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: iScience

Article Title: Superenhancer-driven circRNA Myst4 involves in pulmonary artery smooth muscle cell ferroptosis in pulmonary hypertension

doi: 10.1016/j.isci.2024.110900

Figure Lengend Snippet: CircMyst4 is downregulated in PH (A) Genomic location of circRNA Myst4 (circMyst4). CircMyst4 was formed by the back-splicing of exons 2 of Myst4. And the back-splicing junction site of circMyst4 was identified by Sanger sequencing. (B) Real-time qPCR analysis of genomic DNA (gDNA) and cDNA using divergent and convergent primers of circMyst4 ( n = 6). (C) RNase R degradation analysis was performed to detect the stability of circMyst4 and linear Myst4 mRNA ( n = 6). (D) Real-time qPCR analysis was conducted to detect the amount of circMyst4 and linear Myst4 mRNA in PASMCs after actinomycin (D) treatment ( n = 6). (E and F) Real-time qPCR analysis of circMyst4 expression levels in lung tissues of hypoxic PH mouse ( n = 8 mice/group) and hypoxic PASMCs ( n = 6). (G) Real-time qPCR analysis of circMyst4 expression levels in plasma of hypoxic PH mouse and normoxic mouse ( n = 10). (H) Fluorescence in situ hybridization analysis of circMyst4 location in lung tissues of mouse. CircMyst4 probes were labeled with Cy3 (red). Nucleus were stained with DAPI (blue), pulmonary smooth muscle stained with α-SMA (green). Scale bar, 100 μm ( n = 6 mice/group). (I) Real-time qPCR analysis was using to determine the circMyst4 expression in the nucleus and cytoplasm of PASMCs after exposure to hypoxia for 24 h ( n = 6). (J) RNA FISH analysis of the subcellular localization of circMyst4 in PASMCs. CircMyst4 probes were labeled with Cy3 (red). Nucleus were stained with DAPI (blue). U6 and 18S RNA, used as internal references, were labeled with Cy3 (red). Scale bar, 100 μm ( n = 6). Data are shown as means ± SD. Statistical analysis was performed with Student’s t test. Hyp, hypoxia; Nor, normoxia; ns, not significantly different. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Mouse pulmonary artery smooth muscle cells (PASMCs) were obtained from Procell Life Science & Technology (Wuhan, China), and were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 15% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PEST) and were placed in a 37°C humidified incubator containing 5% CO2.

Techniques: Sequencing, Expressing, Clinical Proteomics, Fluorescence, In Situ Hybridization, Labeling, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: FGF21 attenuates pulmonary arterial hypertension via downregulation of miR‐130, which targets PPARγ

doi: 10.1111/jcmm.17154

Figure Lengend Snippet: FGF21 can significantly downregulate miR‐130 expression in PAH mice, and miR‐130 can directly target PPARγ and inhibit its expression. (A) Heatmap diagram revealing all differentially expressed microRNAs (miRNAs) in the Hyp and Hyp+FGF21 groups compared in lung tissues, ranging from the most upregulated (red) to the most downregulated (blue). (B) Scatter plot demonstrating differential miRNA expression in the two groups. Red = miRNAs with higher expression, green = miRNAs with lower expression and black = miRNAs with equal expression. (C) The top 15 pathways of the target genes of dysregulated miRNA were identified using KEGG analysis according to the number of enriched genes. (D) qRT‐PCR was used to detect miRNA expression of miR‐130 in the Nor, Hyp and Hyp+FGF21 groups mice ( n = 3) and (E) pulmonary arterial smooth muscle cells (PASMCs; n = 4). The miRNA level is normalized to U6 by the 2 −ΔCt method before comparative analysis. (F) Potential targeted binding between miR‐130 and peroxisome proliferator‐activated receptor gamma (PPARγ) was predicted by using miRDB, TargetScan and starBase web tools. (G) The identified complementary binding sites were validated by performing dual‐luciferase reporter ( n = 6). (H) qRT‐PCR was used to detect PPARγ 3ʹ‐UTR expression. The mRNA level is normalized to β‐actin by the 2 −ΔCt method before comparative analysis ( n = 4). (I and J) Western blotting for PPARγ expression in PASMCs transfected with miR‐130 mimic and miR‐130 inhibitor ( n = 4), β‐actin was used as a loading control. (K and L) qRT‐PCR was used to detect mRNA expression of PPARγ in PASMCs transfected with miR‐130 mimic and miR‐130 inhibitor ( n = 4). The mRNA level is normalized to β‐actin by the 2 −ΔCt method before comparative analysis. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: Pulmonary artery smooth muscle cells were isolated from male Sprague‐Dawley rats (weighing 180 ± 10 g) using well‐established methods, as previously described.

