murine tsp1  (Athens Research)

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

Journal:

Article Title: Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-? in Fibrotic Renal Disease in the Rat in Vivo

doi:

Figure Lengend Snippet: Transfer of phosphorothioate ODN against TSP1 in cultured MC inhibits glomerular TSP1 expression. FITC-labeled phosphorothioate ODNs against TSP1 were successfully transferred into nuclei of cultured MC (A). Of 11 non-cross-reacting antisense sequences, five ODNs were able to block FCS-mediated TSP1 induction in cultured MC compared to stimulated cells using transfection reagent without ODNs (pC) (B). Non-stimulated but Effectene-treated MC expressed no TSP1 (nC). The two most effective ODN sequences (number 2 and 11) were chosen for the in vivo experiments.

Article Snippet: 25 Immunostaining for matrix proteins was conducted with polyclonal antibodies to collagen I (rabbit anti-rat collagen I; Quartett, Berlin, Germany), 25 collagen IV (goat anti-human/bovine collagen IV; Southern Biotechnology Associates, Inc., Birmingham, AL), 25 active TGF-β1 (chicken anti-human active TGF-β1; R&D systems, Wiesbaden-Nordenstadt, Germany), 28 TGF-β1 (rabbit anti-human TGF-β1; Santa Cruz Biotechnology Inc., Santa Cruz, CA), 24 TGF-β2 (rabbit anti-human TGF-β2; Santa Cruz Biotechnology), 24 and a murine IgG 1 mAb against TSP1 (Dunn, Labortechnik GmbH, Asbach, Germany), 25 P-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435; Santa Cruz).

Techniques: Cell Culture, Expressing, Labeling, Blocking Assay, Transfection, In Vivo

Journal:

Article Title: Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-? in Fibrotic Renal Disease in the Rat in Vivo

doi:

Figure Lengend Snippet: Transfer of phosphorothioate ODN against TSP1 inhibits glomerular extracellular matrix accumulation and MC activation. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). EDA-fibronectin mRNA, a typical TGF-β-dependent matrix gene, was quantified via quantitative real-time PCR in pooled isolated glomeruli comparing scrambled- with antisense-treated kidneys. Antisense therapy against TSP1 almost completely abolished transcript expression of EDA-fibronectin in isolated glomeruli from nephritic rats on day 7 (A). Glomerular ECM accumulation on day 7 as determined by immunostaining for collagen IV (C and D) or collagen I (G and H) was markedly inhibited by antisense but not by scrambled ODN therapy against TSP1. E: A representative picture of glomerular collagen IV accumulation (dark gray immunostaining) in antisense-treated rats; F shows typical collagen IV expression in scrambled-treated rats on day 7. Glomerular de novo expression of smooth-muscle actin during glomerulonephritis, a marker of MC activation, was decreased by the TSP1 antisense but not by scrambled ODN transfer (B). Matrix accumulation was either evaluated by scoring system (C and G) or by computerized morphometry (B, D, H). * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Article Snippet: 25 Immunostaining for matrix proteins was conducted with polyclonal antibodies to collagen I (rabbit anti-rat collagen I; Quartett, Berlin, Germany), 25 collagen IV (goat anti-human/bovine collagen IV; Southern Biotechnology Associates, Inc., Birmingham, AL), 25 active TGF-β1 (chicken anti-human active TGF-β1; R&D systems, Wiesbaden-Nordenstadt, Germany), 28 TGF-β1 (rabbit anti-human TGF-β1; Santa Cruz Biotechnology Inc., Santa Cruz, CA), 24 TGF-β2 (rabbit anti-human TGF-β2; Santa Cruz Biotechnology), 24 and a murine IgG 1 mAb against TSP1 (Dunn, Labortechnik GmbH, Asbach, Germany), 25 P-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435; Santa Cruz).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Isolation, Expressing, Immunostaining, Marker

Journal:

Article Title: Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-? in Fibrotic Renal Disease in the Rat in Vivo

doi:

