Hypoxia-induced Pulmonary Hypertension in Different Mouse Strains: Relation to Transcriptome.

Patients with pulmonary arterial hypertension (PAH) can harbor mutations in several genes, most commonly in BMPR2. However, disease penetrance in patients with BMPR2 mutations is low. In addition, most patients do not carry known PAH gene mutations, suggesting that other factors determine susceptibility to PAH. To begin to identify additional genomic factors contributing to PAH pathogenesis, we exposed 32 mouse strains to chronic hypoxia. We found that the PL/J strain has extremely high right ventricular systolic pressure (RVSP; 86.58 mm Hg) but minimal lung remodeling. To identify potential genomic factors contributing to the high RVSP, RNAseq analysis of PL/J lung mRNAs and microRNAs (miRNAs) after hypoxia was performed, and it demonstrated that 4 of 43 upregulated miRNAs in the Dlk1-Dio3 imprinting region are predicted to target T cell marker mRNAs. These target mRNAs, as well as the numbers of T cells were downregulated. In addition, C5a and its receptor, C5AR1, were increased. Analysis of Rho-associated protein kinase (Rock) 2 mRNA expression, in the RhoA/Rock pathway, demonstrated a significant increase in PL/J. Inhibition of Rock2 ameliorated a portion of the elevated RVSP. In addition, we identified miR-150-5p as a potential regulator of Rock2 expression. In conclusion, we identified two possible pathways contributing to the hypoxia pulmonary hypertension phenotype of extreme RVSP elevation: aberrant T cell expression driven by hypoxia-induced miRNAs and increased expression of C5a and C5AR1. We suggest that the PL/J mouse will be a good model for seeking mechanism(s) of RVSP elevation in hypoxia-induced PAH.

Epithelial EGF receptor signaling mediates airway hyperreactivity and remodeling in a mouse model of chronic asthma.

Increases in the epidermal growth factor receptor (EGFR) have been associated with the severity of airway thickening in chronic asthmatic subjects, and EGFR signaling is induced by asthma-related cytokines and inflammation. The goal of this study was to determine the role of EGFR signaling in a chronic allergic model of asthma and specifically in epithelial cells, which are increasingly recognized as playing an important role in asthma. EGFR activation was assessed in mice treated with intranasal house dust mite (HDM) for 3 wk. EGFR signaling was inhibited in mice treated with HDM for 6 wk, by using either the drug erlotinib or a genetic approach that utilizes transgenic mice expressing a mutant dominant negative epidermal growth factor receptor in the lung epithelium (EGFR-M mice). Airway hyperreactivity (AHR) was assessed by use of a flexiVent system after increasing doses of nebulized methacholine. Airway smooth muscle (ASM) thickening was measured by morphometric analysis. Sensitization to HDM (IgG and IgE), inflammatory cells, and goblet cell changes were also assessed. Increased EGFR activation was detected in HDM-treated mice, including in bronchiolar epithelial cells. In mice exposed to HDM for 6 wk, AHR and ASM thickening were reduced after erlotinib treatment and in EGFR-M mice. Sensitization to HDM and inflammatory cell counts were similar in all groups, except neutrophil counts, which were lower in the EGFR-M mice. Goblet cell metaplasia with HDM treatment was reduced by erlotinib, but not in EGFR-M transgenic mice. This study demonstrates that EGFR signaling, especially in the airway epithelium, plays an important role in mediating AHR and remodeling in a chronic allergic asthma model.

Early growth response-1 suppresses epidermal growth factor receptor-mediated airway hyperresponsiveness and lung remodeling in mice.

Transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor receptor, are induced after lung injury and are associated with remodeling in chronic pulmonary diseases, such as pulmonary fibrosis and asthma. Expression of TGF-alpha in the lungs of adult mice causes fibrosis, pleural thickening, and pulmonary hypertension, in addition to increased expression of a transcription factor, early growth response-1 (Egr-1). Egr-1 was increased in airway smooth muscle (ASM) and the vascular adventitia in the lungs of mice conditionally expressing TGF-alpha in airway epithelium (Clara cell secretory protein-rtTA(+/-)/[tetO](7)-TGF-alpha(+/-)). The goal of this study was to determine the role of Egr-1 in TGF-alpha-induced lung disease. To accomplish this, TGF-alpha-transgenic mice were crossed to Egr-1 knockout (Egr-1(ko/ko)) mice. The lack of Egr-1 markedly increased the severity of TGF-alpha-induced pulmonary disease, dramatically enhancing airway muscularization, increasing pulmonary fibrosis, and causing greater airway hyperresponsiveness to methacholine. Smooth muscle hyperplasia, not hypertrophy, caused the ASM thickening in the absence of Egr-1. No detectable increases in pulmonary inflammation were found. In addition to the airway remodeling disease, vascular remodeling and pulmonary hypertension were also more severe in Egr-1(ko/ko) mice. Thus, Egr-1 acts to suppress epidermal growth factor receptor-mediated airway and vascular muscularization, fibrosis, and airway hyperresponsiveness in the absence of inflammation. This provides a unique model to study the processes causing pulmonary fibrosis and ASM thickening without the complicating effects of inflammation.

