Muscularization of small pulmonary arteries.

<p><b>A</b>: Representative images of α-SMA immunostaining of non-muscular (NM), partially muscularized (PM) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032468#pone.0032468-McCormack1" target="_blank">[53]</a>, and fully muscularized (FM) small arterioles used to determine percent muscularization of vessels after HDM exposure (40×). <b>B</b>: WT and Bmpr2 ΔE2 (ΔE2) mice demonstrated increases in the number of arterioles that were fully muscularized (FM) and fewer arterioles that were non-muscular (NM) compared to saline controls after 7 weeks (wk) HDM (left). The percentage of FM arterioles was further enhanced after 20 weeks of HDM and the proportion of NM arterioles was further reduced. No differences were detected between groups of WT and Bmpr2 ΔE2 mice exposed to saline or HDM (n = 5–13 mice/group). *P<0.05 vs saline control, ∧P<0.05 vs ΔE2 saline PM.</p

Airway hyperreactivity following chronic HDM exposure.

<p>Bmpr2 ΔE2 mice developed more severe airway respiratory resistance to methacholine after 20 weeks of HDM compared to WT mice (n = 4–12 mice/group). Both HDM groups demonstrated increased airway resistance with methacholine exposure compared to saline controls. *P<0.05 vs saline controls, ∧P<0.05 vs WT HDM.</p

Genotype analysis of Bmpr2 hypomorph allele and levels of P-Smad1/5 in HDM exposed mice.

<p><b>A</b>: Tail DNA from mice carrying the wild-type (WT) and/or the Bmpr2 hypomorph (ΔE2) alleles were genotyped by PCR using the primers Bmpr2 MT Neo R1 to detect the hypomorph allele (ΔE2 450 bp) and Bmpr2 WT R2 to detect the WT allele (WT 230 bp). PCR products were run on a 4% agarose gel containing ethidium bromide and photographed. <b>B</b>: Western blot analysis of P-Smad1/5, a downstream mediator of BMPR-II signaling, was decreased by approximately 50% after HDM exposure compared to saline controls (<i>n</i> = 5 mice/group). *P<0.05 vs saline.</p

Pulmonary arterial hypertension following chronic HDM exposure.

<p><b>A</b>: Right ventricular systolic pressures (RVSP) was increased in both WT and Bmpr2 ΔE2 (ΔE2) mice after HDM exposure for 20 weeks compared to saline controls. There was no difference in RVSP between WT and Bmpr2 ΔE2 mice exposed to HDM (n = 4–11 mice/group in two independent experiments). *P<0.05 vs saline control. <b>B</b>: The ratio of RV weight to LV+S weight was only increased in Bmpr2 ΔE2 mice compared to saline controls (n = 4–11 mice/group in two independent experiments). *P<0.05 vs saline control.</p

Allergic sensitization in HDM exposed mice.

<p>Levels of HDM specific IgG1 and IgE were increased in both WT and Bmpr2 ΔE2 mice after 7 weeks (top panels) and 20 weeks (bottom panels) of HDM exposure. At 7 weeks, HDM specific IgG1 levels in WT mice were slightly higher compared to Bmpr2 ΔE2 mice (n = 4–12 mice/group). *P<0.05 vs saline control, ∧P<0.05 vs HDM WT 7 weeks.</p

Inflammatory cell response in HDM exposed mice.

<p><b>A</b>: Total inflammatory cell counts were increased in BALF in both WT (white bars) and Bmpr2 ΔE2 (ΔE2; black bars) mice after 7 weeks (wk) and 20 weeks of HDM (n = 4–13 mice/group in two independent experiments). *P<0.05 vs saline control, ∧P<0.05 vs saline WT 20 weeks. <b>B</b>: Representative images of cytospins from BALF demonstrating specific inflammatory cell types in the lung after 7 and 20 weeks of saline or HDM exposure. The inset in the top panel shows a high power image of an eosinophil and the bottom panel inset is a representative image of a neutrophil. M = Macrophage, L = Lymphocyte, N = Neutrophil, and E = Eosinophil. Scale bar larger panels = 20 µm. Insets scale bar = 10 µm. <b>C</b>: Changes in macrophage cell numbers were only observed after 20 weeks of HDM and only in BMPR2 ΔE2 mice. Neutrophils and eosinophil numbers were increased in HDM exposed groups at both time points, however, the number of eosinophils was lower after 20 weeks. *P<0.05 vs saline control, ∧P<0.05 vs saline ΔE2 lymphocyte 20 weeks, #P<0.05 vs saline WT macrophage 20 weeks, +P<0.05 vs HDM WT macrophages 20 weeks, @P<0.05 vs HDM WT neutrophil 20 weeks.</p

Vascular remodeling and arterial wall thickness following chronic HDM exposure.

<p><b>A</b>: α-Smooth muscle actin (α-SMA) staining was performed on lung sections from WT (top) and Bmpr2 ΔE2 (ΔE2; bottom) mice exposed to saline or HDM for 7 or 20 weeks (wk). Thickening of the arterial wall (medial layer) in HDM exposed mice is seen (arrows) at both 7 and 20 weeks. Scale bar larger panels = 200 µm. Insets scale bar = 50 µm. <b>B</b>: Image of α-SMA immunostaining of a pulmonary artery demonstrating the method used to measure arterial wall thickness. For each vessel, external diameter and two wall thicknesses were measured along two different axes. Percent wall thickness was calculated as [(1a+1b)/external diameter]×100. This measurement was performed along each axis. The two axes measurements were averaged together to determine the final wall thickness. <b>C</b>: Percent wall thickness of pulmonary arteries (20–150 µm) was increased 1.5 fold in HDM exposed mice after 7 weeks (wk) and 2 fold in mice exposed to HDM for 20 weeks. Differences between WT (white bars) and Bmpr2 ΔE2 (ΔE2) (black bars) mice exposed to saline or HDM were not significant (n = 5–13 mice/group). *P<0.05 vs saline control.</p