Daily rhythms of plasma CORT and 5HT in mice exposed to sub-chronic (10 day) air or CS.

<p>Mice were exposed to CS for 10 days, and plasma samples were collected at 6-h interval for 48 h (starting at ZT12) beginning 24 h after the last exposure. Plasma levels of (A) CORT and (B) 5HT were determined by ELISA. Gray shading indicates the dark phase (ZT12 to ZT24). Data from air- and CS-exposed mice are shown as mean ± SEM (n = 3 mice per group). Data were fit with non-linear regression (multi-order polynomial) analyses. **<i>P</i><0.01 significant compared to air-exposed mice at ZT12; ***<i>P</i><0.001 significant compared to air-exposed mice at ZT6.</p

PPAR-γ ligands inhibit TGFβ-induced Akt phosphorylation and myofibroblast differentiation in a PPAR-γ-independent but electrophilic carbon-dependent manner.

<p>HLF cells were treated with indicated compounds and/or TGFβ (5ng/ml) for 48 hours. Immunoblots were performed to assess the expression of indicated proteins. Protein lysates from all the indicated samples were electrophoretically separated on the same gel, and representative lanes from a single experiment are shown here. <i>A</i>, the ability of PPAR-γ ligands (CDDO (1µM) and 15d-PGJ₂ (5µM)) to reduce p-Akt was not altered upon GW9662-mediated inhibition of PPAR-γ. GW9662 (5µM) inhibits PPAR-γ activity by a covalent bond formation with PPAR-γ protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015909#pone.0015909-Huang1" target="_blank">[36]</a>. R.P. indicates relative changes in Akt phosphorylation compared to control sample, and R.E., relative changes in expression compared to control sample. <i>B</i>, PPAR-γ ligands contain electrophilic carbons. Here, positions of the electrophilic carbons in the structures of the compounds are marked. CAY10410 and PGA₁ are structural analogues of 15-d-PGJ₂. PGA₁ has an electrophilic center but CAY10410 does not. DSPS, like CDDO, has two electrophilic centers. Cells were pre-treated with CAY10410 (5µM), PGA₁ (10µM) and DSPS (10µM) for 30 minutes <i>C</i>, only compounds with an electrophilic carbon are able to reduce Akt phosphorylation, indicating that presence of an electrophilic carbon is essential for the observed reduction in the phosphorylation of Akt. All the experiments were performed in triplicate and representative images are shown here.</p

Daily rhythms of plasma CORT and 5HT in mice exposed to acute (3 day) air or CS.

<p>Mice were exposed to acute CS during the light phase for 3 days, and plasma samples were collected at every 6-h interval (ZT0, ZT6, ZT12, and ZT18) beginning 24 h after the last exposure. Plasma levels of (A) CORT and (B) 5HT were determined by ELISA. Gray shading indicates the subjective dark phase (ZT12 to ZT24). Data were fit with non-linear regression (multi-order polynomial) analyses. Data are shown as mean ± SEM (n = 3–7 mice per group). **<i>P</i><0.01 significant compared to CS-exposed mice at ZT6.</p

Daily rhythms of plasma CORT and 5HT in mice exposed to chronic (6 month) air or CS.

<p>Mice were exposed to chronic CS for 6 months and plasma samples were collected every 6-h interval (ZT0, ZT6, ZT12, ZT18 and ZT24) after 24 h of last CS exposure. Plasma levels of (A) CORT and (B) 5HT and were determined by ELISA. Gray shading indicates the dark phase (ZT12 to ZT24). Data from air- and CS-exposed mice are shown as mean ± SEM (n = 3–4 mice per group). Data were analyzed with non-linear regression (multi-order polynomial) analyses. *<i>P</i><0.05 significant compared to air-exposed mice at ZT0; *<i>P</i><0.05 significant compared to CS-exposed mice at ZT24; ***<i>P</i><0.001 significant compared to CS-exposed mice at ZT18.</p

Inhibition of PI3K-Akt pathway by LY294002 inhibits myofibroblast differentiation.

