murine tsp1 elisa kit  (Cusabio)

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Expression of thrombospondin 1 (TSP1) and CD47 in lungs of 4- and 9- to 11-mo-old Berkeley (BERK) sickling (Sickle) mice. A and B: Western blotting of lung lysates from 4-mo-old female C57BL/6J control (C57BL) and female Sickle mice and 9- to 11-mo-old female Sickle, BERK nonsickling hemizygous control (Hemi), C57BL, CD47 knockout (CD47KO), and TSP1 knockout (TSP1KO) mice (n = 20) for TSP1 and CD47. CD47KO and TSP1KO mice were used as positive and negative controls. Each lane represents an individual animal, and all data are shown. Densitometry results were normalized to β-actin. Values are means ± SE. Unpaired t-test was applied for comparison between each group.

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: Expressing, Western Blot, Control, Knock-Out, Comparison

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Expression of thrombospondin 1 (TSP1) and its receptor CD47 in lungs of 13- to 14-mo-old Berkeley (BERK) sickling (Sickle) mice. Left: Western blots of lung lysates from 13- to 14-mo-old female Sickle, BERK nonsickling hemizygous control (Hemi), C57BL/6J control (C57BL), CD47 knockout (CD47KO), and TSP1 knockout (TSP1KO) mice (n = 26) for TSP1 and CD47. CD47KO and TSP1KO mice were used as positive and negative controls. Each lane represents an individual animal, and all data are shown. Pulmonary TSP1 and CD47 were significantly increased in Sickle compared with Hemi and C57BL mice. Right: densitometry results normalized to β-actin. Values are means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (by one-way ANOVA with multiple comparisons).

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: Expressing, Western Blot, Control, Knock-Out

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Thrombospondin 1 (TSP1) and CD47 are upregulated in lungs of patients with sickle cell disease (SCD)-associated pulmonary hypertension (PH). A and B: TSP1 and CD47 expression was measured by immunofluorescence in lung sections from 6 patients with SCD-associated PH (SCD-PH) and 6 control patients without PH or overt lung disease. A: expression of TSP1 (red; von Willebrand factor stained green, and DAPI stained blue) was increased in patients with SCD-PH, although the signal appeared to originate from intraluminal red blood cells. B: increased levels of CD47 [red; platelet endothelial cell adhesion molecule stained green, and DAPI stained blue) were consistently observed in lung sections from all SCD-PH compared with control patients. ROI, region of interest. Scale bars = 50 µm. C: CD47 or TSP1 SCD-PH expression reported as percentage of control. Values are means ± SE. **P < 0.01 (by unpaired t-test).

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: Expressing, Immunofluorescence, Control, Staining

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Berkeley (BERK) sickling (Sickle) mice develop pulmonary hypertension (PH) and vascular dysfunction. Hemodynamic assessment of male 2- to 8-mo-old Sickle mice (n = 9) and age-matched C57BL/6J control (C57BL) mice (n = 6) was performed by open-chest right heart microcatheterization. A: right ventricular (RV) pressure (RVP) was measured by maximum RVP (RVPmax) and mean pulmonary artery pressure (mPAP). B: afterload measured by pulmonary vascular resistance (PVR) and RV effective arterial elastance (Ea). C: RV systolic function measured by the contractility index [maximum change in pressure over time (dP/dtmax)/RVPmax]. D: RV diastolic function measured by minimum change in pressure over time (RV dP/dtmin), a measure of RV stiffness. E: pressure-volume relations of RV of 2 representative Sickle and C57BL mice. ESP, end-systolic pressure; ESV, end-systolic volume; EDV, end-diastolic volume. F: plasma thrombospondin 1 (TSP1) measured by ELISA at the time of euthanasia. G and H: vascular reactivity of isolated aortic segments of male age-matched Sickle (n = 6) and C57BL (n = 5) mice assessed on the myograph system in response to acetylcholine (ACh) and phenylephrine (PE). Values are means ± SE. Unpaired t-test was applied for comparison between the 2 groups of mice; 2-way ordinary ANOVA with Sidak’s multiple comparisons test was used for myograph data: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Isolation, Comparison

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Generation of chimeras and measurement of engraftment. The thrombospondin 1 (TSP1)-CD47 axis was interrogated in vivo by generation of chimeric animals with a sickle erythropoiesis on a CD47 knockout (CD47KO) background. Bone marrow was harvested from flushed femurs and tibias of adult Berkeley sickling (Sickle) mice, and whole bone marrow (5 × 106 cells) was transplanted into age-matched, lethally myeloablated (10 Gy) 2-mo-old CD47KO (n = 27) and C57BL/6J control (C57BL, n = 24) mice, the background strain of CD47KO mice, by retroorbital sinus injection in 7 separate experiments. A: schematic representation of transplantation protocol. B: Kaplan-Meyer survival curve of transplanted chimeras. C: engraftment assessed by measurement of HbS percentage by HPLC followed by confirmatory capillary zone electrophoresis (CZE) in blood samples obtained at the time of euthanasia (4–6 mo after transplantation). Top left: CZE gel. Top right: human HbA and HbS in a C57BL recipient transplanted with Sickle bone marrow with mixed chimerism. Bottom: CZE of a C57BL recipient (left) and a Sickle mouse donor (right). CSFA, cerebrospinal fluid analysis. D: linear regression of engraftment and spleen size.