Techniques: Expressing, Quantitative RT-PCR, Binding Assay, Luciferase, Western Blot, Transfection, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: FGF21 attenuates pulmonary arterial hypertension via downregulation of miR‐130, which targets PPARγ

doi: 10.1111/jcmm.17154

Figure Lengend Snippet: FGF21 attenuates chronic hypoxia‐induced PAH by inhibiting the negative regulatory effects of miR‐130 on PPARγ. (A) Representative pictures of right ventricular systolic pressure (RVSP) waves in Nor, Hyp, Hyp+FGF21, Hyp+FGF21+NC and Hyp+FGF21+Agomir groups ( n = 5). All hypoxia groups were raised in a normobaric chamber with 10% O 2 for 21 days. (B) Representative images of transthoracic echocardiography. (C) Measurement of RVSP and (D) RV/(LV+S) values in each group ( n = 5). (E) Heart rate (HR, bpm; n = 5). (F) Mice weight in each group ( n = 5). (G) Quantification of pulmonary artery acceleration time (PAAT) and (H) velocity time integral (VTI; n = 5). (I) Representative images of alpha‐smooth muscle actin (α‐SMA; in red) and DAPI (nuclear DNA; in blue) immunostaining of the distal pulmonary arteries in each group (×200, scale bars indicate 50 µm). (J) H&E staining was used to evaluate pulmonary vascular remodelling. Representative photomicrographs showing hypoxia‐induced remodelling in the pulmonary arteries of mice exposed to hypoxia (10% O 2 ) or ambient oxygen levels (21% O 2 ) for 3 weeks (×400; scale bars indicate 50 µm). (K) Quantification of WA/TA (%) and (L) WT/TT (%) ratios in each group ( n = 8). (M) The degree of collagen deposition (blue) in each group was evaluated microscopically by Masson staining and is shown as the ratio of the collagen fibre area (blue) to the pulmonary artery wall area (red; n = 10). (N) Representative photomicrographs showing hypoxia‐induced collagen deposition (blue) around the pulmonary arteries (×200; scale bars indicate 100 µm). Data are presented as the mean ±SD. * p < 0.05, ** p < 0.01

Article Snippet: Pulmonary artery smooth muscle cells were isolated from male Sprague‐Dawley rats (weighing 180 ± 10 g) using well‐established methods, as previously described.

Techniques: Immunostaining, Staining

Journal: BMC Pulmonary Medicine

Article Title: Bmi-1 alleviates adventitial fibroblast senescence by eliminating ROS in pulmonary hypertension

doi: 10.1186/s12890-021-01439-0

Figure Lengend Snippet: Fibroblasts Bmi-1 alters PASMC proliferation by paracrine mode of action. a – b . EDU staining for PASMCs treated with the CM of HLFs. HLFs were infected with adv-Bmi-1 or transfected with si-Bmi-1 following by hypoxia exposure for 96 h, and then the supernatant was collected and added the equal volume of serum-free culture medium to culture PASMCs for 48 h. All data are shown as the mean ± SEM of at least three independent experiments. Statistical significance was assessed using the unpaired two-tailed Student’s t test: * P < 0.05

Article Snippet: The human pulmonary artery smooth muscle cell line (PASMC) and human lung fibroblast cell line (HLF) were purchased from Lonza (Swiss).

Techniques: Staining, Infection, Transfection, Two Tailed Test

Journal: Pulmonary Circulation

Article Title: Increased plasma expression of a disintegrin and metalloproteinase with thrombospondin motifs like 4 in patients with idiopathic pulmonary arterial hypertension and chronic thromboembolic pulmonary hypertension

doi: 10.1002/pul2.12267

Figure Lengend Snippet: ADAMTSL4 expression in PASMCs. The protein expression of ADAMTSL4 in PDGF‐BB‐treated PASMCs were detected using Western blot. * p < 0.05 versus the control group. ADAMTSL4, a disintegrin and metalloproteinase with thrombospondin motifs like 4; IPAH, idiopathic pulmonary arterial hypertension; PASMCs, pulmonary artery smooth muscle cells; PDGF, platelet‐derived growth factor.

Article Snippet: Primary rat pulmonary arterial smooth muscle cells (PASMCs) were isolated from healthy Sprague‐Dawley rats (6–8 weeks, 180–200 g) and cultured in Dulbecco's modified Eagle's medium/nutrient mixture F‐12 containing 20% fetal bovine serum and 1% penicillin and streptomycin.

Techniques: Expressing, Western Blot, Control, Derivative Assay

Journal: bioRxiv

Article Title: A Molecular Pathway for Arterial-Specific Association of Vascular Smooth Muscle Cells