<p>Primary HLFs were treated with the PI3K inhibitor LY294002 (50µM) followed by TGFβ (5ng/ml) for 48 hours and <i>A</i>, immunoblots were performed to detect expression of the indicated proteins, and <i>B</i>, immunofluorescence for αSMA (green) was performed to assess the effects of PI3K inhibition on TGFβ-induced myofibroblast differentiation. DAPI (blue) was used to visualize nuclei. <i>C</i>, HLF cells were transfected with an empty vector or a dominant negative kinase-dead (KD) Akt construct, treated with TGFβ, and assayed for myofibroblast differentiation by Western blot. Protein lysates from all the indicated samples were electrophoretically separated on the same gel, and representative lanes from a single experiment are shown here. These data indicate that a functional PI3K-Akt pathway is essential for the TGFβ-induced myofibroblast differentiation in primary human lung fibroblast.</p

Plasma concentration of CORT and 5HT in non-smokers, smokers and patients with COPD.

<p>(A) The level of plasma CORT increased slightly in smokers but was significantly reduced on patients with COPD relative to non-smokers and smokers (current/ex-smokers). (B) COPD patients show lower 5HT concentration than non-smokers and smokers. The level of plasma 5HT was not affected in smokers when compared with non-smokers. Data are shown as mean ± SEM (n = 10–12 subjects per group). **<i>P</i><0.01 significant when non-smokers were compared to patients with COPD; ^{# #}<i>P</i><0.01 significant when smokers were compared to patients with COPD.</p

Influence of age on daily rhythms of plasma 5HT in air or CS-exposed mice.

<p>Adult mice (8–10 weeks old at start of CS exposure) were exposed to air or CS for 3 days (young), 10 days (young) and 6 months (middle aged). Plasma levels of 5HT were determined by ELISA. Gray shading indicates the dark phase (ZT12 and ZT18). Data from air- and CS-exposed mice are shown as mean ± SEM (n = 3–7 mice per group). Data were analyzed with non-linear regression (multi-order polynomial) analyses. *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001 significant compared to 3 days air- (ZT0–ZT18) or CS- (ZT6) exposed mice; ^{+ +}<i>P</i><0.01; ^{+ + +}<i>P</i><0.001 significant compared to 3 days air-exposed mice (ZT0–ZT18); ^{# #}<i>P</i><0.01; ^{# # #}<i>P</i><0.001 significant compared to 10 days air- (ZT0) or CS- (ZT6) exposed mice.</p

Influence of age on daily rhythms of plasma CORT in air or CS-exposed mice.

<p>Adult mice (8–10 weeks old at start of CS exposure) were exposed to air or CS for 3 days (young), 10 days (young) and 6 months (middle aged). Plasma levels of CORT were determined by ELISA. Gray shading indicates the subjective dark phase (ZT12 and ZT18). Data from air- and CS-exposed mice are shown as mean ± SEM (n = 3–7 mice per group). Data were analyzed with non-linear regression (multi-order polynomial) analyses. ⁺<i>P</i><0.05; ^{+ + +}<i>P</i><0.001 significant compared to 3 days air- or CS-exposed mice; ^{# #}<i>P</i><0.01; ^{# # #}<i>P</i><0.001 significant compared to 10 days air-exposed mice.</p

PPAR-γ ligands inhibit TGFβ-induced phosphorylation of Akt and FAK but not MAPK-p38 and PTEN.

<p>Primary HLFs were pretreated with PPAR-γ ligands; CDDO (1µM) and 15d-PGJ₂ (5µM) followed by TGFβ (5ng/ml). Cells were harvested and lysates analyzed by immunoblots at the indicated time. The ratio of phospho-protein to total protein was measured by densitometric analysis and normalized to untreated cells (untreated = 1.0). TGFβ-induced phosphorylation of <i>A</i>, Akt^S473 and <i>D</i>, FAK^Y397 was inhibited significantly by the PPAR-γ ligands but the phosphorylation of <i>B</i>, PTEN^T308 and <i>C</i>, p38-MAPK ^T180/Y182 was not affected. The statistical significance over TGFβ-treatment alone was calculated either by one way ANOVA on triplicate samples (A, B and C) or using unpaired t-test on duplicate samples (D) and is indicated as * where P≤0.05.</p

A proposed model showing the mechanism of action of electrophilic PPAR-γ ligands on TGFβ-induced myofibroblast differentiation.

<p>TGFβ induces myofibroblast differentiation by activating SMAD, FAK and PI3K-Akt pathways. However, PPAR-γ ligands inhibit the TGFβ-induced PI3K-Akt pathway, partly by targeting FAK induced activation of Akt.</p