<0.01, ***, p<0.001).

Article Snippet: We investigated miR-214 expression in cultured hPASMCs (Lonza) exposed to hypoxia (known to mimic clinical features of PAH). miR-214 was significantly upregulated in hPASMCs exposed hypoxia vs. normoxia ( ).

Techniques: Transfection, Control, Western Blot, Expressing

Journal: PLoS ONE

Article Title: MEF2C-MYOCD and Leiomodin1 Suppression by miRNA-214 Promotes Smooth Muscle Cell Phenotype Switching in Pulmonary Arterial Hypertension

doi: 10.1371/journal.pone.0153780

Figure Lengend Snippet: HPASMCs transfected with anti-miR-214 or scrambled control (Scrmb) were exposed to normoxia (open bars) or hypoxia (filled bars) for 24 hrs and harvested for cell counting by trypan blue exclusion ( A ), trypsinized for flow cytometry analysis using propidium iodide staining ( B ), or lysed for Western blot analysis ( C ). In both cell count and cell cycle analysis, hypoxia significantly increased the number of proliferating cells and hPASMC transfected with anti-miR-214 displayed significantly attenuated hypoxia-induced proliferation ( n = 5–6 ). Also, hPASMC transfected with anti-miR-214 attenuated hypoxia-induced increase in S+ G2/M phase cells as depicted by cell cycle analysis. ( C ) Hypoxia significantly suppressed p21 cip levels in hPASMC and anti-miR-214 significantly reversed hypoxia-induced suppression of p21 cip . HPASMCs were transfected with miR-214 mimic or scrambled control under normoxic conditions for 72 hrs and harvested for cell counting by trypan blue exclusion ( D ), trypsinized for flow cytometry analysis using propidium iodide staining ( E ). miR-214 mimic significantly increased the number of proliferating cells and increased the number of cells in S+ G2/M phase (D, E, respectively; n = 5–6 ). Graphs represent mean ± SEM (*, p<0.05, **, p<0.01, ***, p<0.001).

Article Snippet: We investigated miR-214 expression in cultured hPASMCs (Lonza) exposed to hypoxia (known to mimic clinical features of PAH). miR-214 was significantly upregulated in hPASMCs exposed hypoxia vs. normoxia ( ).

Techniques: Transfection, Control, Cell Counting, Flow Cytometry, Staining, Western Blot, Cell Cycle Assay