primary human pulmonary arterial smooth muscle cells (hpasmcs  (ScienCell)

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 suppress HPASMC proliferation and migration. HPASMCs were cultured with specific microRNAs (miRNAs) as indicated for 48 h under hypoxic culture conditions, followed by qRT‒PCR. ( B , C ) Cells from ( A ) were seeded into 96-well plates, followed by MTT ( B ) and EdU ( C ), the statistical analysis were performed. D Cells from ( A ) were subjected to a transwell assay, the statistical analysis were performed.The data are presented as the means ± SDs ( n = 5). The statistical test used was the paired t test. ***, p < 0.001 versus miR-NC.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Cell Culture, Transwell Assay

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 inhibit the expression of TRIM29. ( A ) HPASMCs were transfected with miR-637 or miR-661, followed by RNA-seq analysis. Venn diagram of gene changes after the overexpression of two different microRNAs (FC: fold change). ( B , C ) HPASMCs were overexpressing miR-637 or miR-661, followed by RNA-seq analysis. The transcript volcano plot shows the differentially expressed genes as indicated. ( D ) HPASMC cells were transfected with indicated plasmids for 48 h, followed by luciferase assay. ( E ) HPASMCs were overexpressing miR-637 or miR-661 under hypoxic culture conditions, followed by RT‒PCR and WB. Original blots are presented in Supplementary Fig. 1.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Transfection, RNA Sequencing, Over Expression, Luciferase

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: TRIM29 attenuates the miR-637- and miR-661-induced inhibition of AKT/mTOR signalling. ( A ) Statistical analysis of the results of the GO enrichment assay was performed after the indicated miRNAs were overexpressed. B-C HPASMCs were cultured for different durations under hypoxic conditions, followed by WB ( B ) and qRT‒PCR ( C ). ( D ) qRT‒PCR was used to detect the expression level of TRIM29 in the serum of patients with pulmonary hypertension and healthy volunteers. ( E ) HPASMCs were overexpressing miR-637 or miR-661 under hypoxic culture conditions, followed by RT‒PCR and WB. ( F ) WB assays were performed after the inhibition of miR-637 and miR-661. ( G ) HPASMCs were transfected with Flag-TRIM29 for 48 h under hypoxic culture conditions, and the cells were subjected to WB analysis. ( H ) TRIM29 was knocked out in HAPSMCs under hypoxic culture conditions, and cell lysates were then prepared for WB analysis. ( I ) HPASMCs were transfected with Flag-TRIM29 or overexpressed with microRNA as indicated for 48 h under hypoxic culture conditions, followed by WB analysis. Original blots are presented in Supplementary Figs. 2–7.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Inhibition, Cell Culture, Expressing, Transfection

Journal: Scientific Reports

Article Title: microRNA-637/661 ameliorate hypoxic-induced pulmonary arterial hypertension by targeting TRIM29 signaling pathway

doi: 10.1038/s41598-024-79769-2

Figure Lengend Snippet: miR-637 and miR-661 attenuate TRIM29-induced HPASMC proliferation and migration. ( A ) HPASMCs were transfected with the indicated plasmids or infected with TRIM29 sgRNA for 48 h of hypoxia, followed by the MTT assay. ( B – E ) HPASMCs were transfected with Flag-TRIM29, infected with TRIM29 sgRNA lentivirus or overexpressed with microRNA as indicated for 48 h under hypoxic culture conditions, followed by the EdU assay ( B ), wound healing assay ( D ) and the statistical analysis were performed ( C , E ). The data are presented as the means ± SDs ( n = 5). The statistical test used was the paired t test. ***, p < 0.001 versus miR-NC.

Article Snippet: Human pulmonary artery smooth muscle cells (HPASMCs) were purchased from American Type Culture Collection (Manassas, VA, USA).

