p53-mediated induction of PAI-1 expression contributes to increased pulmonary MPO levels in mice with PCSE.

<p>(<b>A</b>) WT mice were exposed to ambient air (control) or PCS (n = 5/group) for 20 weeks. LL collected from these mice was subjected to total cell counting. (<b>B</b>) Total protein in the lavage was quantified from the above exposed mice. (<b>C</b>) Paraffin embedded lung sections from WT, p53- and PAI-1-deficient mice (n = 5mice/group) exposed to ambient air (control) or PCS for 20 weeks were subjected to H&E staining. Representative fields from 1 of 3 sections per subject are shown at X 400 magnification. Lung sections were subjected to IHC analysis for neutrophils using anti-MPO antibody and for macrophages using anti-F4/80 antibody. Neutrophils (<b>D</b>) and macrophages (<b>E</b>) were counted in 10 high-power fields (hpf) are shown as bar graph. (<b>F</b>) Lung homogenate from WT, p53- and PAI-1-deficient mice exposed to ambient air or PCS for 20 weeks were immunoblotted for changes in the levels of MPO using anti-MPO antibody. These membranes were later stripped and analyzed for β-actin to assess loading. Data shown in bar graphs are mean ± SD of two independent experiments (n = 5 mice/group). Differences between treatments are statistically significant *(P<0.05).</p

p53 and PAI-1 are prominently linked to PCSE-induced type II AEC apoptosis.

<p>(<b>A</b>) WT, p53- and PAI-1-deficient mice were exposed to ambient air (control) or PCS for 20 weeks. Lung section obtained from these mice were subjected to TUNEL staining and the bar graph represents percent apoptosis in these groups with error bars and significance *(p<0.005) (n = 5 mice/group). (<b>B</b>) Type II AECs isolated from WT, p53- and PAI-1-deficient mice as described in methods were subjected to TUNEL staining and the bar graph represents percent apoptosis in these groups with error bars and significance *(p<0.005) (n = 5 mice/group). (<b>C</b>) Type II AECs isolated from WT, p53- and PAI-1-deficient mice were subjected to flow cytometric analysis after staining with anti-annexin-v antibody and PI to assess apoptosis. NS = the differences are not statistically significant (n = 5 mice/group). (<b>D</b>) Type II AECs isolated from WT and p53- and PAI-1-deficient mice as described above were immunoblotted for SP-C and β-actin as a loading control.</p

Mice lacking PAI-1 expression resist type II AEC apoptosis.

<p>Mice in ambient AIR or exposed to PCS were treated with 50 μl saline or purified IAV in saline via intranasal instillation. One week after IAV infection, the mice were sacrificed. (<b>A</b>) Lung sections were subjected to H & E and IHC staining for M2 antigen. Representative fields from 1 of 3 sections per subject are shown at X 400 magnification (n = 5 mice/group). (<b>B</b>) Lung homogenates were immunoblotted for changes in IAV M2 and active caspase-3antigens to assess severity of IAV infection and apoptosis respectively. The plot represents ratios in the densities of bands normalized against β-actin levels in the same sample (n = 5 mice/group). (<b>C</b>) Lung homogenates were tested for MPO activity by colorimetric assay and represented as a bar graph of two independent experiments (n = 5 mice/group). (<b>D</b>) Lung homogenates from the mice in ambient AIR or exposed to PCS for 20 weeks were immunoblotted for changes in EAR1 with β-actin antibody as a loading control. (<b>E</b>) Total RNA from the mice exposed to AIR or PCS was analyzed for EAR and β-actin mRNA (n = 5 mice/group).</p

Increased PAI-1 expression sensitizes mice to IAV infection and alveolar injury.

<p>(<b>A</b>) Mice (n = 3) treated with saline or 0.5 LD₅₀ of purified mouse-adapted IAV (strain A/PR/8/34) in 50 μl by intranasal instillation. LL fluids were analyzed for PAI-1, and isolated type II AECs lysates were immunoblotted for p53, activation of caspase-3 and β-actin. Data shown in bar graphs are means ± SD of two independent experiments. Differences between treatments are statistically significant *(P<0.05) (n = 3 mice/group). (<b>B</b>) Mice exposed to saline or IAV as described above were analyzed for SP-C and β-actin. Data shown in bar graphs are means ± SD of two independent experiments. Differences between treatments are statistically significant *(P<0.05) (n = 3 mice/group). (<b>C</b>) Lung sections from the mice treated as described above were subjected to IHC analysis for MPO and macrophage antigens, and TUNEL staining to assess inflammation and apoptosis. Neutrophils, macrophages and apoptotic (TUNEL-positive) cells were quantified by counting positive cells in 10 high-powered fields (hpf) are shown as bar graph. (<b>D</b>) WT mice or transgenic mice that over express PAI-1 (PAI-1^+/+) or PAI-1-deficient mice (PAI-1^-/-) were exposed to 50 μl saline or IAV in saline. Lung homogenates were immunoblotted for changes in IAV M2, MPO and active caspase-3 antigen levels to assess severity of IAV infection, inflammation and lung injury. β-actin was tested to gauge similar loading. Bar represents fold changes in the densities of bands (IAV M2) normalized against β-actin levels in the same sample (n = 3 mice/group). (<b>E</b>) Lung homogenates from WT mice or transgenic mice that overexpress PAI-1 (PAI-1^+/+) or PAI-1-deficient mice (PAI-1^-/-) were also tested for MPO activity by colorimetric assay. Data shown in bar graphs are means ± SD of two independent experiments (n = 3 mice/group).</p