Additional file 2: Figure S2. of The role of circulating thrombospondin-1 in patients with precapillary pulmonary hypertension

Pulmonary vascular resistance index was derived from invasive measurements of PAOP, mean PAP and cardiac output by thermodilution method. Data is expressed as mean ± SD [dyn.s.cm−5]. Statistically significance is denoted in Table 2 for readability. (TIFF 270 kb

Additional file 1: Figure S1. of The role of circulating thrombospondin-1 in patients with precapillary pulmonary hypertension

Pulmonary artery pressures were determined invasively. Data is expressed as mean ± SD [mmHg]. Statistically significance is denoted in Table 2 for readability. (TIFF 384 kb

RAGE contributes to maintenance of pulmonary mechanics and structure.

<p>Quantification of mean chord length (A) were performed by stereological analysis of alveolar parenchyma; n ≥ 4 per group. (B) Representative histology (hematoxylin and eosin staining) of ten months old WT and RAGE^-/- mice; scale bars: 200μm. Respiratory system compliance (C) and respiratory system elastance (D) were determined in two, four and ten months old WT and RAGE^-/- mice using invasive pulmonary function measurements; n ≥ 7 per group. (E) Concentration of serum protein albumin in BALF of two months old mice; n = 10. Data are shown as mean ± SEM. *p < 0.05 and **p < 0.01.</p

RAGE promotes the differentiation and formation of an intact barrier function in primary murine alveolar epithelial cells.

<p>Alveolar epithelial cells were isolated from WT and RAGE^-/- mice and analyzed in an air-liquid interface culture system. (A) TEER of isolated alveolar epithelial cells was measured for a period of five days; n = 12 per group. Relative mRNA induction of alveolar epithelial type 2 cell marker surfactant protein C (B), alveolar epithelial type 1 cell marker aquaporin-5 (AQP5) (C) and the tight junction proteins claudin 18 (D), ZO-1 (E) and occludin (F) was determined by qRT-PCR; n = 6 per group. The proteins of cell lysate of alveolar epithelial cells at day 2 and 4 post isolation were separated by SDS-PAGE and stained with antibodies against α-Tubulin (αTub), aquaporin-5 (AQP5), pro-surfactant protein C (Sp-C), and claudin 18 (Cldn18) (G). The bands of the blot were densitometrically analyzed and the results are shown for AQP5 (H), SP-C (I), and Cldn18 (J). Data are shown as mean ± SEM. *p < 0.05; **p < 0.01 and ***p < 0.001.</p

RAGE promotes the development of CS-induced lung damage.

<p>WT and RAGE^-/- mice were exposed to CS or room-air for six months. Total lung capacity (A), quasi-static compliance (B) and inspiratory capacity (C) were determined by invasive pulmonary function measurements; n ≥ 18 per group. (D) Representative histology of alveolar parenchyma (hematoxylin and eosin staining); scale bars: 100μm. Quantification of mean chord length (E) were performed by stereological analysis of alveolar parenchyma; n ≥ 10 per group. Data are shown as mean ± SEM. *p < 0.05; **p < 0.01 and ***p < 0.001.</p

RAGE is expressed on alveolar epithelial cells and alveolar macrophages.

<p>(A) Immunostaining for RAGE protein in WT and RAGE^-/- lung tissue. (B) Isolated alveolar epithelial cells (AEC) (n = 7 per group) and alveolar macrophages (AM) (n = 4 per group) were analyzed for RAGE mRNA expression using qRT-PCR. Data are shown as mean ± SEM. Scale bar: 100μm.</p

CDA-2 reduces development of lung tumor in mice.

<p>(A) Lung appearance (up) and histology (H&E stain; down) in LLC inoculated C57/BL6 mice 10 days after CDA-2 treatment with indicated doses. 2×10⁵ LLC cells were intravenously injected into sex-matched C57/BL6 mice by tail vein, 14 days later, mice were treated with PBS or CDA-2 for 10 days, at day 25, the lungs were removed. (B) Lung tumor multiplicity and maximal tumor sizes were determined by serial sectioning at 350 µm intervals. Results are mean ± SEM, n = 5, significant difference, * p<0.05. (C) Survival curves of mice (p<0,001; Log-rank test for statistic analysis; n = 10).</p

CDA-2 decreases pulmonary inflammation.

<p>Induction of inflammatory cytokine and chemokine mRNA in homogenates of 3 or 5 days 2000 mg/kg CDA-2 (A) or 800 mg/kg PG (B) treated lungs was measured by real time PCR. Results are means ± SEM, n = 5, significant difference,*p<0.05. Concentration of inflammatory cytokines in BALF of 3 or 5 days 2000 mg/kg CDA-2 (C) or 800 mg/kg PG (D) treated mice was evaluated by ELISA. Results are means ± SEM, n = 5, significant difference,*p<0.05.</p

PG inhibits lung tumor promotion.

<p>(A) Lung appearance (up) and histology (H&E stain; down) in LLC inoculated C57/BL6 mice 10 days after PG treatment with indicated doses. 2×10⁵ LLC cells were intravenously injected into sex-matched C57/BL6 mice by tail vein, 14 days later, mice were treated with PBS or PG for 10 days, at day 25, the lungs were removed. (B) Lung tumor multiplicity and maximal tumor sizes were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052117#pone-0052117-g001" target="_blank">Figure 1B</a>. Results are mean ± SEM, n = 5, significant difference, * p<0.05.</p

CDA-2 inhibits LLC-CM-induced activation of TLR2 signaling in BMDMs.

<p>BMDMs were treated for 24 h by serum-free DMEM (SFM) or LLC-CM or combination with CDA-2 (A) or PG (B). Total RNAs were isolated from BMDMs, and gene expression was assessed by real-time PCR. Results are mean fold change ± SEM, n = 3, significant difference, * p<0.05.</p