Journal: Vascular Pharmacology

Article Title: SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells

doi: 10.1016/j.vph.2020.106823

Figure Lengend Snippet: The SARS-CoV-2 spike protein S1 promotes the MEK phosphorylation in human cells. (A) Human pulmonary artery smooth cells ( N = 5) and (B) human pulmonary artery endothelial cells ( N = 3) were treated with recombinant full length S1 subunit of the SARS-CoV-2 spike protein (Val16 - Gln690) at 10 ng/ml for the durations indicated. Cell lysates were prepared and subjected to Western blotting using antibodies against phosphorylated MEK (p-MEK), MEK protein, phosphorylated Akt (p-Akt), and phosphorylated Stat3 (p-Stat3). Bar graphs represent means ± SEM. *Significantly different from 0 min at p < 0.05.

Article Snippet: Human pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and rat pulmonary artery smooth muscle cells were purchased from Cell Applications (San Diego, CA, USA).

Techniques: Phospho-proteomics, Recombinant, Western Blot

Journal: Vascular Pharmacology

Article Title: SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells

doi: 10.1016/j.vph.2020.106823

Figure Lengend Snippet: In rat cells, the SARS-CoV-2 spike protein S1 does not phosphorylate, but rather dephosphorylates MEK. Rat pulmonary artery smooth muscle cells were treated with recombinant full length S1 subunit of the SARS-CoV-2 spike protein (Val16 - Gln690) at 10 ng/ml for the durations indicated. Cell lysates were prepared and subjected to Western blotting using antibodies against phosphorylated MEK (p-MEK) and MEK protein. The bar graph represents means ± SEM ( N = 4). *Significantly different from 0 min at p < 0.05.

Article Snippet: Human pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and rat pulmonary artery smooth muscle cells were purchased from Cell Applications (San Diego, CA, USA).

Techniques: Recombinant, Western Blot

Journal: Vascular Pharmacology

Article Title: SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells

doi: 10.1016/j.vph.2020.106823

Figure Lengend Snippet: The SARS-CoV-2 spike protein S1 does not promote the phosphorylation of MEK in the presence of the neutralizing antibody against ACE2. Human pulmonary artery smooth muscle cells were pre-treated with the ACE2 antibody for 1 h and then treated with recombinant full length S1 subunit of the SARSCoV-2 spike protein (Val16 – Gln690) for 10 min. Cell lysates were prepared and subjected to Western blotting using antibodies against phosphorylated MEK (p-MEK) and MEK protein. The bar graph represents means ± SEM (N = 4).*Significantly different from untreated control at p < 0.05. NS denotes that the two values are not significantly different from each other at p < 0.05.

Article Snippet: Human pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and rat pulmonary artery smooth muscle cells were purchased from Cell Applications (San Diego, CA, USA).

Techniques: Phospho-proteomics, Recombinant, Western Blot, Control

Journal: Vascular Pharmacology

Article Title: SARS-CoV-2 spike protein-mediated cell signaling in lung vascular cells

doi: 10.1016/j.vph.2020.106823

Figure Lengend Snippet: The RBD only containing SARS-CoV-2 spike protein S1 does not activate the MEK phosphorylation. (A) Human pulmonary artery smooth muscle cells and (B) human pulmonary artery endothelial cells were treated with recombinant full length S1 subunit (Val16 - Gln690) or the RBD region of S1 subunit (Arg319 - Phe541) at 100 ng/ml for the durations indicated. Cell lysates were prepared and subjected to Western blotting using antibodies against phosphorylated MEK (p-MEK) and the MEK protein. Bar graphs represent means ± SEM ( N = 3). *Significantly different from 0 min control at p < 0.05.

Article Snippet: Human pulmonary artery smooth muscle cells and human pulmonary artery endothelial cells were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA), and rat pulmonary artery smooth muscle cells were purchased from Cell Applications (San Diego, CA, USA).

