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Figure Lengend Snippet: Standard-dose bleomycin increases pulmonary vascular permeability in EC- S1pr1 −/− mice. ( A ) Evans blue dye assay was performed on Day 0 and Day 7 after standard-dose intratracheal (IT) bleomycin (1.0 U/kg). ( B ) Quantification of vascular permeability was calculated as an Evans blue (EB) index defined as the ratio of lung Evans blue to plasma Evans blue. n = 3–7 mice per group. ( C ) Hematoxylin and eosin and Masson’s trichrome staining of lungs was performed on Day 7 after standard dose bleomycin. Representative images are shown from n = 3 mice per group. Scale bars, 50 μm. ( D ) Survival of mice receiving standard-dose (1.0 U/kg) bleomycin. n = 5 mice per group. * P < 0.05 comparing survival of EC- S1pr1 −/− mice to littermate controls S1pr1 f/f . *** P < 0.001 comparing EB index in EC- S1pr1 −/− mice to littermate controls S1pr1 f/f . H&E = hematoxin and eosin; Trich = trichrome.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Permeability, Clinical Proteomics, Staining

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Figure Lengend Snippet: Low-dose bleomycin induces fibrosis in EC- S1pr1 −/− mice. ( A ) Survival of mice receiving low-dose (0.5 U/kg) bleomycin. n = 5 mice per group. ( B ) Total protein content in the BAL was quantified at Days 0, 7, 14, and 21 after low-dose bleomycin. BAL from EC- S1pr1 −/− mice was compared with intact littermate controls S1pr1 f/f . n = 3–5 mice per group. ( C ) Hematoxylin and eosin and Masson’s trichrome staining of lungs were performed on Day 21 after low-dose bleomycin. Representative images are shown from n = 3 mice per group. Scale bars, 50 μm. ( D ) Fibrosis was quantified at Days 0, 7, 14, 21, and 42 after low-dose IT bleomycin by measuring hydroxyproline levels in the mouse lungs. n = 3–15 mice per group. ( E ) Ashcroft scoring performed on trichrome stained lung sections from EC- S1pr1 −/− mice at Days 0 and 14 or 21 was compared with intact littermate controls S1pr1 f/f . n = 3–4 mice per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of total protein or hydroxyproline in EC- S1pr1 −/− mice compared with intact littermate control mice S1pr1 f/f . OHP = hydroxyproline.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Staining, Control

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Figure Lengend Snippet: Endothelial-specific S1pr1 deletion leads to reduced intercellular junction proteins and increased extravascular coagulation. ( A ) Mice were injected with FITC-labeled albumin to identify albumin extravasated into the lung tissue after bleomycin. Sequential sectioning and staining of the lung tissue were performed with Masson’s trichrome stain. Representative images are shown from n = 3 mice per group. Scale bars, 100 μm. ( B ) D-dimer ELISA was performed on BAL fluid collected from mice on days 0, 7, 14, and 21 after low-dose IT bleomycin (0.5 U/kg). n = 4 mice per group. ( C ) D-dimer ELISA was performed on homogenized lung tissue collected from mice on days 0, 7, 14, and 21 after low-dose IT bleomycin (0.5 U/kg). n = 4 mice per group. ( D ) Junctional proteins were assessed through immunoblotting for VE-Cadherin in lung tissue homogenates harvested at days 0 and 14 after low-dose bleomycin challenge. Densitometry was performed to quantify differences in protein expression at each time point. n = 2–3 mice per group. * P < 0.05 and *** P < 0.001 for comparisons of D-dimer in the BAL and lung tissue of EC- S1pr1 −/− mice as compared with intact littermate controls S1pr1 f/f .

