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: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunocytochemistry images of pSPHK2 (pink), actin (green, cytoplasmic marker) and DAPI (blue, nuclear) coimmunostaining in EMAP II treated (2 hr) or vehicle treated fixed hPASMCs, scale bar is 20 μm, n=3. (B) Representative immunoblot probed for pSPHK2, tubulin and lamin B in cytoplasmic and nuclear fractions of hPASMCs following EMAP II treatment for 0, 2 and 4 hours, n=3. (C) Representative immunoblot probed for pSPHK2 and lamin B in nuclear fractions of hPASMCs following EMAP II treatment (150 minutes) with or without SPHK2 inhibitor (D) quantification of nuclear pSPHK2/lamin B, n=3. (E) ELISA-nuclear C18-S1P levels normalized against 1 μg of nuclear proteins in the nuclear fractions of hPASMCs following EMAP II for 15 or 150 minutes with or without SPHK2 inhibitor, n=3 or 4/group. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Immunocytochemistry, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Schematic diagram representing the collection of vascular endothelial cells (ECs) conditioned media (ECM) from ECs grown in 1%O2 or room air to treat vascular smooth muscle cells (SMCs) and, (B) representative dot blot probed for secreted EMAP II expression in ECM. (C) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing and, (D) quantification of KLF4/Tubulin, n=3 and (E) quantification of Ac-H3K9/total histone H3, n=3. (F) KLF4 expression levels normalized against 18S rRNA in normoxia or hypoxia ECM with or without EMAP II neutralizing antibody treated hPASMCs pre-transfected with siRNA mediated SPHK2 or scramble silencing, n=3–4. (G) EMAP II secreted by vascular ECs promote SPHK2/Ac-H3K9/KLF4 signaling in vascular SMCs that may promote PASMCs proliferation. P values are calculated using Kruskal-Wallis against Hy ECM+Scr or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Dot Blot, Expressing, Western Blot, Transfection

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for AIMP1 (precursor form of EMAP II) and Tubulin in protein lysates of human iPAH or FDL, n=19–20/group and, (B) quantitation of AIMP1 (AIMP1/Tubulin) in protein lysates of human iPAH or FDL, n=19–20/group. (C) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPASMCs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (D) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPASMCs, n=4. (E) Representative immunoblot probed for Ac-H3K9, total H3 or tubulin in hPMVECs following EMAP II treatment for 0, 1, 2, 4 and 6 hours and (F) quantitation of Ac-H3K9 expression levels normalized against total H3 in hPMVECs, n=3. P values are calculated using unpaired t-test or Kolmogorov-Smirnov non-parametric testing and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) Representative immunoblot probed for Ac-H3K9, total H3, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 4 hours and (B) quantitation of Ac-H3K9/total H3 and (C) quantification of SPHK2/tubulin, n=4. (D) Volcano plot showed the log2-fold changes and statistical significance of hyperacetylated H3K9 regions calculated after differential binding analysis of EMAP II treated vs control hPASMCs. Pink points indicate significantly hyperacetylated H3K9 regions in EMAP II (right to 0) or in control (left to 0). FDR=0.05, n=2 (E) Genome wide distribution of differentially enriched hyperacetylated H3K9 peaks (log2-fold change > 1, p value < 0.05) n=2. (F) Number of peaks of Ac-H3K9 normalized to IgG in with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs, n=2. (G) Gene Ontology results using differentially enriched Ac-H3K9 peaks in EMAP II treated hPASMCs, n=2. (H) Cell proliferation rate in hPASMCs treated with vehicle or EMAP II following SPHK2 inhibitor treatment for 24 hours, n=3. P values are calculated using Kruskal-Wallis against control or Kolmogorov-Smirnov non-parametric test and results are shown as means ± SEM or median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Western Blot, Transfection, Quantitation Assay, Binding Assay, Control, Genome Wide

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) The Venn’s diagram of differential acetylated sites in control vs EMAP II (total) (purple), EMAP II vs iSPHK2+EMAP II (yellow) and control vs EMAPII only in 5’UTR and upstream with fold enrichment greater than 2 (green). The red circle indicates the potential gene set with potential upstream candidate regulatory elements that would be differentially acetylated by EMAP II through SPHK2 in hPASMCs. Venn diagram is created using Venny 2.1 (an online interactive tool), n=2/group (B) Snapshot of IGV view of KLF4 gene in Ac-H3K9 CUT&RUN data of with or without SPHK2 inhibitor and EMAP II treated (2–3 hours) hPASMCs. (cCRE= candidate Cis-Regulatory Elements) n=2/group (C) Representative immunoblot probed for KLF4, SPHK2 and tubulin in whole cell lysates of hPASMCs following siRNA mediated SPHK2 silencing and post-transfection EMAP II treatment for 6–8 hours, and (D) quantitation of KLF4/tubulin and (E) KLF4 expression levels normalized against 18S rRNA in hPASMC cells following siRNA mediated SPHK2 silencing and EMAP II treatment for 6 hours, n=4. P values are calculated using Kolmogorov-Smirnov non-parametric testing and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: Control, Western Blot, Transfection, Quantitation Assay, Expressing

Journal: Circulation research

Article Title: Altered Smooth Muscle Cell Histone Acetylome by the SPHK2/S1P Axis Promotes Pulmonary Hypertension

doi: 10.1161/CIRCRESAHA.123.322740

Figure Lengend Snippet: (A) RNA-seq data of SPHK2, KLF4 and AIMP1 in iPAH: PASMCs and non-iPAH:PASMCs in log2-fold of count per million (cpm). Following two-way ANOVA, Sidak’s multiple comparisons test for logarithmic values, n=4. (B) Representative immunoblot probed for KLF4, SPHK2, tubulin, Ac-H3K9 and total histone H3 in whole cell lysates of non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection and, quantification of (C) Ac-H3K9/total histone H3 and, (D) KLF4/Tubulin, n=3 (E) KLF4 expression levels normalized against 18S rRNA in non: iPAH or iPAH PASMCs with scramble or SPHK2 siRNA transfection, n=4. (F) Cell proliferation rate of non: iPAH or iPAH PASMC with or without iSPHK2 pretreatment for 24 hours, n=4. (G) The proposed model: Endothelial monocyte activating polypeptide II (EMAP II) plays a key role in reawakening pluripotency factor, KLF4 in human pulmonary artery smooth muscle cells (PASMCs) through stimulation of the nuclear SPHK2/S1P epigenetic modulating axis, suggesting that cooperation between SPHK2 and EMAP II could be a major driving force for epigenetic-mediated vascular PASMCs reprogramming and remodeling in PH. Ablation of SPHK2 expression confers protection against PH by rescuing the global and local transcription machinery from histone acetylation and activation of the pluripotency factor, KLF4. P values are calculated using Kruskal-Wallis against iPAH or Kolmogorov-Smirnov non-parametric test if not mentioned otherwise, and results are shown as median and inter-quartile range.

Article Snippet: Primary human PASMCs (hPASMCs), pulmonary microvascular endothelial cells (hPMVECs) purchased from Lonza (Walkersville, MD) and iPAH: PASMCs were cultured in complete growth medium or conditioned media in a humidified atmosphere or 1% O 2 with 5% CO 2 at 37° C. For all studies, passages 5–10 were used for hPASMCs and hPMVECs.

Techniques: RNA Sequencing, Western Blot, Transfection, Expressing, Activation Assay