Techniques: Phospho-proteomics, Recombinant, Western Blot, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 2. Effects of Bcl-xL on pulmonary vas- cular SMC apoptosis. (A) BPASMC were in- fected with adenovirus expressing Bcl-xL (AdBcl-xL) for 48 h, then treated with SNP (300 M) for 17 h. Cells were washed with PBS, trypsinized, incubated with Trypan Blue, and the number of viable cells was counted on a hematocytometer. The values represent means SE. Letters (a and b) denote that the values with the same letter are significantly different from each other at P 0.05. Top panel shows the expression level of Bcl-xL with or without adenovirus (adv)-mediated over- expression. (B) BPASMC were infected with AdBcl-xL or control adenovirus (AdCont) for 48 h, then treated with SNP (100 M) for 17 h. Mitochondrial membrane potentials were measured by DePsipher Kit. In healthy mitochondria, the DePsipher dye aggregates and forms red fluorescence. When the mem- brane potential is disrupted during the early stage of apoptosis, the dye cannot cross the mitochondrial membrane and is visualized as a monomeric form with green fluorescence in the cytosol. Fluorescence signals were ob- tained using a red/green dual filter, and un- der these conditions, green fluorescence was not observed in cells without staining with the dye. Green apoptotic cells are indicated by the arrows. (C) BPASMC were pretreated with 5-HT or ET-1 for 2 h, then treated with SNAP (100 M) or DNR (2 M) for 24 h. Percent of apoptotic cells were monitored by neutral comet assay. Values represent means SE. The values denoted by the same letter (a, b, or c) are significantly different from each other at P 0.05.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Expressing, Incubation, Over Expression, Infection, Control, Membrane, Fluorescence, Staining, Neutral Comet Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 3. Effects of SNP on Bcl-xL expression. (A) Rats were subjected to chronic hypoxia with 10% O2 in an OxyCycler Oxygen Profiler for 2 wk to elicit pulmonary vascular remodeling. Remodeled pulmonary arteries were surgically isolated, cut into 2-mm segments, and placed in Dulbecco’s modified Eagle’s medium (DMEM). Arterial segments were treated with SNP (300 M) for 20 h. Tissues were homogenized in Trizol and total RNA was prepared. RT-PCR was performed with primers for bcl-xL and g3pdh mRNA. The bar graph represents means SE of the intensity of the bcl-xL band expressed in arbitrary units (a.u.). (B) BPASMC were treated with SNP for 24 h at various concentra- tions. Cell lysates (10 g protein) were subjected to Western blot to monitor levels of Bcl-xL. ERK was used as a loading control. The bar graph represents means SE of the intensity of Bcl-xL band from cells untreated or treated with 100 M SNP (n 3). (C) BPASMC were transfected with the luciferase gene controlled by the 0.6-kb bcl-xL promoter region without (open circles) or with (solid triangles) SNP. Cell lysates were prepared at various time points after transfection and luciferase activities were measured and expressed in resonance units (R.U.). (D) Cells were co-transfected with firefly luciferase gene con- trolled by 0.6-kb bcl-xL promoter (pBcl-xL) and Renilla luciferase con- trolled by thymidine kinase promoter (pTK), and treated with various concentrations of SNP. Cell lysates were prepared and luciferase activi- ties were measured. The values represent means SE of the ratio of firefly luciferase activity to Renilla luciferase activity. Asterisk denotes a significantly different value compared with control at P 0.05.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Expressing, Isolation, Modification, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Transfection, Luciferase, Activity Assay