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: In Vivo, Knock-Out, Control, Injection, Transplantation Assay, Electrophoresis

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Absence of tissue-resident thrombospondin 1 (TSP1)-CD47 signaling improves pulmonary hemodynamics and arterial vasodilator responsiveness in chimeric mice with a sickle erythropoiesis. Full hemodynamic assessment of Sickle-to-CD47 knockout (CD47KO) and Sickle-to-C57BL chimeras was performed by open-chest right heart microcatheterization. A: right ventricular (RV) pressure (RVP), including maximum RVP (RVPmax) and mean pulmonary artery pressure (mPAP). B: afterload measured as pulmonary vascular resistance (PVR) and RV effective arterial elastance (Ea). C: RV systolic function measured by the contractility index [maximum change in pressure over time (dP/dtmax)/RVPmax]. D: RV diastolic function measured by minimum change in pressure over time (dP/dtmin). E: pressure-volume relations of the RV of 2 representative Sickle-to-C57BL and Sickle-to-CD47KO chimeras. F: plasma TSP1 measured by ELISA at the time of euthanasia. G and H: vascular reactivity of isolated aortic segments of Sickle-to-C57BL (n = 5) and Sickle-to-CD47KO (n = 13) chimeras assessed on the myograph system in response to acetylcholine (ACh) and phenylephrine (PE). Values are means ± SE. Unpaired t-test was applied for comparison between the 2 groups of mice; 2-way ordinary ANOVA with Sidak’s multiple-comparisons test was used for myograph data: *P < 0.05.

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: Knock-Out, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Isolation, Comparison

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Thrombospondin 1 (TSP1) augments reactive oxygen species (ROS) in human pulmonary endothelial cells via CD47. A and B: commercially available human pulmonary artery endothelial cells (n = 1 donor) were treated with vehicle or TSP1 (0–10 nmol/l) for 60 min, and ROS production was measured using 2 independent complementary assays: O2•− generation by total cellular homogenates measured by cytochrome c reduction (A) and endothelial cell homogenate H2O2 production measured using the Amplex red assay (B). Values are means ± SE of 3 experiments. **P < 0.01 (by one-way ANOVA followed by Sidak’s multiple-comparisons test). C: commercially available human pulmonary artery endothelial cells (n = 2 donors) were established in a 96-well plate and directly exposed to the following treatment for 60 min: vehicle, TSP-1 (10 nM, 2.2 nM, or 0.2 nM), or 2.2 nM TSP-1 + 2 µg/ml CD47 blocking antibody (clone B6H12.2). Select wells received 1,000 U/ml bovine liver catalase to act as a negative control. Coumarin boronic acid probe detection of H2O2 production in the cells was measured kinetically for 2 h. Average rate of fluorescence generation was normalized to vehicle control and displayed as fold change in H2O2 production. RFU, relative fluorescence units. Values are means ± SE of 3–4 experiments per donor (total n = 6–7). *P < 0.05, **P < 0.01, ****P < 0.0001 (by unpaired t-test).

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: Amplex Red Assay, Blocking Assay, Negative Control, Fluorescence, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Vascular TSP1-CD47 signaling promotes sickle cell-associated arterial vasculopathy and pulmonary hypertension in mice

doi: 10.1152/ajplung.00302.2018

Figure Lengend Snippet: Thrombospondin 1 (TSP1) augments pulmonary oxidative damage and promotes vascular congestion in chimeric mice with a sickle erythropoiesis. A: lung tissue sections from chimeras were stained for 4-hydroxynonenal (4-HNE) or 3-nitrotyrosine (3-NT) and examined by immunofluorescence to determine whether in vivo sickle cell disease-mediated pulmonary reactive oxygen species production (4-HNE) and secondary reactive oxygen species-mediated protein (3-NT) were modified by the absence of TSP1-CD47 signaling. 3-NT is shown as green fluorescence; DAPI stained blue (n = 3 per group, 2 sections per animal). Representative 4-HNE deposition is also shown by green fluorescent immunohistochemical staining; DAPI stained blue (n = 2 mice per group, 3 sections per animal). Fluorescence signal was quantified using ImageJ software and reported as 3-NT or 4-HNE intensity per cell (DAPI signal). Vascular congestion on hematoxylin-eosin-stained slides from chimeras (n = 3 mice per group) was scored by 3 blinded, independent readers, who used a semiquantitative, relative scale of 0–4, where 0 = absence of red blood cells in the lumens of pulmonary blood vessels. BERK, Berkeley; CD47KO, CD47 knockout. B: increasing lumen congestion and number of affected vessels scored on a scale of 1–4. Images were taken using a Nikon A1 confocal microscope or Nikon 90i upright microscope at ×20. Scale bars = 50 µm. Results are shown as representative slides. Values are means ± SE. *P < 0.05, ****P < 0.0001 (by unpaired t-test).

Article Snippet: An aliquot was set aside for determination of engraftment, and the plasma was batch-tested with a murine TSP1 ELISA kit (Cusabio, College Park, MD), as previously described ( 56 ).

Techniques: Staining, Immunofluorescence, In Vivo, Modification, Fluorescence, Immunohistochemical staining, Software, Knock-Out, Microscopy