Expression of cytokines and extracellular matrix proteins in the right ventricle: effects of the hypomorphic BMPR2 transgene and antigen exposure.

<p>Bar graphs show means and standard error of the means (SEM) of mRNA expression in right ventricles relative (rel.) to β-actin (×10,000) or ratio of expression of cytokine to cytokine receptor. Data are shown for the cytokines RELMα (<b>A</b>), RELMγ (<b>B</b>), and IL-33 (<b>C</b>); the IL-33 receptor ST2-IL1RL1 (<b>D</b>); the ratio of IL-33 to ST2-IL1RL1 expression (<b>E</b>); and the extracellular matrix protein biglycan (<b>F</b>). Data of individual mice are represented as dots. Data were pooled from two independent experiments (n = 6 to 12 per group). Pair-wise comparisons between groups were calculated with the Mann-Whitney U test (p<0.05; a: transgene-negative saline (PBS) vs. antigen (OVA); b: transgene-positive saline vs. antigen; c: saline exposed transgene-negative vs. transgene–positive; d: antigen exposed transgene-negative vs. transgene-positive).</p

Body and heart weights: effects of the hypomorphic BMPR2 transgene and antigen exposure.

<p>(<b>A, B</b>) Diamond mean comparison plots show group means, upper and lower 95% confidence limits, overlap marks, for body weight (<b>A</b>) and right ventricular weight relative to the weight of the left ventricle and septum, RV/LV+S (<b>B</b>). Data are shown for groups of saline (PBS) versus antigen (OVA) exposed mice stratified by transgene-negative (tg−) versus -positive (tg+). The dashed line represents the overall mean. Dots represent data from individual mice. Data were pooled from three independent experiments (n = 7 to 19). Pair-wise comparisons were performed with the Mann-Whitney U test (b: transgene-positive saline vs. antigen, p<0.05). (<b>C–F</b>) Dot plots show individual data points for right ventricular systolic pressure (RVSP, mmHg) plotted against body weight (<b>C</b>), right ventricular weight relative to the weight of the left ventricle and septum (<b>D</b>), right ventricular weight relative to body weight (<b>E</b>), and weight of left ventricle and septum relative to body weight (<b>F</b>). Data were pooled from three independent experiments (n = 7 to 18 per group). Combined data from saline (PBS) or antigen (OVA) exposed groups of transgene-negative or trangene-positive mice (indicated by color code) were analyzed with the Spearman's Rank Order Correlation Coefficient test (p- and r- values are indicated).</p

Expression of BMPR2^R899X transgene, experimental schedule and groups of animals studied.

<p>(<b>A</b>) Notched box and whisker plots extending to the 10^th and 90^th percentiles showing expression of mRNA for transgenic BMPR2^R899X (relative to β-actin; ×10,000) in heart tissues from animals aged 1 to 85 days old and transgene-negative (tg−) controls (n = 3–12). Data of individual mice are represented as dots. At 85 days old, mice were from the saline (PBS) or antigen (OVA) exposed groups as indicated. There were no statistically significant differences between pairs of groups (Mann Whitney U test, p>0.05). (<b>B</b>) Schematic representation of the experimental schedule for antigen (Ovalbumin, OVA) priming by intraperitoneal (i.p.) injections of OVA-Alum followed by intranasal (i.n.) challenge with soluble OVA. Control animals were given saline (PBS). (<b>C</b>) Study groups of animals as defined by BMPR2^R899X transgene expression and exposure to antigen or saline.</p

Histological changes in the lungs: effects of the hypomorphic BMPR2 transgene and antigen exposure.

<p>(<b>A–D</b>) Notched box and whisker plots extending to the 10^th and 90^th percentiles showing scores (1-normal to 4-severe change) for (<b>A</b>) pulmonary arterial remodeling, (<b>B</b>) perivascular inflammation, (<b>C</b>) alveolar inflammation, and (<b>D</b>) goblet cells. Data of individual mice are represented as dots. Data were pooled from three independent experiments (n = 7 to 20 per group). Pair-wise comparisons between groups were calculated with the Mann-Whitney U test (p<0.05; a: transgene-negative saline (PBS) vs. antigen (OVA); b: transgene-positive saline vs. antigen; d: antigen exposed transgene-negative vs. transgene-positive). (<b>E–H</b>) Scanned representative images show sections of lungs from saline, transgene-negative (<b>E</b>), saline, transgene-positive (<b>F</b>), antigen, transgene-negative (<b>G</b>), or antigen, transgene-positive (<b>H</b>) mice. The images of the antigen exposed mice show pulmonary arterial remodeling, perivascular inflammation and alveolar inflammation. For reference the airways (airway) and pulmonary arteries (*) are indicated; the scale bar represents 100 µm. Sections were stained with hematoxylin and eosin.</p

Sequences of Primers and Probes.