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 4. Effects of SNP on GATA-4. (A) BPASMC were treated with SNP for 20 h. Nuclear extracts were prepared and the GATA DNA-binding activity was moni- tored by EMSA. The bar graph represents means SE of the intensity of GATA activ- ity from cells untreated or treated with SNP (300 M) (n 3). Asterisk denotes a value significantly different from the control value at P 0.05. (B) BPASMC were infected with control adenovirus (AdCont) or adenovirus expressing wild- type GATA-4 (AdGATA4) for 24 h, then treated with SNP (100 M) for 24 h. Cell lysates were prepared and levels of Bcl-xL expression were monitored by Western blot. The ERK antibody was used as a load- ing control. The values in the bar graph represent means SE. Asterisk denotes values significantly different from SNP- treated values at P 0.05. (C) HPASMC were treated with SNP (100 M) for dura- tions indicated. Total RNA was isolated and mRNA expression levels of gata4 and g3pdh were determined by RT-PCR. The bar graph shows the ratio of gata4 to g3pdh bands. Similar results were ob- tained in three separate experiments. (D) Rats were subjected to chronic hypoxia with 10% O2 in an OxyCycler Oxygen Profiler for 2 wk to elicit pulmonary vascu- lar remodeling. Remodeled pulmonary arteries were surgically isolated, cut into 2-mm segments, and plated in DMEM. Arterial segments were treated with SNP (300 M) for 20 h. Arterial segments were homogenized in Trizol and total RNA was prepared. RT-PCR was performed with primers for gata4 and g3pdh mRNA. The bar graph represents means SE of the intensity of the gata4 band. Asterisk de- notes a value significantly different from the control value at P 0.05.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Binding Assay, Activity Assay, Control, Infection, Expressing, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 5. Effects of SNP on the gata4 gene promoter activity. (A) A scheme depicting the mouse gata4 gene structure with the major tran- scriptional start site of mouse gata4 gene identified by 5RACE to occur 4.1 kb upstream of the translational start site. The 1,000 bp upstream from the identified transcriptional start site (shaded area) that is con- served among various species was cloned into a luciferase reporter vector. (B) BPASMC were co-transfected with the firefly luciferase con- struct controlled by the 1,000-bp proximal region of the gata4 promoter (pGATA4) and Renilla luciferase construct controlled by the thymidine kinase promoter (pTK). Cells were then treated with SNP (300 M) for 24 h, cell lysates were prepared, and luciferase activities were measured. Values represent means SE of the ratio of pGATA4-luciferase to pTK luciferase activities (n 4). Asterisk denotes values significantly different from the untreated control value at P 0.05. (C) The region of the gata4 promoter 1,000 bp proximal to the transcriptional start site was truncated to generate regions of the promoter 500 or 250 bp upstream from the transcriptional start site. BPASMC were transfected with lucifer- ase constructs controlled by these regions of the gata4 promoter. Values represent means SE of the ratio of pGATA4-luciferase and pTK lucifer- ase activities (n 6–11). (D) BPASMC were co-transfected with the firefly luciferase construct controlled by the 1,000, 500 or 250 bp fragment of the gata4 promoter and pTK-Renilla luciferase construct. Cells were then treated with SNP (200 M) for 24 h, cell lysates were prepared, and luciferase activities were measured. Values represent means SE of percent of the ratio of pGATA4-luciferase and pTK luciferase activities relative to untreated controls.

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Activity Assay, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Construct, Control

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Regulation of Bcl-xLExpression in Lung Vascular Smooth Muscle

doi: 10.1165/rcmb.2006-0359oc

Figure Lengend Snippet: Figure 6. Effects of SNP on transcription factors which bind to the proximal 250 bp gata4 promoter. (A) The sequence of the 250-bp gata4 promoter proximal to the transcriptional start site. Putative binding sites for transcription factors are in- dicated. (B) Nuclear extracts were prepared from untreated BPASMC, and the DNA- binding activity toward the 32P-labeled dou- ble-stranded 250 bp gata4 promoter probe was monitored by EMSA. Supershift experi- ments were performed with antibodies (ab) indicated. No ab indicates controls for supershift experiments without the in- clusion of any antibodies in nuclear ex- tracts from untreated cells. The letter A indicates the DNA-protein complex with- out supershift. The letter B indicates the free probe. The gel at the bottom shows that the band A can be eliminated by in- creasing amounts of the cold 250-bp gata4 promoter probe. (C) BPASMC were treated with SNP (100 or 300 M) for 2 h, and nuclear extracts were prepared. The DNA-binding activity toward the

Article Snippet: Bovine pulmonary artery SMC (BPASMC) (19) from mid-size pulmonary arteries and human pulmonary artery SMC (HPASMC) (Cell Applications, San Diego, CA) at 2–6 passages were maintained in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 0.5% fungisone at 5% CO2 and 37 C. Cells were treated with SNP (Sigma Chemical, St. Louis, MO), S-nitroso-N-acetylpenicillamine (SNAP; Calbiochem, San Diego, CA), daunorubicin (DNR; Sigma), serotonin (5-hydroxytryptamine, 5-HT; Sigma) and endothelin-1 (ET-1; Sigma) in media supplemented only with antibiotics.

