Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMP9 and BMP10 selectively activate SMAD1/5/8 signaling and induce proliferation in pulmonary artery endothelial cells but not pulmonary artery smooth muscle cells. ( A ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PAECs treated with the indicated TGF-β superfamily ligands (0.8 nM) or untreated control (UT); β-actin serves as a loading control. ( B ) PAEC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. ( C ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PASMCs treated with the indicated ligands (0.8 nM); β-actin serves as a loading control. ( D ) PASMC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. Data are shown as mean ± SD ( n = 3 replicate wells). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, BrdU Incorporation Assay

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMPR2 dosage-dependent model for BMP9/10 signaling output in pulmonary artery endothelial cells. Schematic illustrating how BMPR2 abundance constrains BMP9/10 (ALK1-dependent) canonical signaling output and downstream cellular programs in PAECs. ( A ) BMPR2-sufficient (~100%) state: BMP9/10 predominantly signal through ALK1–BMPR2 complexes, generating pSMAD1/5/8 output consistent with a threshold-like requirement for proliferation; bimagrumab (BiMab) produces no effect detected under BMPR2-replete conditions. ( B ) BMPR2-limiting (~50%) state: Reduced BMPR2 attenuates BMP9/10-induced canonical output and is associated with reduced proliferation and increased caspase-3/7 activity consistent with stress/injury. Under BMPR2-limiting conditions, residual canonical output becomes bimagrumab-sensitive, consistent with context-dependent contribution of Activin type II receptors (predominantly ACVR2A in PAECs; see for BMP10 affinity comparisons) to the remaining pSMAD1/5/8 signal. A putative non-canonical stress-signaling arm is shown as a proposed intermediate. Solid arrows denote observed relationships; dashed arrows and dashed-outline boxes denote proposed steps. Node shading and output gauges depict relative canonical signaling output.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay

Journal: Cardiovascular Research

Article Title: Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

doi: 10.1093/cvr/cvr356

Figure Lengend Snippet: Pulmonary TSP1 and CD47 are upregulated in human subjects with PAH and in experimental PAH. (A) Lung tissue from a historical cohort of non-PAH, IPAH or SCD was probed by western blot for TSP1 and β-actin. (western blot is the only representative). Densitometry is the result of 10 non-PAH patients, five SCD, 5 IPAH patients and is presented as mean ratio of TSP1 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with non-PAH patients. (B) Western blot analysis of lung samples from a prospective cohort of non-PAH and PAH patients probed for CD47 and β-actin. Densitometry is the result of three non-PAH and five PAH patients. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with non-PAH patients. (C) TSP1 and CD47 mRNA expression levels in lung samples from the prospective cohort. Representative data from three independent experiments are presented. mRNA levels are expressed as fold change over non-PAH and normalized with 18S ribosomal subunit levels as endogenous non-PAH. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with control. Hash (#) indicates statistically significant difference (P < 0.05) compared with non-PAH. (D) Representative western blot of lung tissue from normoxic and chronically hypoxic (21 days normobaric 10% O2) C57BL/6 wild-type and TSP1 null mice probed against TSP1 and β-actin. Densitometry is presented as mean ratio of TSP1 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxic mice and is based on an analysis of eight mice per group. (E) Representative western blot probed against TSP1 and α-tubulin from lung samples from normoxic and acute hypoxia (normobaric 7.5% O2) challenged wild-type mice. Representative data from three independent experiments are presented. Densitometry is presented as mean ratio of TSP1 to α-tubulin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared to normoxia and is based on four mice at each time point. (F) TSP1 mRNA expression levels in lung samples from wild-type and TSP1 null mice exposed to normoxia or hypoxia (normobaric 7.5% O2) for the indicated time points. Representative data from three independent experiments are presented. mRNA levels are expressed as fold change over normoxic conditions and normalized with HPRT levels as endogenous control. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (G) Representative western blot of wild-type and TSP1 null pulmonary microvascular endothelial cells exposed to normoxia or hypoxia (normobaric 1% O2) for 12 h probed against CD47 and α-tubulin. Representative data from three independent experiments are presented. Densitometry is presented as mean ratio of CD47 to α-tubulin (± SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (H) Representative western blot from wild-type mice exposed to acute hypoxia for the indicated time points probed against CD47 and β-actin. Densitometry from analysis of western results from four mice in each treatment group is presented as mean ratio of CD47 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia.

Article Snippet: 26 Human pulmonary arterial and microvascular endothelial cells were purchased from Lonza (Switzerland).

