TGFβ signaling in primary mouse fibroblasts from different sites of lung.

<p>A. Expression of phosphorylated and nonphosphorylated Smad2 protein, as a measure of TGFβ signaling, and Smad4 in primary fibroblasts. B. Densitometry for pSmad2/Smad2 and Smad4. Ratios of pSmad2/Smad2 are higher in PF compared to LAF and MAF and are increased by cigarette smoke as well as exogenous TGFβ. The findings suggest that PF have higher levels of intrinsic TGFβ signaling and that smoke leads to TGFβ release. C-Control - fibroblasts from animals exposed to air; S- Smoke - fibroblasts from animals exposed to cigarette smoke. Data are mean+/−SD.</p

Gene expression of Thy-1.

<p>Thy-1 is involved in control of fibroblast to myofibroblast differentiation. Basal levels are not statistically different among the 3 sample sites, but cigarette smoke tends to drive expression down in MAF and up in PF, a process that might eventually lead to failure of PF to produce matrix (see text). Control - fibroblasts from animals exposed to air; Smoke – fibroblasts from animals exposed to cigarette smoke. Data are mean+/−SD. *P<0.05.</p

Expression of TGFβ signaling related genes.

<p>Gene expression for CTGF, a downstream mediator of TGFβ signaling, and for TGFβ receptors 1 (Alk5), 2, and 3. Smoke increases CTGF expression in PF, suggesting that smoke acts to release TGFβ. TGFβ receptor gene expression is quite variable but tends to be highest in MAF. Smoke and exogenous TGFβ tend to decrease expression of all 3 receptors. Data are mean+/−SD. *P<0.05, **P<0.01, ***P<0.001.</p

Expression levels of genes for matrix proteins.

<p>Cells are derived from air (Control) or smoke exposed (Smoke) animals and some have been exposed to exogenous TGFβ1 <i>in vitro.</i> Gene expression varies considerably among the three sites. Decorin is the only gene which is much more highly expressed in PF in the basal state. Smoke increases expression of procollagen 1α1 and decreases expression of decorin. Although there is site to site and gene to gene variability, exogenous TGFβ1 has relatively little effect (except for procollagen 1α1 and fibrillin-1 in MAF, and the combination of smoke plus TGFβ1 on elastin expression in MAF) suggesting that PF in particular normally have high intrinsic TGFβ signaling. Overall, these data show that PF can repair by producing matrix molecules after a single <i>in vivo</i> smoke exposure. *P<0.05, **P<0.01, ***P<0.001. Data are mean+/−SD.</p

Primary cells from mouse lung display the morphology and phenotype of fibroblasts.

<p>A. Appearance of fibroblasts from LAF, MAF, and PF exposed to air (control) or smoke. All are morphologically similar and their appearance is not altered by smoke exposure. B. Representative western blot of lineage markers. All lines express the fibroblast/myofibroblast markers smooth muscle actin (SMA) and vimentin and are negative for the hematopoietic marker CD45, the epithelial markers e-cadherin and cytokeratins. MCF-7 and Jurkat cells are included as positive epithelial and hematopoietic controls. C. Quantification of Western blot results for SMA . Data are mean+/−SD. *P<0.05 D. Immunofluorescent staining for F-actin and SMA showing prominent stress fibers and the typical microfilament array of fibroblasts.</p

Wound healing assay.

<p>A. Representative wound healing images at 0, 16, and 38 hours. Wounds were made with a pipette tip in confluent monolayers. Wound healing rates decrease from LAF to MAF to PF and are decreased by smoke exposure in cells from each site. B. Quantification of wound healing rates. Data are mean+/−SD. *P<0.05, **P<0.01, ***P<0.001.</p

Measures of emphysema.

<p>1A: Mean airspace size is increased by a similar amount in young and old animals. 1B. Surface to volume ratio is decreased by a similar amount in young and old animals. Data are mean ± SD. N = 5 to 7 animals/group. P values as shown.</p

Comparison of telomere length between smoked and control mice segregated by age.

<p>Telomere length is greatest in young control animals and is decreased by smoke. Telomere length is also decreased by age but not affected by smoke in old animals. Data are shown as median and interquartile range (box). N = 5 animals/group. * P = 0.0055; † P = 0.0452.</p

Gene expression of MIP-2 in laser capture microdissected airways (bronchioles) (13A) and parenchyma (13B).

<p>MIP-2 is a neutrophil chemoattractant and is upregulated by smoke in both young and old animals. Data are mean ± SD. N = 5 animals/group. P values as shown.</p

Gene expression of GM-CSF in laser capture microdissected airways (bronchioles) (11A) and parenchyma (11B).

<p>GM-CSF acts as an inflammatory cell chemoattractant in the lung and is upregulated by smoke in young animals, but not affected by smoke in old animals. Data are mean ± SD. N = 5 animals/group. P values as shown.</p