Chronic Mixed-Allergen Exposure Induces Pulmonary Arterial Remodeling and Vascular Dysfunction in Mice

Abstract

Chronic asthma can be associated with secondary pulmonary vascular dysfunction, however, the detailed functional profiling of the asthmatic pulmonary artery remains understudied. In this study, we characterized cardiopulmonary and vascular dysfunction in a chronic mixed allergen (MA) mouse model of asthma. Wild-type mice were intranasally challenged with MA or PBS for four weeks, followed by in vivo respiratory mechanics (flexiVent), ex vivo pulmonary artery wire myography, structural morphometry, and right ventricular analysis (Fulton Index). Chronic MA exposure induced significant airway hyperresponsiveness, evidenced by elevated airway resistance (Rrs) and elastance (Ers) with reduced compliance (Crs). Lung histology revealed increased pulmonary artery medial wall thickness and smooth muscle hyperplasia. Wire myography demonstrated enhanced serotonin (5-HT)-induced vasoconstriction and endothelial dysfunction in MA mice, evidenced by blunted endothelium-dependent relaxation to acetylcholine (ACh) with preserved endothelium-independent relaxation to sodium nitroprusside (SNP). Lastly MA mice had significant right ventricular hypertrophy by Fulton index. Collectively, chronic allergic airway inflammation drives vascular remodeling, endothelial dysfunction, and smooth muscle hyperreactivity in pulmonary arteries. This study demonstrates substantial structural and functional consequences of MA exposure to the pulmonary vasculature that models asthmatic pulmonary vascular dysfunction.

New & Noteworthy

Chronic mixed-allergen exposure causes not only airway hyperresponsiveness but also strong pulmonary vascular remodeling and dysfunction. Using a mouse model, we show increased vasoconstriction, endothelial dysfunction, and right ventricular hypertrophy. These findings highlight the bronchovascular axis as an important site of disease interaction and provide a platform to unveil localized mechanisms and therapeutic targets linking asthma to pulmonary vascular disease.

Graphical Abstract

1. Introduction

In asthma, chronic inflammation causes structural changes in the airways, including smooth muscle hypertrophy and hyperplasia, subepithelial fibrosis, and goblet cell hyperplasia, collectively termed airway remodeling (1–3). Airway structural and functional changes have been widely studied in asthma, but there is less data on impacts of asthma on the pulmonary vasculature. Mice exposed to 7 weeks of house dust mite extract have increased pulmonary vascular collagen deposition, endothelial and smooth muscle hyperplasia, and the presence of proliferating myofibroblasts in the medial wall (4). A comparison of nontranspantable donor lungs from patients with and without asthma found increased pulmonary artery intimal and adventitial thickness and increased vascular collagen deposition in patients with asthma (5). Even less data is available on the prevalence of clinically significant pulmonary vascular disease in asthma. An analysis of United States hospital admissions from 2016–2020 with the primary diagnosis of asthma found that about 1.5% of patients also had a diagnosis of pulmonary hypertension (PH), and these patients were more than twice as likely to die during admission review of hospital admissions (6). However, computational estimates of the amount of vascular obliteration required for measurable hemodynamic changes and symptom presentation range from 50% to 70% (7, 8). Thus, the 1.5% of asthma admissions with a PH diagnosis may vastly underestimate the number of asthmatic patients with vascular remodeling. This gap in knowledge presents a need for more robust models to characterize the functional consequences of asthma on the pulmonary vasculature.

Anatomically, the pulmonary arterial tree undergoes branching morphogenesis to provide matching circulation to the conducting airways, which together form the bronchovascular bundle (9). During chronic asthma, this structural proximity exposes the pulmonary vasculature to a sustained inflammatory environment (10, 11). This inflammatory environment induces hypertrophy and hyperplasia of pulmonary vascular smooth muscle cells, termed vascular remodeling (10–12). Previous studies utilizing allergic models have reported histological evidence of vascular medial wall thickening and inflammatory cell infiltration (13, 14). Others have established that allergic airway inflammation can drive pulmonary vascular remodeling and hyperresponsiveness. Hogaboam and colleagues demonstrated that a Th2 immune response induces distinct pulmonary arterial remodeling (15), while Grunig and colleagues showed that inhaled antigen coupled with fine particulate matter drives an IL-13- and IL-17A-dependent PH phenotype (16). Furthermore, experiments by Witzenrath et al. show that allergic lung inflammation induces pulmonary vascular hyperresponsiveness in isolated, perfused lungs (17).

Despite these structural and in vivo findings, gaps remain in our understanding of the consequences of chronic asthma on pulmonary vascular remodeling and dysfunction. To address this gap, we utilized the mixed allergen (MA) mouse model of asthma to characterize asthma-induced cardiopulmonary and vascular dysfunction. The MA model was employed because it induces early persistent inflammation along with structural and functional airway changes as early as 2 weeks, and it produces significant inflammation and airway remodeling in C57BL/6 mice (18, 19). In this study, we integrated in vivo respiratory mechanics (flexiVent) with ex vivo pulmonary artery wire myography, structural morphometry, and right ventricular analysis (Fulton Index). We found that MA mice exhibit vascular remodeling, hyperreactivity, and right ventricular hypertrophy. These findings provide a key foundation for identifying specific local mediators of pulmonary vascular remodeling in asthma and developing targeted therapeutics.

