Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Constitutively active form of natriuretic peptide receptor 2 ameliorates experimental pulmonary arterial hypertension

doi: 10.1038/mtm.2016.44

Figure Lengend Snippet: Transduction of the constitutively active mutant of natriuretic peptide receptor 2 (caNPR2) by Sendai virus (SeV) vectors in human pulmonary arterial smooth muscle cells (PASMCs). (a) Immunocytochemical analyses show that SeV vectors carrying Azami-Green control, wild-type NPR2 (WT- NPR2 ), and ca NPR2 efficiently infect PASMCs. The infectious ability of the Azami-Green-expressing control SeV vector (purchased as ready to use) is higher than that of the WT- NPR2 - or ca NPR2 -expressing vectors, with no significant difference between WT- NPR2 - and ca NPR2 -expressing SeV vectors ( n = 3). Bar = 100 µm. * P < 0.05. N.S., not significant. (b) A quantitative PCR analysis shows no significant difference in NPR2 expression levels between WT- NPR2 - and ca NPR2 -transduced PASMCs ( n = 3). N.S., not significant. (c) Western blotting demonstrates that NPR2 expression levels were not different between WT- NPR2 - and ca NPR2 -transduced PASMCs, as detected by anti-HA tag antibody ( n = 3). N.S., not significant. (d) The overexpression of ca NPR2 induces the synthesis of larger amounts of cyclic guanosine monophosphate (cGMP) in PASMCs with and without the C-type natriuretic peptide (CNP) stimulation ( n = 3). Note that ca NPR2 has the ability to produce large amounts of cGMP, regardless of the CNP stimulation. Even under the unphysiologically high concentration of CNP (10 −6 mol/l), cGMP levels are higher in ca NPR2 -expressing cells than in WT- NPR2 -expressing cells. * P < 0.05. (e) The effects of riociguat (100 µmol/l) or sildenafil (5 µmol/l) on cGMP concentrations in PASMCs ( n = 3 for riociguat; n = 8 for sildenafil). * P < 0.05. (f) The EdU incorporation assay reveals that ca NPR2 has the ability to suppress the proliferation of PASMCs ( n = 6). * P < 0.05. (g) The EdU incorporation assay reveals that riociguat (100 µmol/l) and sildenafil (5 µmol/l) did not have significant effects, although we found a small tendency toward attenuation of cell proliferation of PASMCs ( n = 4). N.S., not significant. (h) The TUNEL assay demonstrates that ca NPR2 does not induce apoptosis in PASMCs ( n = 3). The left panels show representative phase contrast images. Bar = 500 µm. The right panel shows corresponding quantifications. N.S., not significant.

Article Snippet: A healthy human pulmonary arterial smooth muscle cell line (PASMCs) was purchased from Lonza (Walkersville, MD).

Techniques: Transduction, Mutagenesis, Virus, Control, Expressing, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Over Expression, Concentration Assay, TUNEL Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Constitutively active form of natriuretic peptide receptor 2 ameliorates experimental pulmonary arterial hypertension

doi: 10.1038/mtm.2016.44

Figure Lengend Snippet: Sendai virus (SeV) vector–mediated transduction of constitutively active mutant of natriuretic peptide receptor 2 (caNPR2) suppresses the proliferation of pulmonary arterial hypertension (PAH) patient-derived pulmonary artery smooth muscle cells (PHSMCs). (a) The infection of SeV vectors carrying ca NPR2 strongly induces the production of cyclic guanosine monophosphate (cGMP) in PHSMCs without the C-type natriuretic peptide (CNP) stimulation ( n = 3). * P < 0.05. (b) The 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay reveals that ca NPR2 suppresses the proliferation of PHSMCs, consistent with healthy PASMCs ( n = 6). * P < 0.05. (c) ca NPR2 does not induce apoptosis in PHSMCs. The left panels show representative images of the TUNEL assay ( n = 3). Bar = 500 µm. The right panel shows corresponding quantifications. N.S., not significant.

Article Snippet: A healthy human pulmonary arterial smooth muscle cell line (PASMCs) was purchased from Lonza (Walkersville, MD).

Techniques: Virus, Plasmid Preparation, Transduction, Mutagenesis, Derivative Assay, Infection, TUNEL Assay

Journal: BMC Pulmonary Medicine

Article Title: Bmi-1 alleviates adventitial fibroblast senescence by eliminating ROS in pulmonary hypertension

doi: 10.1186/s12890-021-01439-0

Figure Lengend Snippet: Fibroblasts Bmi-1 alters PASMC proliferation by paracrine mode of action. a – b . EDU staining for PASMCs treated with the CM of HLFs. HLFs were infected with adv-Bmi-1 or transfected with si-Bmi-1 following by hypoxia exposure for 96 h, and then the supernatant was collected and added the equal volume of serum-free culture medium to culture PASMCs for 48 h. All data are shown as the mean ± SEM of at least three independent experiments. Statistical significance was assessed using the unpaired two-tailed Student’s t test: * P < 0.05

Article Snippet: The human pulmonary artery smooth muscle cell line (PASMC) and human lung fibroblast cell line (HLF) were purchased from Lonza (Swiss).

Techniques: Staining, Infection, Transfection, Two Tailed Test