Body weights of apo E-knockout mice treated with PBS or 15d-PGJ₂ from 8 to 16 weeks of age.

<p>From the 8th week to 16th week, female mice were randomized to receive a Western-type diet and PBS or 1 mg/kg/day of 15d-PGJ₂ (n = 15 animals for each group). At 16th week, body weight of 15d-PGJ2 treated mice tended to be higher than controls, but it was not significantly different (21.6±4.2 g and 21.2±3.9 g, respectively, <i>p</i> = 0.6). Statistical analyses were performed with Student's t test.</p

Serum lipid levels in the controls and the 15d-PGJ₂ group.

<p>Blood was collected from the cardiac cavity of mice aged 16 wk and analyzed for the lipid profile. The plasma chylomicron (CM) (B), very low density lipoprotein (VLDL) (C), low density lipoprotein (LDL) (D), and high density lipoprotein (HDL) (E) levels were determined by use of a high-sensitivity lipoprotein-profiling system by high-performance liquid chromatography. The total serum cholesterol level (A) was significantly lower in the15d-PGJ₂ group than in the control group (795.5±39.31 mg/dl vs 944.1±49.04 mg/dl, <i>p</i> = 0.029). Especially LDL was significantly reduced in the 15d-PGJ₂ group (186.9±13.49 mg/dl vs 234.3±16.60 mg/dl, <i>p</i> = 0.0397). CM, VLDL and HDL were not different between the control and 15d-PGJ₂ groups, 36.96±4.999 mg/dl vs 68.13±23.98 mg/dl, 553.5±26.67 mg/dl vs 622.7±28.02 mg/dl, 18.14±1.264 mg/dl vs 19.01±2.562 mg/dl, respectively. *<i>p</i><0.05, with Student's <i>t</i> test.</p

Representative sections with immunohistochemical analysis.

<p>Apo E-knockout mice were fed a Western-type diet and treated with PBS (control group) (n = 10) or 1 mg/kg/day 15d-PGJ₂ (15d-PGJ₂ group) (n = 10) for 2 mo. Representative cross-sections of the aortic sinus were stained with MOMA-2 (A), which detected macrophages, and MCP-1 Abs (B), MIF Abs (C), TNF-α Abs (D), MMP-9 Abs (E), PPARγ Abs (F), and counterstained with hematoxylin. Right sections are control group and left ones are 15d-PGJ₂ group in each figure. Black arrows indicate the positive lesions.</p

Knock Out of S1P3 Receptor Signaling Attenuates Inflammation and Fibrosis in Bleomycin-Induced Lung Injury Mice Model

<div><p>Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein–coupled receptors (S1P1–5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.</p></div

Collagen levels in bronchoalveolar lavage fluid (BALF).

<p>Collagen levels in BALF collected on the seventh and 28th day after intratracheal administration of bleomycin or saline. Knockout (KO) mice had significantly less pulmonary fibrosis compared with wild-type (WT) mice on the seventh day (<i>n</i> = 5 in each genotype, *<i>p</i><0.05) and the 28th day (<i>n</i> = 5 in each genotype, **<i>p</i><0.01). In saline-treated control mice, collagen levels were undetectable on both the seventh and 28th days.</p

Histopathological findings of pulmonary inflammation.

<p>Representative results of H&E staining of lung tissue from mice in the bleomycin-induced lung injury model (A, B, C and D). H&E staining of lung tissue from (A) wild-type (WT) and (B) knockout (KO) mice on the seventh day after intratracheal administration of bleomycin. H&E staining of lung tissue from (C) WT and (D) KO mice on the 28th day after intratracheal administration of bleomycin. All scale bars  = 100 µm. The window shows an area of increased magnification revealing inflammatory cell infiltration. Representative results of H&E staining of lung tissue from mice in the saline-treated control group (E, F, G and H). H&E staining of lung tissue from (E) WT and (F) KO mice on the seventh day after intratracheal administration of saline. H&E staining of lung tissue from (G) WT and (H) KO mice on the 28th day after intratracheal administration of saline. All scale bars  = 100 µm. The window shows an area of increased magnification revealing hyperplasia of the alveolar/bronchiolar epithelium.</p

Concentration of S1P in bronchoalveolar lavage fluid (BALF).

<p>S1P concentration in BALF collected on the seventh day after intratracheal administration of bleomycin or non-treated control. There were no significant differences in S1P concentration in BALF between wild-type (WT) and knockout (KO) mice at baseline or on the seventh day after bleomycin administration, nor were there significant differences in the S1P concentrations in BALF collected before and after bleomycin administration in WT (baseline vs. seventh day; <i>p</i> = 0.14) or KO (baseline vs. seventh day; <i>p</i> = 0.50) mice.</p

ELISA analyses of bronchoalveolar lavage fluid (BALF).

<p>A. CTGF concentrations in BALF collected on the seventh day after intratracheal administration of bleomycin or saline. The CTGF concentrations in BALF after administration of bleomycin were significantly decreased in S1P3 knockout (KO) mice compared with wild-type (WT) mice (<i>n</i> = 5 in each genotype, *<i>p</i><0.05). Values are presented as the mean ± SD. B. TGF-β1 concentrations in BALF collected on the seventh day after intratracheal administration of bleomycin or saline. No significant differences between WT and KO mice were observed in the concentration of TGF-β1 in BALF collected after administration of bleomycin (<i>n</i> = 5 in each genotype, <i>p</i> = 0.30). Values are presented as the mean ± SD. C. MCP-1 concentrations in BALF on the seventh day after intratracheal administration of bleomycin or saline. No significant differences between WT and KO mice were observed in the concentration of MCP-1 measured in BALF collected after administration of bleomycin (<i>n</i> = 5 in each genotype, <i>p</i> = 0.23). Values are presented as the mean ± SD.</p

Histopathological findings of fibrosis induced by bleomycin.

<p>Representative results of Masson's trichrome staining of lung tissue from mice in the bleomycin-induced lung injury model (A, B, C and D). Masson's trichrome staining of lung tissue from (A) wild-type (WT) and (B) knockout (KO) mice on the seventh day after intratracheal administration of bleomycin. Masson's trichrome staining of lung tissue from (C) WT and (D) KO mice on the 28th day after intratracheal administration of bleomycin. All scale bars  = 100 µm. The window shows an area of increased magnification revealing a part of lung fibrosis. Representative results of Masson's trichrome staining of lung tissue from mice in the saline-treated control group (E, F, G and H). Masson's trichrome staining of lung tissue from (E) WT and (F) KO mice on the seventh day after intratracheal administration of saline. Masson's trichrome staining of lung tissue from (G) WT and (H) KO mice on the 28th day after intratracheal administration of saline.</p

Changes in body weight in mice after administration of bleomycin.

<p>Time course of changes in body weight in wild-type (WT) (<i>n</i> = <i>9</i>) and in S1P3 knockout (KO) (<i>n</i> = <i>8</i>) mice after administration of bleomycin. S1P3 KO mice exhibited decreased body weight loss compared to WT mice. The weight of the WT mice gradually decreased, reaching their lowest point on the seventh day after treatment and increasing afterwards. Values are presented as the mean ± SD (*<i>p</i><0.05, **<i>p</i><0.01).</p