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

Journal: International Journal of Molecular Medicine

Article Title: Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3β signalling

doi: 10.3892/ijmm.2018.3745

Figure Lengend Snippet: Effect of DCA, LY294002 or combination of DCA and LY294002 on the growth of human PASMCs. The human PASMCs were seeded in 96-well plates in RPMI-1640 medium supplemented with 10% FBS followed by 48 h serum starving prior to exposure to 20 ng/ml PDGF or with increased concentrations of (A) DCA or (B) LY294002 in fresh culture medium with 0.5% foetal bovine serum for 72 h. * P<0.05, ** P<0.01 and *** P<0.001 vs. PDGF-treated cells; #P<0.05 and ## P<0.01 vs. cells treated with 5 mM DCA or 5 µ M LY294002. (C) Cells were exposed to PDGF or 5 µ M LY294002, 10 or 20 mM DCA and combination of 5 µ M LY294002 and DCA (10 mM) for 24 h. * P<0.05, ** P<0.01 as compared with PDGF cells. The data are presented as the mean ± standard deviation of 6 duplicated wells in three separate experiments. PASMCs, pulmonary arterial smooth muscle cells; DCA, dichloroacetate; PDGF, platelet-derived growth factor; D5, D10, D20 and D50, cells treated with dichloroacetate at 5, 10, 20, 50 mM, respectively, following PDGF exposure. L5, 10 and 20, cells treated with LY294002 at 5, 10, 20 µ M, respectively, following PDGF exposure. L5D10, cells treated in a combination of 5 µ M LY294002 and 10 mM dichloroacetate following PDGF exposure.

Article Snippet: Human PASMCs were purchased from Wuxi BioHermes Biomedical Technology Co., Ltd (Wuxi, China).

Techniques: Standard Deviation, Derivative Assay

Journal: International Journal of Molecular Medicine

Article Title: Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3β signalling

doi: 10.3892/ijmm.2018.3745

Figure Lengend Snippet: Effect of DCA, LY294002 or combination of DCA and LY294002 on the apoptosis and mitochondria membrane potential of human PASMCs. The PASMCs were seeded into 25 cm 2 tissue culture flask at a density of 5×10 5 cell/flask and cultured in RPMI-1640 complete culture medium for 16 h followed by serum starvation for 24 h. The cells were then exposed to PDGF alone or 5 µ M LY294002, DCA at 10 mM or a combination of 5 µ M LY294002 and 10 mM DCA for 48 h prior to (A) apoptosis or (B) JC-1 assay. (C) The expression levels of caspase-3 and cleaved caspase-3 were analyzed with western blot analysis. The representative change of one of the three experiments is presented, as all assays exhibited identical results. (D) Results were pooled from three separate experiments and are presented as mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 vs. control cells. ## P<0.01 and ### P<0.001 vs. cells treated with PDGF. C, control; PASMCs, pulmonary arterial smooth muscle cells; DCA, dichloroacetate; PDGF, platelet-derived growth factor; D5 and D10, cells treated with DCA at 5 and 10 mM, respectively, following PDGF exposure; L5, cells treated with LY294002 at 5 µ M following PDGF exposure; L5D10, cells treated with a combination of 5 µ M LY294002 and 10 mM DCA following PDGF exposure.

Article Snippet: Human PASMCs were purchased from Wuxi BioHermes Biomedical Technology Co., Ltd (Wuxi, China).

