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

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Microtubule-disturbing drugs, including DTX, are effective in killing proliferating PASMCs. Proliferating/synthetic phenotype (A) and differentiated/contractile phenotype of human PASMCs (B) were treated with various antitumor drugs at 1 µM for 24 hours. Cell number was determined by counting on a hemocytometer. Equal amounts of water (for daunorubicin) and 0.1% dimethylsulfoxide (DMSO; for other drugs) were used as vehicle controls. Symbols a and b denote significantly different from DMSO and water, respectively (n = 6–9) at P < 0.05. (C) Representative photographs of control and DTX-treated PASMCs. (D–F) Proliferating/synthetic human PASMCs were treated with DTX, paclitaxel, or vincristine for 24 hours. The number of viable cells was monitored by using Cell Counting Kit-8.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Cell Counting

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Inhibition of autophagy potentiates DTX-induced death of PASMCs. (A) Proliferating/synthetic human PASMCs were pretreated with dimethylsulfoxide (DMSO; 0.5%), SBI-0206965 (50 μM), or Z-VAD-FMK (50 μM) for 30 minutes and then treated with DMSO (0.1%) or DTX (50 nM) for 22 hours. The number of viable cells was monitored by using Cell Counting Kit-8 at absorbance 450 nm (A450). (B) Cells were transfected with siRNA for beclin-1 or control scrambled siRNA for 2 days. Cells were then treated with DMSO or DTX (50 nM) for 22 hours, and cell number was counted using a hemocytometer. Western blotting results demonstrate the extent of siRNA knockdown of Beclin-1 (n = 6–9). (C) Cells were transfected with siRNA for LC3B or control scrambled siRNA. Cells were then treated with DMSO or DTX, and cell number was counted. Western blotting results demonstrate the extent of siRNA knockdown of LC3B (n = 5). (D) Cells were transfected with siRNA for p62 or control scrambled siRNA. Cells were then treated with DMSO or DTX, and cell number was counted. Western blotting results demonstrate the extent of siRNA knockdown of p62 (n = 6–9). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Inhibition, Cell Counting, Transfection, Western Blot

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Effects of DTX on autophagy in PASMCs. (A and B) Human PASMCs were treated with dimethylsulfoxide (DMSO; 0.1%) or DTX (50 nM) for 22 hours, and cell lysates were subjected to Western blotting to monitor LC3B-II and p62 levels (n = 5–7). (C) Rats were treated with SU5416/hypoxia and injected with saline or DTX. Protein levels of p62 were monitored by Western blotting in isolated PA homogenates (n = 7). *Significant difference between each other at P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Western Blot, Injection, Isolation

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: Effects of DTX on Beclin-1. (A) Proliferating/synthetic human PASMCs were treated with dimethylsulfoxide (DMSO; 0.1%) or DTX (50 nM) for 22 hours. Beclin-1 protein expression was monitored by Western blotting (n = 6). *Values that are significantly different from each other at P < 0.05. (B) Rats with PAH and control rats were treated with saline or DTX, and Beclin-1 protein expression was monitored in the homogenates of isolated PAs (n = 7). *Values that are significantly different from each other at P < 0.05. (C) Proliferating/synthetic human PASMCs were treated with DMSO or DTX (50 nM) for 22 hours. The beclin-1 mRNA expression was monitored by reverse-transcription polymerase chain reaction (n = 6). ns, values are not significantly different from each other at P < 0.05. (D) Human PASMCs were infected with adenovirus expressing Beclin-1 for 48 hours. Cells were then treated with DMSO (0.1%) or DTX (50 nM) for 22 hours. Beclin-1 protein expression was monitored by Western blotting (n = 3). *Values that are significantly different from each other at P < 0.05. (E) Human PASMCs were pretreated with MG132 (250 nM) for 6 hours and then treated with DMSO (0.1%) or DTX (50 nM) for 22 hours. Beclin-1 protein expression was monitored by Western blotting in cell lysates (n = 6). The symbol “a” denotes values that are significantly different from the DTX value at P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: The Journal of Pharmacology and Experimental Therapeutics

Article Title: Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

doi: 10.1124/jpet.117.239921

Figure Lengend Snippet: The identification of a protein that interacts with Beclin-1 in response to DTX. (A) Proliferating/synthetic human PASMCs were treated with dimethylsulfoxide (DMSO; 0.1%) or DTX (50 nM) for 24 hours. Cell lysates were subjected to immunoprecipitation with rabbit Beclin-1 IgG or normal rabbit IgG, SDS-PAGE, and Coomassie Blue staining. The arrow indicates a band that is consistently upregulated by DTX. (B) Immunoprecipitated (IP) samples with the Beclin-1 IgG were Western blotted (WB) with goat MYH9 IgG (n = 6). *Values that are significantly different from each other at P < 0.05. (C) PA homogenates from rats with PAH treated with saline or DTX were immunoprecipitated with goat MYH9 IgG and subjected to Western blotting with rabbit Beclin-1 IgG (n = 4). *Values that are significantly different from each other at P < 0.05. (D) Human PASMCs were transfected with siRNA for MYH9. The extent of the MYH9 knockdown was determined by Western blotting. (E) Human PASMCs with MYH9 knocked down were treated with DMSO or DTX. Cell number was counted on a hemocytometer (N = 6). The symbol “a” denotes values that are significantly different from the control siRNA + DTX value at P < 0.05. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Human PASMCs and PAECs were purchased from ScienCell Research Laboratories (Carlsbad, CA) and Cell Applications, Inc. (San Diego, CA) and were cultured in accordance with the manufacturers’ instructions in 5% CO 2 at 37°C.

Techniques: Immunoprecipitation, SDS Page, Staining, Western Blot, Transfection