Techniques: Sequencing, Binding Assay, Activity Assay, Labeling

Journal: The American Journal of Pathology

Article Title: Exploratory Study of Prognostic Plasma Biomarkers in Patients with Pulmonary Arterial Hypertension

doi: 10.1016/j.ajpath.2025.04.018

Figure Lengend Snippet: Incremental predicted performance of identified biomarkers and expression in animal models. A: Performance of the logistic regression models with 2015 European Respiratory Society (ERS)/European Society of Cardiology (ESC) risk score, REVEAL 2.0 risk score, and the refined four-strata risk assessment method in patients with PAH for death and lung transplantation. Graph of C-statistics with 95% CI for each biomarker and their combinations. C-statistics were compared using the DeLong test with each reference. B: Heat map showing RNA-sequencing expression data of the indicated genes [in transcripts per million (TPM)] obtained from the Genotype-Tissue Expression (GTEx version 8) database ( https://gtexportal.org/home , last accessed August 13, 2024) in lung, heart, artery, kidney, liver, and adipose tissue. C: Western blot analyses and corresponding quantifications of WFDC2 (HE4) in right ventricles and dissected pulmonary arteries (PAs) from monocrotaline (MCT)–injected and Sugen/hypoxia (Su/Hx)–exposed rats. D: Western blot analyses and corresponding quantifications of WFDC2 (HE4) in kidney from MCT-injected and Su/Hx-exposed rats. E: Wfdc2 mRNA expression in right ventricle (RV), lung, and kidney of control (CTRL) and MCT-injected or Su/Hx-exposed rats. F: Representative fluorescent images of 5-ethynyl-2′-deoxyuridine (EdU)–labeled control pulmonary artery smooth muscle cells (PASMCs) and control cardiac fibroblasts (CFs) exposed or not to recombinant human WFDC2 (rhWFDC2; 40 nmol/L) for 24 and 48 hours, respectively. Ar row s indicate EdU-positive cells. The corresponding quantifications are shown. Scatter dot plots show individual values. Protein expression was normalized to amido black (AB). Statistical analyses were performed using t -test. Data are given as means ± SEM ( A and E ). n = 6 to 16 ( E ); n = 3 control PASMCs ( F ); n = 4 control CFs ( F ). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001. Scale bars = 100 μm ( F ). Veh, vehicle.

Article Snippet: Control pulmonary artery smooth muscle cells (PASMCs; n = 5 cell lines) were either purchased from Cell Applications (San Diego, CA) or isolated from patients without PAH.

Techniques: Expressing, Transplantation Assay, Biomarker Discovery, RNA Sequencing, Western Blot, Injection, Control, Labeling, Recombinant

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Expression Profiles of circRNAs and Identification of hsa_circ_0007608 and hsa_circ_0064656 as Potential Biomarkers for COPD-PH Patients

doi: 10.2147/COPD.S424712

Figure Lengend Snippet: qRT-PCR validation of circRNAs expression in serum and pulmonary vascular cell. ( A and B ) Two significantly up-regulated circRNAs in serum, n=12. ( C ) hsa_circ_0007608 was constantly up-regulated with the prolongation of hypoxia in HPAECs, n=3. ( D and E ) hsa_circ_0064656 decreased significantly in HPAECs, while increased in HPASMCs, n=3. (*P<0.05, **P<0.01, *** P<0.001).

Article Snippet: Pulmonary arterial smooth muscle cells (HPASMCs) were purchased from iCell Research Laboratory cultured in smooth muscle culture medium.

Techniques: Quantitative RT-PCR, Biomarker Discovery, Expressing

Journal: International Journal of Chronic Obstructive Pulmonary Disease

Article Title: Expression Profiles of circRNAs and Identification of hsa_circ_0007608 and hsa_circ_0064656 as Potential Biomarkers for COPD-PH Patients

doi: 10.2147/COPD.S424712

Figure Lengend Snippet: Identification and validation of hub mRNAs. ( A ) Correlation heat map of 35 key mRNA. ( B ) The protein–protein interaction (PPI) of key mRNAs.( C – E ) VCAM1, VCAN and THBS1 were up-regulated in HPAECs after 48 hours of hypoxia treatment, n=3 (**P<0.01, *** P<0.001). ( F – H ) VCAM1, VCAN and THBS1 were up-regulated in HPASMCs after 48 hours of hypoxia treatment, n=3 (*** P<0.001). ( I and J ) Representative immunohistochemistry staining and quantitative analysis of VCAN in two groups of human lung tissue, ×200, scale bar: 100μm, n=3. (***P< 0.001, normal vs COPD-PH group, Student t test).

Article Snippet: Pulmonary arterial smooth muscle cells (HPASMCs) were purchased from iCell Research Laboratory cultured in smooth muscle culture medium.

Techniques: Biomarker Discovery, Immunohistochemistry, Staining