Vascular growth and remodeling in compensatory lung growth following right lobectomy.

Studies in animal models have shown that, following lobectomy (LBX), there is compensatory growth in the remaining lung. The vascular growth response following right LBX (R-LBX) is poorly understood. To test the hypothesis that arterial growth and remodeling occur in response to LBX, in proportion to the amount of right lung tissue removed, two (24% of lung mass; R-LBX2 group) or three right lobes (52% of lung mass; R-LBX3 group) were removed via thoracotomy from adult rats. Sham control animals underwent thoracotomy only. Arteriograms were generated 3 wk after surgery. The areas of the left lung arteriogram, arterial branching, length of arterial branches, arterial density, and arterial-to-alveolar ratios were measured. To determine whether R-LBX causes vascular remodeling and pulmonary hypertension, muscularization of arterioles and right ventricular hypertrophy were assessed. Lung weight and volume indexes were greater in R-LBX3. Arterial area of the left lung increased 26% in R-LBX2 and 47% in R-LBX3. The length of large arteries increased in R-LBX3 and to a lesser extent in R-LBX2. The ratio of distal pulmonary arteries to alveoli was similar after R-LBX2 compared with sham but was 30% lower in R-LBX3. Muscularization of arterioles increased after R-LBX3, but not in R-LBX2. Right ventricular hypertrophy increased 50-70% in R-LBX3, but not in R-LBX2. Whereas removal of three right lung lobes induced arterial growth in the left lungs of adult rats, which was proportionate to the number of lobes removed, the ratio of distal pulmonary arteries to alveoli was not normal, and vascular remodeling and pulmonary hypertension developed.

Rapamycin decreases airway remodeling and hyperreactivity in a transgenic model of noninflammatory lung disease.

Airway hyperreactivity (AHR) and remodeling are cardinal features of asthma and chronic obstructive pulmonary disease. New therapeutic targets are needed as some patients are refractory to current therapies and develop progressive airway remodeling and worsening AHR. The mammalian target of rapamycin (mTOR) is a key regulator of cellular proliferation and survival. Treatment with the mTOR inhibitor rapamycin inhibits inflammation and AHR in allergic asthma models, but it is unclear if rapamycin can directly inhibit airway remodeling and AHR, or whether its therapeutic effects are entirely mediated through immunosuppression. To address this question, we utilized transforming growth factor-α (TGF-α) transgenic mice null for the transcription factor early growth response-1 (Egr-1) (TGF-α Tg/Egr-1(ko/ko) mice). These mice develop airway smooth muscle thickening and AHR in the absence of altered lung inflammation, as previously reported. In this study, TGF-α Tg/Egr-1(ko/ko) mice lost body weight and developed severe AHR after 3 wk of lung-specific TGF-α induction. Rapamycin treatment prevented body weight loss, airway wall thickening, abnormal lung mechanics, and increases in airway resistance to methacholine after 3 wk of TGF-α induction. Increases in tissue damping and airway elastance were also attenuated in transgenic mice treated with rapamycin. TGF-α/Egr-1(ko/ko) mice on doxycycline for 8 wk developed severe airway remodeling. Immunostaining for α-smooth muscle actin and morphometric analysis showed that rapamycin treatment prevented airway smooth muscle thickening around small airways. Pentachrome staining, assessments of lung collagen and fibronectin mRNA levels, indicated that rapamycin also attenuated fibrotic pathways induced by TGF-α expression for 8 wk. Thus rapamycin reduced airway remodeling and AHR, demonstrating an important role for mTOR signaling in TGF-α-induced/EGF receptor-mediated reactive airway disease.