doi: 10.1101/2019.12.27.889782

Figure Lengend Snippet: A,B, Schematic diagrams illustrating the experimental design for using the mrc1a promoter to drive ectopic mosaic expression of cxcl12b in veins. A, A Tol2(mrc1a:cxcl12b-2a-mCherry) DNA construct co-translationally expressing cxcl12b and mCherry under the control of the mrc1a promoter is injected into Tg(tagln:eGFP) transgenic zebrafish embryos at the 1 cell stage. B, At 4 dpf tol2(mrc1a:cxcl12b-2a-mCherry) -injected zebrafish larvae are analyzed for vSMC (eGFP) association at sites of mCherry (i.e. cxcl12b ) expression in the dorsal aorta and cardinal vein. C,D , Representative confocal images of the mid-trunk of 4 dpf Tg(tagln:eGFP) transgenic larvae injected with either control Tol2(mrc1a) “empty vector” (C) or Tol2(mrc1a:cxcl12b-2a-mCherry) (D). eGFP-expressing vSMCs are shown in green, cxcl12b-2a-mCherry expression in dorsal aorta (DA) or cardinal vein (CV) endothelium is shown in magenta. E , Quantification of eGFP-positive vSMC associated with the dorsal aorta (DA) or cardinal vein (CV) in 4 dpf Tg(tagln:eGFP) transgenic zebrafish injected with either control Tol2(mrc1a) “empty vector” (black columns) or Tol2(mrc1a:cxcl12b-2a-mCherry) (green columns), showing strongly increased association of vSMCs with the cardinal vein. F , Schematic diagrams showing potential models for direct (left) versus indirect (right) mechanisms for promoting arterial recruitment of vSMC via CXCL12. G , Schematic diagram illustrating the 3D pulmonary artery smooth muscle cell (PASMC) motility assay. CXCL12, PDGFB, or nothing (control) is placed within the collagen gel to determine if PASMCs migrate towards these potential chemoattractants. H, Representative lateral images of 3D collagen gels showing PASMCs within the collagen matrix for each gel condition. I , Quantification of the relative number of PASMCs invading the collagen gel. The control is set to 100% and the CXCL12 and PDGFB conditions normalized to this level of invasion. Scale bars = 75 µm (panels C,D), 200 µm (panel H). Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers. P-values are indicated above statistically significant datasets.

Article Snippet: Human coronary artery smooth muscle cells (PASMC, Lonza) were cultured in 10% FBS in Advanced DMEM base media (Gibco) on 1mg/ml gelatin coated tissue culture flasks.

Techniques: Expressing, Construct, Control, Injection, Transgenic Assay, Plasmid Preparation, Motility Assay

Journal: bioRxiv

Article Title: A Molecular Pathway for Arterial-Specific Association of Vascular Smooth Muscle Cells

doi: 10.1101/2019.12.27.889782

Figure Lengend Snippet: A,B, PDGFB transcript (A) and protein (B) in HUVEC cells cultured in vitro in a confluent cell monolayer for up to 8 hours with (“+CXCL12”) or without (“CTRL”) added recombinant CXCL12. Relative PDGFB transcript levels (A) and protein levels (B) were measured by qPCR and Western blot, respectively, showing an upregulation of both PDGFB transcript and PDGFB protein levels in response to stimulation by CXCL12. C-E , PDGFB transcript (C) and protein levels (D,E) in HUVEC cells cultured in vitro in a confluent cell monolayer and treated with either control, CXCR4, or CXCL12 siRNAs. Relative PDGFB transcript (C) and protein (E,F) levels were measured by qPCR and Western blot, respectively, showing suppression of both PDGFB transcript and protein in response to either CXCR4 or CXCL12 knockdown. Values in A, C, and E are averaged from three individual experiments and expressed as a percentage of control. Error bars ± s.d. (A,C). F, Confocal images of immunohistochemically stained transverse sections through the dorsal aorta of E12.5 Cxcr4+/- heterozygous sibling (F) and Cxcr4-/- mutant (G) mice, probed for platelet derived growth factor B (PDGFB; green) and for smooth muscle 22 alpha (SM22, aka transgelin) for vascular smooth muscle cells (vSMC, red). G, Quantification of relative PDGFB protein expression in Cxcr4+/- heterozygous embryos versus Cxcr4-/- homozygous mutant embryos. Values are expressed as a percentage of heterozygous control and averaged from five individual mice per condition. H , Schematic diagram of a zebrafish larva with the red box highlighting the area imaged in panels I and J. I,J, Whole mount in situ hybridization of the mid-trunk of 2.5 dpf zebrafish injected with control (I) or cxcl12b (J) RNA, showing upregulation of pdgfb transcript in response to exogenous cxcl12b . Red and blue brackets in panel J indicate the dorsal aorta and cardinal vein, respectively. K, Western blot of whole embryo protein lysate from 2.5 dpf zebrafish injected with either control (left) or cxcl12b (right) RNA, probed for pdgfb (top) or alpha tubulin (bottom), showing upregulation of pdgfb protein levels in response to exogenous cxcl12b . Images are representative of data from three individual experiments. M , Schematic diagram illustrating the proposed model for endothelial-autonomous chemokine signaling driving increased endothelial PDGFB ligand production, thereby indirectly promoting vSMC acquisition by arteries. Putative upstream regulators of CXCL12 and CXCR4 are noted in red. Scale bars = 50 µm (panel F). Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers. P-values are indicated above statistically significant datasets.

Article Snippet: Human coronary artery smooth muscle cells (PASMC, Lonza) were cultured in 10% FBS in Advanced DMEM base media (Gibco) on 1mg/ml gelatin coated tissue culture flasks.

Techniques: Cell Culture, In Vitro, Recombinant, Western Blot, Control, Knockdown, Staining, Mutagenesis, Derivative Assay, Expressing, In Situ Hybridization, Injection