Techniques: Migration, Transfection, Infection, MTT Assay, EdU Assay, Wound Healing Assay

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: Cyclin D-CDK4 (cyclin-dependent kinase 4) forms an inducible intermolecular disulfide dimer. A , An Affymetrix GeneChip microarray with gene enrichment pathway analysis of approximately the top 1000 genes of defined biological function with an altered expression between mice exposed to chronic hypoxia (10% oxygen) or normoxia (21% oxygen) for 3 days, n=6 mice per group. The top 20 gene-enriched pathways are shown based on pathway analysis performed in R studio using the gseGO function of the clusterProfiler package. B , CDK4, cyclin D1, and cyclin D3 form oxidant-induced intermolecular disulfide bonds. Monomeric and disulfide dimeric (indicated by black arrows) CDK4, cyclin D1, or cyclin D3 were detected by nonreducing immunoblotting in human pulmonary arterial smooth muscle cells (HPASMCs) after treatment with H 2 O 2 . Vinculin was used as a loading control. C , The percentage of CDK4, cyclin D1, and cyclin D3 observed as a disulfide dimer in response to H 2 O 2 treatment. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons to compare H 2 O 2 -induced disulfide formation with 0 µmol/L control (n=5 independent experiments); the results are shown as means±SEM. D , Cyclin D expression is higher in G1 phase cells than quiescent cells, causing an increase in disulfide cyclin D-CDK4. Monomeric and disulfide dimeric CDK4 were detected by nonreducing immunoblotting of HPASMCs synchronized into the G0/1 phase by serum starvation (0.1% FBS) for 72 hours, followed by stimulation with 10% FBS for 6 or 12 hours. HPASMCs were then treated with H 2 O 2 for 15 minutes. Cyclin D1, cyclin D3, and vinculin (loading control) were detected by immunoblotting under reducing conditions. E , Quantification of the percentage of CDK4 observed as a disulfide dimer. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons to compare H 2 O 2 -induced disulfide formation with 0 µmol/L control within cell cycle phase (n=3 independent experiments); the results are shown as means±SEM. F , Auranofin potentiates accumulation of the oxidant-induced cyclin D1-CDK4 disulfide bond. Monomeric and disulfide dimeric (indicated by black arrows) CDK4 and cyclin D1 were detected by nonreducing immunoblotting in HPASMCs after pretreatment with the thioredoxin reductase inhibitor, auranofin, for 25 minutes followed by the addition of H 2 O 2 for 15 minutes. GAPDH was used as a loading control. G , The proportion of CDK4 and cyclin D1 observed as a disulfide dimer after treatment with auranofin and H 2 O 2 . P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons to compare disulfide formation in response to auranofin treatment within 0, 50, 100 or 200 µM H 2 O 2 -treatment group (n=5 independent experiments); the results are shown as means±SEM.

Article Snippet: Primary human pulmonary arterial smooth muscle cells (HPASMCs) were purchased from ScienCell Research Laboratories (No. 3110).