Figure Lengend Snippet: Transfer of phosphorothioate ODN against TSP1, but not scrambled ODN, into glomerulonephritic rats inhibits glomerular TSP1 expression. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). On day 2 after disease induction, successful transfer of Cy3-labeled phosphorothioate ODN against TSP1 was demonstrated in almost 100% of glomeruli (A) of the left kidney, but none of the right kidney (not shown). Transfer of antisense oligonucleotides selectively inhibited de novo expression of glomerular TSP1 protein in the perfused left kidney on day 7 by more than 60% compared to the non-perfused right kidney, while gene transfer of scrambled control oligonucleotides did not alter glomerular TSP1 expression (B, evaluated by scoring; C, evaluated by computerized morphometry). Representative examples of unaltered TSP1 expression by scrambled ODNs (D) versus reduced TSP1 expression by antisense ODNs (E) are demonstrated by the extent of dark gray immunostaining within glomeruli. The * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Article Snippet: 25 Immunostaining for matrix proteins was conducted with polyclonal antibodies to collagen I (rabbit anti-rat collagen I; Quartett, Berlin, Germany), 25 collagen IV (goat anti-human/bovine collagen IV; Southern Biotechnology Associates, Inc., Birmingham, AL), 25 active TGF-β1 (chicken anti-human active TGF-β1; R&D systems, Wiesbaden-Nordenstadt, Germany), 28 TGF-β1 (rabbit anti-human TGF-β1; Santa Cruz Biotechnology Inc., Santa Cruz, CA), 24 TGF-β2 (rabbit anti-human TGF-β2; Santa Cruz Biotechnology), 24 and a murine IgG 1 mAb against TSP1 (Dunn, Labortechnik GmbH, Asbach, Germany), 25 P-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435; Santa Cruz).

Techniques: Expressing, Labeling, Immunostaining

Journal:

Article Title: Antisense Oligonucleotides Against Thrombospondin-1 Inhibit Activation of TGF-? in Fibrotic Renal Disease in the Rat in Vivo

doi:

Figure Lengend Snippet: Transfer of phosphorothioate ODN against TSP1 decreases activation, but not expression of TGF-β in nephritic glomeruli. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). Glomerular TGF-β1 or TGF-β2 protein (by brown immunostaining) was not changed in any group of nephritic rats (A and B). In agreement, equal levels of total TGF-β levels were determined using the PAI-1 luciferase assay. Active TGF-β levels were significantly reduced in the antisense-treated kidneys (C). Additionally, active TGF-β in nephritic glomeruli was determined by an antibody specifically recognizing the active form of TGF-β1 (D, gray cytoplasmic staining) and by an antibody specific for the phosphorylated form of the TGF-β signal-transduction molecule Smad 2/3 (H, black nuclear staining; arrowheads indicate examples for P-Smad2/3-positive nuclei). Inhibition of TSP1 expression after antisense ODN therapy but not scrambled ODN therapy was associated with a markedly decreased glomerular TGF-β activity in the left kidney as reflected by immunostaining for active TGF-β1 (E, evaluated by scoring system; F, evaluated by computerized morphometry) and by a marked reduction of glomerular cells showing positive nuclei for the TGF-β signaling molecule phospho-Smad2/3 (G). * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Article Snippet: 25 Immunostaining for matrix proteins was conducted with polyclonal antibodies to collagen I (rabbit anti-rat collagen I; Quartett, Berlin, Germany), 25 collagen IV (goat anti-human/bovine collagen IV; Southern Biotechnology Associates, Inc., Birmingham, AL), 25 active TGF-β1 (chicken anti-human active TGF-β1; R&D systems, Wiesbaden-Nordenstadt, Germany), 28 TGF-β1 (rabbit anti-human TGF-β1; Santa Cruz Biotechnology Inc., Santa Cruz, CA), 24 TGF-β2 (rabbit anti-human TGF-β2; Santa Cruz Biotechnology), 24 and a murine IgG 1 mAb against TSP1 (Dunn, Labortechnik GmbH, Asbach, Germany), 25 P-Smad2/3 (rabbit anti-human Smad2 peptide phosphorylated at Ser-433/435; Santa Cruz).

Techniques: Activation Assay, Expressing, Immunostaining, Luciferase, Staining, Transduction, Inhibition, Activity Assay