Techniques: Membrane, Cell Culture, Expressing, Western Blot, Standard Deviation, Control, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin inhibits mTORC1 and mTORC2. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-p70S6k, p70S6k, p-AKT (S473), p-AKT (T308), AKT. (B) immunoblotting analyses of p-p70S6k, p70S6k, p-AKT (S473), and AKT in hPASMCs, which were stimulated with 5 μg/ml insulin for 24h before treatment with 100 nM rapamycin. (C) hPASMCs were treated with 100 nM rapamycin for the indicated times, and then cell lysates were prepared for and immunoprecipitation (IP) with mTOR antibody. The elution from IP was analyzed by immunoblotting for the levels of mTOR and Rictor. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Western Blot, Immunoprecipitation, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin in hPASMCs. (A) hPASMCs were treated with 100 nM rapamycin for the indicated times and analyzed by immunoblotting for the proteins level of p-PDGFRα/β, PDGFRα, PDGFRβ. (B) Immunoblotting analyses of p-PDGFRα/β, PDGFRα, PDGFRβ, p-AKT (S473), p-AKT (T308), p-S6 and S6 in hPASMCs treated with vehicle (Control), 100 nM rapamycin (Rap), 5 uM imatinib (Ima) and 100 nM rapamycin + 5 uM imatinib (Rap + Ima) for 48 h. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means not significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on the viability, proliferation and migration of hPASMCs. (A) Cell viability was determined by measuring the absorbance at 0, 24, 48 and 72 h after different drug treatments. (B) A scratch was applied to cell monolayers, and migration of the cells towards the wound was recorded by photomicrographs at 0, 4, and 8h ( n = 3); summarized data showing percent wound closure [(0h wound area–4h or 8h wound area)/0h wound area] * 100%. (C) BrdU assay was performed to determine hPASMCs proliferation under normoxia and hypoxia (3% 0 2 ) for 24 and 48 h. (D) BrdU assay was performed to determine hPASMCs proliferation at 24 and 48 h after different drug treatments. Data are presented as the mean ± SE. Two-way ANOVA was used for statistical analysis. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Rap; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Ima.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Migration, BrdU Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by MCT. (A) H&E staining in lung tissue sections. Summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus MCT with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus MCT with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Rapamycin combined with imatinib attenuates PASMC proliferation and remodeling induced by Hypoxia/Sugen. (A) H&E staining of lung tissue sections and summarized data showing pulmonary artery media wall thickness. (B) Lung sections were stained with α-SMA (red) and PCNA (green). Yellow arrowheads point at PCNA positive PASMCs and white arrowheads show the vessels. For each of the 5 groups, approximately 600 PASMC nuclei and 100 fields were analyzed. Summarized data showing PCNA positive cells and muscularization. Data are presented as the mean ± SE. One-way ANOVA was used for statistical analysis. NS means no significant. *** p < 0.001, ** p < 0.01, * p < 0.05 versus control; ### p < 0.001, ## p < 0.01, # p < 0.05 versus Hypoxia/Sugen with vehicle; $$$ p < 0.001, $$ p < 0.01, $ p < 0.05 versus Hypoxia/Sugen with rapamycin.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Combination Therapy With Rapamycin and Low Dose Imatinib in Pulmonary Hypertension

doi: 10.3389/fphar.2021.758763

Figure Lengend Snippet: Effects of rapamycin combined with imatinib on mTOR and PDGFR signaling pathways in pulmonary artery. (A) Pulmonary artery vessels of were isolated for protein extraction, and the expression of mTORC 1, mTORC 2 and PDGFR signaling pathway related proteins were detected by immunoblotting. Data are presented as the mean ± SE. One-way ANOVA followed by Graphpad prism was used for statistical analysis. NS means no significant. *** p < 0.001; ** p < 0.01; * p < 0.05 versus control. ### p < 0.001; ## p < 0.01; # p < 0.05 versus MCT with vehicle. (B) The schematic representation of the findings of this study: rapamycin chronic treatment in hPASMCs induced the highly expression of phosphorylation of PDGFRs. Imatinib inhibits phosphorylation of PDGFRα/β induced by rapamycin. Abbreviations: GF, growth factors; RTK, receptor tyrosine kinase; PDGF, platelet derived growth factor; PDGFR, platelet derived growth factor receptor; PI3K, phosphoatidylinositol 3-kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-bisphosphate; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2.

Article Snippet: Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1).

