human pulmonary arterial smooth muscle cells pa vsmc  (Lonza)

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

Journal: Cardiovascular Research

Article Title: TSP1–CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction

doi: 10.1093/cvr/cvw218

Figure Lengend Snippet: TSP1, via CD47, suppresses cMyc to increase ET-1/ETA signaling in pulmonary vascular cells. (A) Representative immunofluorescence image of cultured rat-1a Myc/ER fibroblast cells expressing the c-Myc/ER fusion protein display nuclear c-Myc (green), DAPI (blue), phalloidin (red); scale bar 50 μm, original magnification ×63. Rat-1a fibroblasts were treated with 4-hydroxytamoxifen at the designated concentrations for 12 h. mRNA expression of CD47, ET-1, and ETA was determined. Data from n = 4 experiments with each run in triplicate are presented as mean ± SD, ****P < 0.0001. (B) Murine pulmonary endothelial cells were isolated from WT and CD47−/− mice. mRNA expression for CD47, cMyc, and ET-1 was determined. Data from n = 6 independent experiments are presented as mean ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001. (C) Human pulmonary arterial endothelial cells (hPAEC) (passage 3–6) were cultured to 80% confluence then treated with normoxia (Nx), hypoxia (Hx, FiO2 1%), or hypoxia + CD47 blocking antibody (1 μg/ml) for 24 h. mRNA expression of CD47, ET-1, and ETA was determined. ET-1 level in cell culture supernatants was measured by ELISA. Representative data from four experiments are presented as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (D) hPAEC were transfected with control (CTRL) or cMyc siRNA for 72 h and transcript levels of CD47, cMyc, ET-1, and ETA measured. ET-1 level in cell culture supernatants was measured by ELISA. Data are represented as mean ± SD from n = 5 experiments; **P < 0.01, ***P < 0.001, ****P < 0.0001. (E) Human pulmonary vascular smooth muscle cells (hPVSMC) were subjected to Nx or Hx (FiO2 1%, 24 h) and protein expression for TSP1 and cMyc determined. Representative blots are shown and data from n = 4 independent experiments are presented as mean ± SD, *P < 0.05. (F) hPVSMC were subjected to Nx or Hx (FiO2 1%, 24, 48, and 72 h) and mRNA transcript levels determined for TSP1, CD47, and ETA. Data from n = 4–5 experiments are presented as mean ± SD, *P < 0.05 Nx versus Hx at indicated time point. (G) Pulmonary VSMC from WT, TSP1−/−, and CD47−/− mice were isolated and subjected to Nx or Hx (FiO2 1%, 24 h). Cell lysate was prepared and protein and mRNA analysis performed. Representative western immunoblots for the indicated proteins are shown. Densitometry from n = 4 independent experiments for cMyc and n = 4–5 independent experiments for TSP1 is presented as the mean ratio of target protein to tubulin ± SD, *P < 0.05, **P < 0.01, ****P < 0.0001. mRNA data are presented as the mean ratio of target protein to the housekeeping gene (± SD), *P < 0.05 Nx versus Hx. (H) hPVMSC were incubated with ET-1 (1 μM) with or without a CD47 blocking antibody (clone B6H12, 1 μg/ml) for 24 h. Cell size was analyzed by FACS. Data from n = 3–5 independent experiments are presented as mean ± SD, *P < 0.05, **P < 0.01.

Article Snippet: Human pulmonary arterial smooth muscle cells (PA VSMC) and endothelial cells (hPAEC) were purchased from Lonza (Basel, Switzerland) and grown in proprietary medium.

Techniques: Immunofluorescence, Cell Culture, Expressing, Isolation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Transfection, Control, Western Blot, Incubation

Journal: Cardiovascular Research

Article Title: TSP1–CD47 signaling is upregulated in clinical pulmonary hypertension and contributes to pulmonary arterial vasculopathy and dysfunction

doi: 10.1093/cvr/cvw218

Figure Lengend Snippet: TSP1–CD47 signaling limits pulmonary cMyc to upregulate ET-1/ETA and promote PH. Hypoxia-mediated induction of TSP1 in the pulmonary vasculature activates endothelial CD47 to suppress cMyc allowing upregulation of ET-1/ETA. ET-1 then targets the pulmonary arterial vascular smooth muscle cell (VSMC) compartment to promote vascular remodeling and cell hypertrophy, decreased vasodilation and increased vasoconstriction. Conversely, treatment with a CD47 antibody, that blocks TSP1 activation of CD47, limits ET-1-mediated VSMC hypertrophy and improves PA sensitivity to NO pathway activators.

Article Snippet: Human pulmonary arterial smooth muscle cells (PA VSMC) and endothelial cells (hPAEC) were purchased from Lonza (Basel, Switzerland) and grown in proprietary medium.

Techniques: Activation Assay