Table 1.
Antibodies and probes used for flow cytometric assay.
Table 2.
Study population characteristics.
Fig 1.
Alprenolol-biotin binding of human PBCs specific for the βAR.
A) Titration of the alprenolol-biotin probe demonstrated an optimal concentration of 25 μg/mL. Values represent MFI for WBC population. B) The titration curve shows that binding is a saturable and specific process. C) Decay curve for alprenolol binding. Values represent mean ± SE of 2 experiments each with N = 3. D) Isoproterenol, another ligand for βAR, displaced alprenolol from the receptor in a dose-dependent fashion. E) Losartan, an angiotensin II receptor antagonist (scramble competitor) did not affect binding of the alprenolol probe. Mean ± SE of 2 experiments are shown.
Fig 2.
Gating strategy for flow cytometry assay.
A) Gating strategy for hematopoietic peripheral blood cell subsets. Artifact exclusion included time gating (1), aggregate exclusion (2), and DAPI gating for cells in G0/G1 (3). FSC vs SSC gating was used to discriminate polymorphonuclear cells (PMN) and mononuclear cells (MNC) (4). CD3 was used as a T-cell maker (5) and CD19 as a B-cell marker (6). CD45 gating of the CD3-CD19- MNC (7) based on fluorescence-minus one (FMO) control (8), followed by CD34 and CD133 gating (9) based on FMO control (10) were used to select hematopoietic progenitor cells (HPC). Alprenolol binding was then assessed based on FMO control (11). B) Gating strategy for circulating endothelial cells (CEC). Artifact exclusion included time gating (1), aggregate exclusion (2), and DAPI gating for cells in G0 and G1 (3). CD45- gating of the CD3-CD19- population was based on FMO control (4). CD34+ gating was used to define CEC (5).
Table 3.
Percentage of PBC subsets in healthy controls (CTRL) and PAH patients.
Fig 3.
Alprenolol-biotin binding in subsets of peripheral blood cells is decreased in PAH as compared to controls.
p-value represents Student’s t test comparison of study groups.
Fig 4.
Alprenolol-biotin binding in circulating endothelial cells (CEC) correlates with TAPSE.
ρ represents Spearman's rank correlation coefficient with corresponding p value.
Fig 5.
Gating strategy for urinary endothelial cell microparticles.
Sequential gating was used to quantify the percentage of endothelial-derived microvesicles among annexin-V positive microparticles in the urine. Small Angle Light Scatter (SALS) and Large Angle Lights Scatter (LALS) were used to gate the microvesicle population (A). Annexin-V+ events were selected on a LALS/Green fluorescence channel plot (B). Annexin-V+ region was defined based on unstained sample (D). CD144 (VE-cadherin)+ events in the annexin-V+ gate were defined on an LALS/orange fluorescence channel (C) based on an FMO control missing CD144 (E) and isotype matched IgG staining (F). Samples were acquired at a flow rate of 6.01 μL/min for 2 minutes. At least 5000 annexin-V+ CD144-PE+ microparticles were acquired.
Fig 6.
Increased endothelial microparticles in pulmonary arterial hypertension (A) and correlation with TAPSE (B).
A) Percentage of endothelial microparticles (Ec-MPs) as defined by CD144+ microparticles (MPs) among annexin-V+ MPs was increased in PAH. p-value represents Student’s t test comparison of study groups. B) Percentage of Ec-MPs correlates with TAPSE. ρ represents Spearman's rank correlation coefficient with corresponding p value.