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Coagulation, Injection, Labeling, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

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Figure Lengend Snippet: Immune cell populations in the BAL of mice lacking endothelial S1pr1 after bleomycin. ( A ) Total cell counts were quantified from BAL obtained on Day 0 and Day 7 after low-dose IT bleomycin. n = 3–4 mice per group. Flow cytometry was performed on BAL collected 7 days after bleomycin challenge in EC- S1pr1 −/− and control mice to assess the number of ( B ) alveolar macrophages, ( C ) dendritic cells, ( D ) NK cells, ( E ) monocyte-derived dendritic cells (DCs), ( F ) inflammatory monocytes, ( G ) CD4 + T cells, ( H ) CD8 + T cells, and ( I ) T regulatory cells. In contrast, no differences were detected in the quantities of BAL ( J ) B cells, ( K ) eosinophils, ( L ) γδ T cells, or ( M ) neutrophils. n = 3–4 mice per group. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of BAL cells in EC- S1pr1 −/− mice as compared with intact control mice S1pr1 f/f . γδ T cells = GD T cells.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Flow Cytometry, Control, Derivative Assay

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Figure Lengend Snippet: Bleomycin-induced lung injury reduces endothelial cell populations in the lung. ( A ) Immunoblotting was performed using protein from lung homogenates of C57BL/6 wild-type mice (WT) at Days 0, 1, 3, 7, and 14 after IT bleomycin (0.5 U/kg). Blots were probed for S1PR1, and protein expression levels were compared using GAPDH as a loading control. n = 2–3 mice per group. Densitometry was performed using Image J software (NIH). ( B ) Flow cytometry to identify CD31 + cells was performed on lung tissue from WT mice on Days 0, 7, and 14 after standard-dose IT bleomycin. n = 4 mice per group. ( C ) Flow cytometry of CD31 + cells expressing S1PR1. n = 4 mice per group. ( D ) The percentage of endothelial cells expressing S1PR1 at each time point after bleomycin was quantified. ( E ) Mean fluorescence intensity of S1PR1 in CD31 + cells after bleomycin was quantified. ( F ) Plasma S1P levels in WT mice were quantified at Days 0, 3, 7, 10, and 14 after IT bleomycin. n = 3–4 mice per group. ( G ) Immunoflourescence co-staining on lungs from naive and bleomycin treated mice at Day 14 was performed for S1PR1 (red), endothelial CDH5/VE-cadherin (green), and nuclear DAPI (blue). White box indicates a fibrotic region. Scale bars, 50 μm. * P < 0.05, ** P < 0.01, and *** P < 0.001 for comparisons of cells in WT mice at various time points after bleomycin injury.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Western Blot, Expressing, Control, Software, Flow Cytometry, Fluorescence, Clinical Proteomics, Staining

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Figure Lengend Snippet: Increased circulating S1P is insufficient to protect mice from bleomycin-induced pulmonary fibrosis. ( A ) Transgenic mice overexpressing human apolipoprotein M (ApoM Tg + ) were bred, and Evans blue dye assay was performed at Day 7 after standard-dose IT bleomycin. ( B ) Evans blue indices were calculated using Evans blue dye indices in the lungs and plasma of ApoM Tg + mice and littermate controls (WT). n = 4 mice per group. ( C ) BAL total protein was quantified at Day 14 after standard dose bleomycin. ( D ) Histology at Days 0 and 14 after standard-dose IT bleomycin were stained with hematoxylin and eosin and Masson’s trichrome staining. Representative images are shown from n = 3 mice per group. Scale bars, 100 μm. ( E ) Quantification of fibrosis was performed with hydroxyproline assay. ( F ) S1P levels were quantified by mass spectrometry from circulating plasma at baseline (Day 0), Day 7, and Day 14 after standard-dose bleomycin. n = 3–6 mice per group. n = 3–8 mice per group. *** P < 0.001, and **** P < 0.0001 for comparisons of S1P between ApoM-Tg + and control mice.

Article Snippet: Fibrosis was induced by single-dose intratracheal (IT) injection of bleomycin (Fresenius Kabi) at a standard dose of 1.0 units/kg and also at a reduced dose of 0.5 units/kg, and quantified by measuring hydroxyproline content, per our usual methods ( 20 ).

Techniques: Transgenic Assay, Clinical Proteomics, Staining, Hydroxyproline Assay, Mass Spectrometry, Control