Techniques: Protein-Protein interactions, Isolation, Protein Extraction, Expressing, Western Blot, Control, Phospho-proteomics, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Prostanoid EP 2 Receptors Are Up-Regulated in Human Pulmonary Arterial Hypertension: A Key Anti-Proliferative Target for Treprostinil in Smooth Muscle Cells

doi: 10.3390/ijms19082372

Figure Lengend Snippet: Differential role of prostanoid IP and EP 2 receptors in mediating the anti-proliferative effects of treprostinil and MRE-269 in PAH cells. The anti-proliferative effects of MRE-269, treprostinil and butaprost in the absence and presence of the IP receptor antagonist, RO1138452 ( A,B ), the EP 2 receptor antagonist, PF-04418948 ( C , D ) or in combination ( D ). Human PASMCs were left untreated (Control) or treated with 10 nM (log −8) and 1000 nM (log −6) of either treprostinil ( E ) or MRE-269 ( F ) in the absence or presence of the IP receptor antagonist, RO1138452 (RO11; 1 µM), the EP 2 receptor antagonist, PF-04418948 (PF-04; 1 µM) or a combination (BOTH). Antagonists (1 µM) were added 30 min prior to the receptor agonists. Cell proliferation was assessed in HPASMCs from PAH patients after 4 days of drug treatment using an MTS assay kit ( A – D ) or by cell counting ( E , F ). Data expressed as % cell proliferation relative to the growth response. Significance was tested using one or two-way ANOVA with Bonferroni post-hoc correction ( A – D ) or Newman–Keuls multiple comparison test ( E , F ). * p < 0.05 when compared to receptor agonist alone ( A – D ), control ( E , F ) or as indicated. Each comparative data-set was acquired using cells from the same patients (4–6 isolates, passage 3–9).

Article Snippet: Human PASMCs or human umbilical vein endothelial cells (HUVECs; Cellworks, Buckingham, UK) were seeded into 8-chambered slides (BD Falcon, Oxford, UK) and grown in DMEM/F-12 or RPMI 1640 (Life Technologies, UK) containing serum.

Techniques: Control, MTS Assay, Cell Counting, Comparison

Journal: Biomedicines

Article Title: H 2 S Donor Therapy Reverses Established Pulmonary Arterial Hypertension and Pulmonary Vascular Structural Remodeling in Rats

doi: 10.3390/biomedicines14040760

Figure Lengend Snippet: H 2 S donors restored the expression of key H 2 S-producing enzymes and antagonized hypoxia-induced proliferation of hPASMCs. ( A ). Schematic diagram of cell grouping and treatment regimen. ( B ). Immunoblotting assays and quantitative analysis showing the effects of hypoxia exposure and NaHS treatment on the protein expression levels of CSE, CBS, and MPST in hPASMCs. ( C ). Immunoblotting assays and quantitative analysis showing the effects of hypoxia exposure and GYY4137 treatment on the protein expression levels of CSE, CBS, MPST, PCNA, and phospho-ERK1/2 in hPASMCs. All data are presented as mean ± SEM, n = 6. * p < 0.05, ** p < 0.01.

Article Snippet: The hPASMCs (Procell, Wuhan, China) were cultured in specialized medium at 37 °C and 5% CO 2 .

Techniques: Expressing, Western Blot

Journal: Biomedicines

Article Title: H 2 S Donor Therapy Reverses Established Pulmonary Arterial Hypertension and Pulmonary Vascular Structural Remodeling in Rats

doi: 10.3390/biomedicines14040760

Figure Lengend Snippet: H 2 S inhibited hPASMC proliferation via upregulation of ETAR persulfidation. ( A ). Biotin-switch assay (BSA) quantitation of ETAR persulfidation and total ETAR in rat lung tissues. ( B ). Schematic diagram of cell grouping and treatment regimen. ( C ). BSA quantification of ETAR persulfidation and total ETAR in cultured hPASMCs. ( D ). Western blot detection of ERK1/2 phosphorylation and PCNA in hPASMCs. All data are expressed as mean ± SEM, n = 6. * p < 0.05, ** p < 0.01, ns: not statistically significant.

Article Snippet: The hPASMCs (Procell, Wuhan, China) were cultured in specialized medium at 37 °C and 5% CO 2 .

Techniques: Biotin Switch Assay, Quantitation Assay, Cell Culture, Western Blot, Phospho-proteomics