Techniques: Western Blot, Expressing, Control

Journal: Cardiovascular Research

Article Title: Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

doi: 10.1093/cvr/cvr356

Figure Lengend Snippet: Activated CD47 regulates hypoxic eNOS by altering Cav-1CD47 co-association. (A) Western blot of lung tissue from wild-type and TSP1 null mice exposed to chronic hypoxia or room air probed against total eNOS protein, eNOS phosphorylation at serine-1176 (murine residue), Cav-1, and β-actin. Quantification of densitometric analysis of the ratio of (B) total eNOS to β-actin, (C) p-eNOS to total eNOS, (D) Cav-1 to β-actin. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with wild-type normoxic. Hash (#) indicates statistically significant difference (P < 0.05) compared with wild-type normoxic. All ratios represent mean ± SD and are the result of the total cohort (n = 8 animals per group). (E) Co-immunoprecipitation in human pulmonary microvascular endothelial cells of Cav-1 and CD47. Immunoprecipitation was with monoclonal antibodies to Cav-1, CD47, or an isotype-matched control IgG antibody. Results presented are representative of four separate experiments. (F) Human pulmonary microvascular endothelial cells were serum starved and then treated with TSP1 (2.2 nmol/L) ± hypoxia (1% O2 for 4 h), lysate prepared and immunoprecipitation for CD47 performed with protein blotted for Cav-1. Results are presented as the quantification of densitometric analysis from three separate experiments of the ratio of Cav-1 to CD47 following immunoprecipitation. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (G) Western blot of pulmonary Cav-1 from lung lysate from chronically hypoxic (21 days normobaric 10% O2) wild-type (n = 3) and CD47 null (n = 4) mice. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with wild-type.

Article Snippet: 26 Human pulmonary arterial and microvascular endothelial cells were purchased from Lonza (Switzerland).

Techniques: Western Blot, Phospho-proteomics, Residue, Immunoprecipitation, Bioprocessing, Control

Journal: Cardiovascular Research

Article Title: Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

doi: 10.1093/cvr/cvr356

Figure Lengend Snippet: Blocking CD47 activation upregulates Cav-1 to inhibit eNOS-derived superoxide production in hypoxic pulmonary arterial endothelial cells. (A) Representative western blot of hPAEC exposed to normoxia, hypoxia (1% O2 for 12 h), or hypoxia plus a CD47-blocking antibody (αCD47, clone B6H12, 1 µg/mL). Quantification of densitometric analysis of the ratio of (B) TSP1 to β-actin and (C) Cav-1 to β-actin. Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic control. (D) Superoxide production in hPAEC exposed to normoxia, hypoxia (1% O2 for 12 h), hypoxia plus L-NAME (100 mmol/L), or hypoxia plus a CD47-blocking antibody (clone B6H12, 1 µg/mL). Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic control. (E) Western blot analysis for Cav-1 expression in hPAEC transfected with control or Cav-1 siRNA. (F) Superoxide production in hPAEC transfected with control or Cav-1 siRNA then exposed to 24 h hypoxia (1% O2) with or without a CD47-blocking antibody. Data represent three independent experiments. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with hypoxia control alone. Hash (#) indicates statistically significant difference (P < 0.05) compared with hypoxia alone. (G) Wild-type and TSP1 null pulmonary microvascular endothelial cells harvested from 10-week-old male animals were grown to 80% confluence, treated with normoxia or hypoxia (1% O2) ± TSP1 (2.2 or 22 nmol/L) as indicated for 24 h, cell lysate prepared and western blot analysis for Cav-1 expression performed. A representative blot and accompanying densitometry from three separate experiments are presented. Quantification of densitometric analysis of the mean ratio of Cav-1 to α-tubulin. Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic untreated. Hash (#) indicates statistically significant difference (P < 0.05) from normoxic wild-type + 22 nmol/L TSP1 and hypoxic wild-type. (H) Western blot analysis for phospho-Cav-1Tyr14 expression in normoxic hPAEC treated with the indicated amounts of TSP1 (2.2 nmol/L) ± a CD47 antibody (B6H12, 1 µg/mL) for 30 m. Quantification of densitometric analysis of mean ratio p-Cav-1 to total Cav-1 of three separate experiments. Asterisk (*) indicates statistically significant difference (P < 0.05) from untreated and antibody alone. Hash (#) indicates statistically significant difference (P < 0.05) from TSP1 treated.

Article Snippet: 26 Human pulmonary arterial and microvascular endothelial cells were purchased from Lonza (Switzerland).

Techniques: Blocking Assay, Activation Assay, Derivative Assay, Western Blot, Control, Expressing, Transfection