1<p>Abbreviations: F- forward, R-reverse.</p

Inflammatory cell responses in the airways: effects of the hypomorphic BMPR2 transgene and antigen exposure.

<p>Bar graphs show means and standard error of the means (SEM) for cellular analysis of bronchoalveolar lavage (BAL): numbers per sample of (<b>A</b>) neutrophils, (<b>B</b>) eosinophils, (<b>C</b>) lymphocytes (sum of CD4+ and CD8+ cells) and (<b>D</b>) MHCII expression by CD11c+ cells (mean fluorescence intensity, MFI). Data of individual mice are represented as dots. Data were pooled from three independent experiments (n = 7 to 20 per group). Pair-wise comparisons between groups were calculated with the Mann-Whitney U test (p<0.05; a: transgene-negative saline (PBS) vs. antigen (OVA); b: transgene-positive saline vs. antigen; c: saline exposed transgene-negative vs. transgene-positive).</p

Increased right ventricular systolic pressures in mice that express the hypomorphic BMPR2 transgene and are exposed to antigen.

<p>(<b>A</b>) Diamond mean comparison plots show group means, upper and lower 95% confidence limits, overlap marks, for right ventricular systolic pressures (RVSP, mmHg) of saline (PBS) versus antigen (OVA) exposed mice stratified by transgene-negative (tg−) versus -positive (tg+). The dashed line represents the overall mean. Dots represent data from individual mice. Data were pooled from three independent experiments (n = 7 to 18). Pair-wise comparisons between groups were calculated with the Mann-Whitney U test (p<0.05; b: transgene-positive saline vs. antigen). (<b>B</b>) Bar graphs show the frequency of RVSP measurements that were lower or higher than 26 mmHg for each of the groups of animals. Chi-Square analysis of the data distribution between all groups was p = 0.0039; pair-wise comparisons calculated p<0.05 for a: transgene-positive antigen (OVA) vs. transgene negative saline (PBS); b: transgene-positive antigen vs. transgene-positive saline).</p

Muscularization of small blood vessels in the lungs: effects of the hypomorphic BMPR2 transgene and antigen exposure.

<p><b>A</b>) Stacked bar graphs represent the percentage of non-muscularized, partially muscularized, and fully muscularized blood vessels that are 50–100, or <50 µm in diameter. <b>B</b>) Stacked bar graphs represent the percentage of bloods vessels (50–100 µm in diameter) for which the smooth muscle actin positive area was <10%, 10–30%, or >30% relative to the area of the total vessel. Data were pooled from two independent experiments (n = 4–9 per group with 60–1000, mean 400 vessels analyzed per mouse). Pair-wise comparisons were calculated with the t test with Welch's correction (p<0.05; a: transgene-negative saline (PBS) vs. antigen (OVA); b: transgene-positive saline vs. antigen; c: saline exposed transgene negative vs. transgene-positive). (<b>C, D</b>) Individual data points from two independent experiments (n = 4–7 per group) are shown. Each data point plots the percentage of fully muscularized blood vessels sized 50–100 µm (<b>C</b>) or <50 µm (<b>D</b>) in diameter against the right systolic ventricular pressure (RVSP, mmHg). Combined data from saline or antigen exposed groups of transgene-negative or trangene-positive mice (indicated by color code) were analyzed with the Spearman's Rank Order Correlation Coefficient test (p- and r- values are indicated).</p

Experimental Design.

<p>Schematic representation of the timing of sensitization by intraperitoneal injection of OVA-Alum; intranasal administration of saline or antigen and PM_2.5 (OVA & PM_2.5); and the antibody injections administered intraperitoneally.</p

List of markers, their regulation, function related to tissue remodeling, and relation to T helper responses.

<p>List of markers, their regulation, function related to tissue remodeling, and relation to T helper responses.</p