oxLDL activates the PI3k/Akt pathway in a PAFR and Gαi coupled protein-dependent manner.

<p>Adherent monocytes/macrophages were treated with WEB2170 (50 µM, 30 min) (A) or PTX (600 ng/mL, 18 h) (B) and stimulated with oxLDL (30 µg/mL) for 10 min. Western blot analysis of cell lysates were performed using antibodies to phosphorylated and non-phosphorylated forms of Akt. Data are presented as mean ± SEM of four donors. Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). The autoradiographs show one representative experiment. * p<0.05 comparing oxLDL-stimulated with the non-stimulated cells (cont). <b>#</b> p<0.05 comparing cells treated with WEB2170 or PTX with non-treated cells.</p

PI3K/Akt pathway is required for oxLDL induced upregulation of CD36 expression (A) oxLDL uptake (B) and IL-8 production (C).

<p>Adherent human monocytes/macrophages were treated with LY294002 (10 µM, 30 min), and then stimulated with oxLDL (30 µg/mL) for 24 h. The expression of CD36 was measured by FACS and IL-8 production by ELISA. For the uptake evaluation, stimulated cells were washed and incubated with FITC-oxLDL for 1 h and uptake was measured by FACS. Data are presented as mean ± SEM of the mean fluorescence Intensity of five donors. * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). # p<0.05 comparing cells treated with LY294002 with non-treated cells.</p

oxLDL induces IL-8 and MCP-1 in a PAFR-dependent manner.

<p>Adherent human monocytes/macrophages were treated with WEB2170 (50 µM) or CV3988 (10 µM) for 30 min and stimulated with oxLDL (30 µg/mL). The mRNA levels for MCP-1 and IL-8 were evaluated after 5 h (n = 4) (A). The IL-8 concentration was measured in the supernatants after 24 h (n = 3) (B). THP-1 cells stimulated with different concentrations of oxLDL or LDL, the IL-8 concentration was measured after 24 h (C). Data are presented as mean ± SEM. * p<0.05 comparing oxLDL stimulated with non-stimulated cells (cont). <b>#</b> p<0.05 comparing cells treated with WEB2170 or CV3988 with non-treated cells.</p

PI3k/Akt pathway is required for p38 and JNK MAPK activation induced by oxLDL.

<p>Adherent monocytes/macrophages were treated with LY294002 (10 µM, 30 min) and then stimulated with oxLDL (30 µg/mL) for 10 min. The MAPK Western blot analysis of cell lysates were performed using antibodies to phosphorylated and non-phosphorylated forms of ERK1/2, p38 and JNK (A, B and C, respectively). The autoradiographs show one representative experiment. Graph data are presented as mean ± SEM of four donors. Protein expression was quantified by the AlphaEaseFC™ software V3.2 beta (Alpha Innotech). * p<0.05 comparing oxLDL-stimulated with the non-stimulated cells (cont). <b>#</b> p<0.05 comparing cells treated with LY294002 with non-treated cells.</p

CD36 and PAFR cooperatively mediate FITC-oxLDL uptake and IL-8 production.

<p>HEK 293t cells were transiently transfected with hCD36 and/or hPAFR, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036632#s4" target="_blank">methods</a> (A–B). Transfected cells were incubated with FITC-oxLDL (30 µg/mL) for 1 h at 37°C. Cells were washed and fixed with paraformaldehyde 4%. Fluorescence was visualized by microscopy (C). Transfected cells were stimulated with oxLDL (30 µg/mL) and the IL-8 concentration in the supernatant was measured after 24 h (D). * p<0.05 comparing with cDNA3 plasmid control transfected cells.</p

Based on these results we conclude that the addition of oxLDL to human monocytes/macrophages activates the PI3K/Akt pathway which in turn activates the p38 and JNK MAPK.

<p>Phosphorylation of Akt requires engagement of PAFR and a Gαi-coupled protein. This should lead to NFκB and/or AP-1 activation and gene transcription. We found increased expression of CD36, IL-8 and MCP-1 mRNA in cells stimulated with oxLDL in a PAFR-dependent manner. Moreover, we observed increased expression of CD36 protein on macrophage membrane and increased uptake of oxLDL by these cells upon exposure to oxLDL which were also dependent on PAFR. By transfecting CD36 and PAFR to HEK 293t cells, we found that whereas each receptor <i>per se</i> was able to induce oxLDL uptake, both receptors were required for IL-8 production. These results point to an important role for PAFR in atherosclerosis by promoting foam cells formation because of the increased uptake of oxLDL and also by contributing to macrophage infiltration due to IL-8 and MCP-1 production. Thus, antagonists of PAFR might be a promising target for atherosclerosis treatment. Dashed lines represent data obtained from the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036632#pone.0036632-Hoffmann1" target="_blank">[21]</a>.</p

BMP-7 inhibits SMADs phosphorilation in mouse lung fibroblasts from chronic asthmatic mice.

<p>Mouse lung fibroblasts were treated with TGF-β1 (5 ng/ml) alone or in combination with BMP-7 (300 ng/ml). After 15 min, samples were analyses for SMAD 2 and 3 or p-p38 expression. Proteins were analyzed by western blot and normalized by their respective total proteins. Data are representative of 5 animals in each of 3 independent experiments. *p<0.05 comparing with the control group; Δ p<0.05 comparing TGF-β1-treated with TGF-β1/BMP-7-treated groups.</p

Intranasal treatment with BMP-7 reduces collagen-I expression in chronic asthmatic lungs.

<p>Lung slides were stained with Masson's Trichrome (blue) and the morphometric analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095959#s2" target="_blank">methods</a>. Control (A), Chronic asthmatic (B), Chronic asthmatic treated with BMP-7 (300 µg/kg) by intranasal route (C). Morphometric analysis of the stained area (blue)(D). Data are representative of 5 animals in each of 3 independent experiments. Bars  = 50 µm. *p<0.05 comparing with the non asthmatic control; Δ p<0.05 comparing asthmatic mice treated or not with BMP-7.</p

BMP-7 inhibits TGF-β1-induced synthesis of collagen-I by lung fibroblasts.

<p>Mouse lung fibroblasts from control animals (A) were stimulated with TGF-β1 (5 ng/ml) alone or in combination with BMP-7 (30, 100 ou 300 ng/ml) during 18 hours. Mouse lung fibroblasts from asthmatic animals (B) were treated with TGF-β1 (5 ng/ml) alone or in combination with BMP-7 (300 ng/ml). Type I collagen was analyzed by western blot and normalized by β-actin expression. Results are presented as percentage of the control or as arbitrary units. Data are representative of 5 animals in each of 3 independent experiments. *p<0.05 comparing with the control group.</p

Fibrosis markers in the asthmatic lung.

<p>Lung sections were taken from animals with asthma (6 challenges), stained with PAS for mucus (A) or prepared for immunohistochemical and morphometric analysis of α-SMA (B) and collagen type I (C). Data are representative of 5 animals in each of 3 independent experiments. Bars  = 50 mm. *p<0.05 comparing with control group.</p