Techniques: Microarray, Expressing, Western Blot

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: The intermolecular disulfide bond forms between CDK4 (cyclin-dependent kinase 4) C135 and cyclin D1 C7/8. A , The cyclin D1-CDK4 crystal structure (PDB 2W96) showing the locations of CDK4 C135 and cyclin D1 C7/8 in black. The distance (Å) between cysteine residues was measured using PyMOL. The cyclin D1 protein structure and CDK4 protein structure are shown in gray and green, respectively. B , C7/8A cyclin D1 and C135A CDK4 are redox-dead. Immunoblots show disulfide dimeric cyclin D1 and CDK4 (indicated by black arrows) in human pulmonary arterial smooth muscle cells (HPASMCs) overexpressed with wild-type (WT) or C135A CDK4-FLAG and WT or C7/8A cyclin D1-HA and treated with H 2 O 2 . Cyclin D1 (HA-tag) and CDK4 (FLAG-tag) were detected by nonreducing immunoblotting. Red arrows indicate additional artificial bands. C , The percentage of cyclin D1 and CDK4 observed as a disulfide dimer in overexpressed HPASMCs. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using unpaired 2-tailed nonparametric Mann-Whitney U test to compare between vehicle and H 2 O 2 treatment for each mutant; nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons was used to compare disulfide formation in response to 200 µM H 2 O 2 treatment between each mutant and WT (n=5 independent experiments); the results are shown as means±SEM. D , BLAST alignments show CDK4 C135 is not conserved between cell cycle CDK proteins. E , BLAST alignments show cyclin D1 C7/8 is conserved between cyclin D subtypes. All sequences were obtained from the UniProt database with identification codes presented in brackets. F , C5/6A cyclin D3 and C135A CDK4 are redox-dead. Disulfide dimeric cyclin D3 and CDK4 (indicated by black arrows) in HPASMCs overexpressed with WT or C135A CDK4-FLAG and WT or C5/6A cyclin D3-HA. Cells were treated with H 2 O 2 for 15 minutes. Cyclin D3 and CDK4 (FLAG-tag) were detected by nonreducing immunoblotting. Red arrows indicate additional artificial bands. G , The percentage of cyclin D3 and CDK4 observed as a disulfide dimer in overexpressed HPASMCs. P values are calculated using unpaired 2-tailed nonparametric Mann-Whitney U test to compare between H 2 O 2 treatment and vehicle for each mutant; nonparametric Kruskal-Wallis test followed by Dunn multiple comparisons was used to compare disulfide formation in response to 200 µM H 2 O 2 treatment between each mutant and WT (n=5 independent experiments); the results are shown as means±SEM. H and I , Analysis of an extracted ion chromatogram from an LC-MS/MS precursor ion survey scan identified an ion of ( H ) m/z 681.55 4+ and ( I ) m/z 656.82 4+ , which correspond with the peptide 127 GLDFLHANCIVHR 139 in CDK4 bound through a disulfide bond to the peptide 1 MELLCCEGTR 10 in cyclin D3, as shown in the red boxes. The cyclin D3 peptide was identified with dioxidation of the free cysteine residue, and acetylation of the N-terminal, either with the N-terminal methionine ( H ) present, or ( I ) absent.

Article Snippet: Primary human pulmonary arterial smooth muscle cells (HPASMCs) were purchased from ScienCell Research Laboratories (No. 3110).

Techniques: Western Blot, FLAG-tag, MANN-WHITNEY, Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Residue

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: Oxidation decreases cyclin D-CDK4 (cyclin-dependent kinase) substrate phosphorylation and induces G1 phase cell cycle arrest. A , Oxidation of human pulmonary arterial smooth muscle cells (HPASMCs), induced by acute H 2 O 2 treatment, decreased. Rb (retinoblastoma) phosphorylation at S780, S795, or S807-811, and total Rb were detected by immunoblotting under reducing conditions. B , Quantification of Rb phosphorylation, normalized to total Rb. As the data sample size with an n<6 cannot be reliably tested for normality, P values are calculated using a nonparametric Kruskal-Wallis test followed by Dunn post hoc multiple comparisons to compare Rb phosphorylation in response to H 2 O 2 with the 0 µmol/L control (n=5 independent experiments); the results are shown as means±SEM. C , Histograms show cell cycle analysis using flow cytometry of propidium iodide-stained HPASMCs. Continuously cycling cells were maintained in DMEM growth medium (10% FBS; upper left), while G0/1 phase synchronized cells were maintained in starvation media (0.1% FBS) for 40 hours (upper right). Synchronized cells were stimulated to enter the cell cycle with 10% FBS alone for 24 hours (middle left), FBS plus 50 µmol/L H 2 O 2 for 24 hours (middle right), FBS plus DMSO vehicle (bottom left), or FBS plus 10 µmol/L palbociclib for 24 hours (bottom right). The proportion of cells in each phase of the cell cycle at this time point was analyzed by the Watson Pragmatic cell cycle model and an average from 4 to 5 independent experiments is provided. D , H 2 O 2 causes G1 phase cell cycle arrest. Graph shows the proportion of synchronized cells in each cell cycle phase with time after stimulation with 10% FBS alone (solid lines) or 10% FBS plus 50 µmol/L H 2 O 2 (dashed lines). P values are calculated using unpaired 2-tailed nonparametric Mann-Whitney U test to compare between vehicle and H 2 O 2 treatment in cell cycle phases at each time point (0 and 8 hours, n=5 experimental sample per group; 16, 24, or 28 hours, n=4 experimental samples per group); the results are shown as means±SEM. E , Proliferation of HPASMCs treated with 50 µmol/L H 2 O 2 or vehicle. Cells were seeded in xCELLigence real-time cell analysis (RTCA) E-plates at 3×10 4 cells/well in starvation media (0.1% FBS). After 40 hours, cells were stimulated with 10% FBS with or without H 2 O 2 . Proliferation rate of HPASMCs after treatment was quantified using area under the curve. As the data sample size with an n<6 cannot be reliably tested for normality, the P value is calculated using an unpaired 2-tailed nonparametric Mann-Whitney U test to compare between area under the curve for 0 and 50 µmol/L (n=4 independent experiments/ biological replicates per group, including n=2 performed in technical replicates for 0 µmol/L, altogether comprising n=6 replicates for 0 µmol/L and n=4 replicates for 50 µmol/L); the results are shown as means±SEM.

