Journal: Molecular Medicine Reports

Article Title: Rs41291957 polymorphism in the promoter region of microRNA-143 serves as a prognostic biomarker for patients with intracranial hemorrhage

doi: 10.3892/mmr.2021.11928

Figure Lengend Snippet: TLR2 is a direct target gene of miR-143. (A) Computational analysis of the regulatory relationship between miR-143 and TLR2 mRNA. (B) Luciferase assay of THP-1 cells co-transfected with wild-type or mutant TLR2 mRNA, and miR-143 or miRNA controls. (C) Luciferase assay of HPASMC cells co-transfected with wild-type or mutant TLR2 mRNA, and miR-143 or miRNA controls. (D) Relative expression of miR-143 in THP-1 cells transfected with miR-143 mimics. (E) Relative expression of miR-143 in HPASMC cells transfected with miR-143 mimics. n=3. *P<0.05 vs. negative control group. TLR2, Toll-like receptor 2; miR-143, microRNA-143; miR-cont, microRNA-control; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant; NC, negative control.

Article Snippet: THP-1 (human monocytic cells) and human pulmonary artery smooth muscle cells (HPASMC) obtained from American Type Culture Collection were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Luciferase, Transfection, Mutagenesis, Expressing, Negative Control, Control

Journal: Molecular Medicine Reports

Article Title: Rs41291957 polymorphism in the promoter region of microRNA-143 serves as a prognostic biomarker for patients with intracranial hemorrhage

doi: 10.3892/mmr.2021.11928

Figure Lengend Snippet: IL-16 mRNA is not a target of miR-143. (A) Computational analysis of the regulatory relationship between miR-143 and IL-16 mRNA. (B) Luciferase assay of THP-1 cells co-transfected with wild-type or mutant IL-16 mRNA, and miR-143 or miRNA controls. (C) Luciferase assay of HPASMC cells co-transfected with wild-type or mutant IL-16 mRNA, and miR-143 or miRNA controls. IL-16, interleukin-16; miR-143, microRNA-143; 3′-UTR, 3′-untranslated region; WT, wild-type; MUT, mutant.

Article Snippet: THP-1 (human monocytic cells) and human pulmonary artery smooth muscle cells (HPASMC) obtained from American Type Culture Collection were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Luciferase, Transfection, Mutagenesis

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: Cell culture and treatments 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: Cell culture and treatments 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: Cell culture and treatments 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: Cell culture and treatments 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: Cell culture and treatments 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: Cell culture and treatments 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: Frontiers in Cell and Developmental Biology

Article Title: Activation and Contraction of Human “Vascular” Smooth Muscle Cells Grown From Circulating Blood Progenitors

doi: 10.3389/fcell.2021.681347

Figure Lengend Snippet: Morphology and comparative calcium responses to U46619 of blood outgrowth smooth muscle cells (BO-SMCs), vascular smooth muscle cells (VSMCs), and fibroblasts (HPFs). Phase contrast images of BO-SMCs, VSMCs, and fibroblasts were obtained using Zeiss Axio Observer WF1 or WF3 microscope at ×20 magnification (A) . Represented fluorescence images of cells stained with Flou-4 treated with U46619 (10 –6 M) (B) . Fluorescence intensity (fluorescence minus basal fluorescence taken at t = 0; F – F 0 ) tracings from n = 10 individual cells per donor imaged at 5–10 frames/s stained with Flou-4 treated with U46619 (10 –6 M) (C) . Pooled data showing mean ± SEM for the average intensity tracings obtained following U46619 (10 –6 M) treatment (BO-SMCs; n = 40 cells comprising 10 individual cells each from four separate donors, VSMCs and HPF; n = 30 cells comprising 10 individual cells from three separate donors) or untreated time controls (BO-SMCs; n = 30 cells comprising 10 individual cells each from three separate donors, VSMCs, and HPF; n = 20 cells comprising 10 individual cells from two separate donors) (D) . Scale bars represent 100 μm.

Article Snippet: Human primary pulmonary artery VSMCs and pulmonary fibroblasts were obtained from PromoCell (Germany) and cultured in SMC and fibroblast growth media (5% FBS) respectively, following suppliers’ protocols (PromoCell, Heidelberg, Germany).

Techniques: Microscopy, Fluorescence, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Activation and Contraction of Human “Vascular” Smooth Muscle Cells Grown From Circulating Blood Progenitors

doi: 10.3389/fcell.2021.681347

Figure Lengend Snippet: Contraction responses to U46619 of blood outgrowth smooth muscle cells (BO-SMCs), vascular smooth muscle cells (VSMCs), and fibroblasts (HPFs). Representative images of contracting cells captured at 10 min after addition of cumulative concentrations of U46619 (1 × 10 –9 to 10 –6 M) (A) and pooled data (B) . Data in (B) are mean ± SEM; for BO-SMCs, n = 23 using cells from four separate donors; for VSMCs, n = 14 using cells from three separate donors; and for HPFs, n = 9 fields using cells from three separate donors. Filled symbols represent treated cells and open symbols represent untreated “time-control” (TC) cells. Statistical differences were tested using two-way ANOVA with a Sidak-multiple comparison post hoc test comparing individual U46619 concentration responses with cell-relevant time controls (* p < 0.05) or two-way ANOVA comparing the absence vs. presence of U46619 over time ( # p < 0.05). Data in panel 4 (bottom right hand graph) which include all cell types were tested using a two-way ANOVA ( # p < 0.05) comparing VSMCs or HPFs with BO-SMCs. Scale bars represent 100 μm.

Article Snippet: Human primary pulmonary artery VSMCs and pulmonary fibroblasts were obtained from PromoCell (Germany) and cultured in SMC and fibroblast growth media (5% FBS) respectively, following suppliers’ protocols (PromoCell, Heidelberg, Germany).

Techniques: Concentration Assay