Article Snippet: Primary human pulmonary arterial smooth muscle cells (HPASMCs) were purchased from ScienCell Research Laboratories (No. 3110).

Techniques: Western Blot, Cell Cycle Assay, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Circulation Research

Article Title: Cyclin D-CDK4 Disulfide Bond Attenuates Pulmonary Vascular Cell Proliferation

doi: 10.1161/CIRCRESAHA.122.321836

Figure Lengend Snippet: Idiopathic pulmonary arterial hypertension (PAH; IPAH) pulmonary arteries and human pulmonary arterial smooth muscle cells (HPASMCs) have less CDK4 (cyclin-dependent kinase 4) disulfide than donor controls. A , Formation of the CDK4 disulfide bond is decreased in pulmonary arteries of IPAH patients compared with healthy volunteer controls. Nonreducing immunoblots show monomeric and disulfide dimeric CDK4, while reducing immunoblots show CDK4 expression with vinculin used as a loading control. Quantification is provided for the percentage of CDK4 observed as a disulfide dimer. The data passed the normality test performed by the Shapiro-Wilk test. P values were calculated using the parametric unpaired 2-tailed t test to compare between 2 groups (control, n=8; IPAH, n=9), and the results are shown as means±SEM. B , IPAH HPASMCs have an attenuated response to oxidant treatment compared with donor control cells. HPASMCs were treated with H 2 O 2 for 15 minutes in DMEM growth medium. The formation of disulfide cyclin D-CDK4 was detected by nonreducing immunoblotting probed for CDK4, cyclin D1, and cyclin D3. The graph shows quantification of the percentage of CDK4 that is detected as a disulfide dimer. The data passed the normality test performed by the Shapiro-Wilk test. P values were calculated using the parametric 2-way ANOVA followed by Sidak post hoc multiple comparisons test (n=6 per group); the results are shown as means±SEM. C , IPAH HPASMCs accumulate less disulfide CDK4 than donor control cells in response to auranofin treatment. HPASMCs were treated with auranofin for 1 hour in serum-free DMEM. Monomeric and disulfide dimeric CDK4, cyclin D1, and cyclin D3 were detected by nonreducing immunoblotting. The graph shows quantification of the proportion of CDK4 that was observed to form a disulfide (n=6 per group). The data did not pass the normality test performed by the Shapiro-Wilk test. P values are calculated using an unpaired 2-tailed nonparametric Mann-Whitney U test to compare 0.5 or 1 μM Aur-treatment between Control and IPAH groups (n=6 per group). The results are shown as means±SEM. D , HPASMCs isolated from patients with IPAH have an increased proliferation rate compared with donor control HPASMCs. Proliferation was measured by electrical impedance in real time using xCELLigence (representative of n=6 independent biological replicates per group, in technical duplicate). Proliferation rate was then analyzed using area under the curve (AUC). The data passed the normality test performed by the Shapiro-Wilk test. P value is calculated using the parametric unpaired 2-tailed t test to compare between 2 groups (n=6 independent biological replicates per group, in technical duplicate), and the results are shown as means±SEM.

Article Snippet: Primary human pulmonary arterial smooth muscle cells (HPASMCs) were purchased from ScienCell Research Laboratories (No. 3110).

Techniques: Western Blot, Expressing, MANN-WHITNEY, Isolation