2. Materials and Methods

2.1. Experimental animals

The NIH Guide for the Care and Use of Laboratory Animals (20) was followed for all animal experiments. The study was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Oklahoma Health Campus. Wild-type (WT) C57BL/6J breeding pairs (male and female) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). Animals were bred in-house, and offspring aged 6–8 weeks (20–25 g) were utilized for the experiments. They were housed under controlled environmental conditions, including a 12-hour light/dark cycle and constant temperature, with ad libitum food and water. Both male and female mice were randomly assigned (minimum n = 6 per group) to the following experimental groups: PBS (PBS-challenged) and MA (Mixed allergen-challenged). All animal experiments were conducted and reported in accordance with the ARRIVE guidelines.

2.2. Mixed allergens and reagents

Mixed allergen (MA) components- Alternaria alternata, Aspergillus fumigatus, and Dermatophagoides farinae (house dust mite), were sourced from Greer Laboratories (Lenoir, NC, USA), whereas ovalbumin was purchased from Sigma (St. Louis, MO, USA). The α-SMA antibody was obtained from Cell Signaling Technology (Danvers, MA, USA; Cat# 19245S). Fluorescent secondary antibodies and Dulbecco’s PBS were sourced from Thermo Scientific (Waltham, MA, USA).

2.3. Mixed allergen challenge

Male and female mice were exposed by intranasal administration to MA (10 μg each allergen, suspended in 25 μL PBS), on alternate days for 4 weeks, as described previously (18). Control animals receive 25 μL PBS intranasally on alternate days (3 days/week). All administrations were performed under isoflurane anesthesia. The experimental design is illustrated in Figure 1A.

Figure 1. Chronic mixed allergen (MA) exposure induces parallel airway and cardiovascular dysfunction.

(A) Schematic representation of the experimental design showing MA sensitization followed lung function assessment. (B-D) Lung function parameters including total respiratory system resistance (Rrs), elastance (Ers), and compliance were measured in response to increasing concentrations of methacholine (MCh, 0–50 mg/mL) using the flexiVent. (E-G) Maximal Rrs (Max Rrs), Ers (Max Ers), Crs (Max Crs) at 50 mg/mL MCh was used to compare treatment efficacy. (H-J) Cumulative Area Under the Curve (AUC). (K) Right ventricular hypertrophy quantified gravimetrically via the Fulton Index [RV / (LV + Septum)]. (L) Representative transverse mid-ventricular heart cross-sections stained with H&E. Dose-response curves were analyzed using two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. All other data were analyzed using an unpaired Welch’s t-test. Data are presented as mean ± SD (n = 6 mice per group; biological replicates). In bar graphs, male mice are denoted by circular symbols and female mice by square symbols. *P < 0.05, **P < 0.01, **P < 0.001. RV, right ventricle; LV, left ventricle.

2.4. Lung function analysis

On day 29, pulmonary function was evaluated using the flexiVent FX1 system (Scireq, Montreal, Canada). Mice were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). Respiratory parameters, including total system resistance (Rrs), elastance (Ers), and compliance (Crs) were measured at baseline (0 mg/mL methacholine, MCh) and after exposure to increasing doses of nebulized MCh (6.25 to 50 mg/mL). The maximum values at 50 mg/mL MCh were used for inter-group comparisons (18, 19). Further, the Area Under the Curve (AUC) across the full MCh dose-response range was calculated for Rrs, Ers, and Crs.

2.5. Fulton Index

Following euthanasia, the heart and pulmonary vasculature were perfused in situ with cold PBS injected into the right ventricle (RV) to clear luminal blood. The heart was excised, blotted, and the RV wall was dissected and weighed. The LV and attached interventricular septum (IVS) were then weighed. The Fulton index (FI) was calculated as the ratio of RV weight to the LV + IVS weight (RV/LV + IVS)(21).

2.6. Histopathology analyses

Following euthanasia, the pulmonary vasculature was cleared of blood by in situ perfusion with cold PBS via the right ventricle. Heart and left lung tissues were excised and immersion-fixed in Carnoy’s solution (60% absolute ethanol, 30% chloroform, and 10% glacial acetic acid containing ferric chloride; Sigma-Aldrich) for several hours at room temperature to preserve structural morphometry and antigenicity. Fixed tissues were subsequently transferred to 70% ethanol and dehydrated through graded ethanol series. Paraffin-embedded tissues were sectioned (5 μm) and stained with H&E (Sigma-Aldrich), following standard procedures. Slides were scanned using an Epredia Pannoramic MIDI II (3DHISTECH, Budapest, Hungary). To determine pulmonary vascular remodeling, images were analyzed using ImageJ (NIH, https://imagej.net/software/fiji/). For small intra-acinar pulmonary arteries (<100 μm in external diameter), medial wall thickness was quantified by measuring the medial wall area as a percentage of the total vessel area. This was calculated using the formula: [(Total Vessel Area −Luminal Area) / Total Vessel Area] × 100 (22). In addition to Fulton Index, right ventricular hypertrophy was qualitatively assessed from transverse sections of H&E-stained heart.

2.7. Immunofluorescence

Immunofluorescence staining was conducted as previously described (23, 24). Briefly, 5-μm lung sections were baked at 56 °C for 2 h, then deparaffinized and rehydrated. Tissue sections were permeabilized with 0.1% Triton X-100, blocked using 10% goat serum, and incubated overnight at 4 °C with α-SMA antibody (1:100). Following washes with TBS-T, sections were incubated with Alexa Fluor 488-conjugated secondary antibodies (1:200) for 2 h at room temperature. Nuclei were counterstained with DAPI, and images were acquired using confocal mode on a Cytation C10 imaging system (Agilent BioTek).

2.8. Multi-wire myography

Isolated vascular force studies were performed using a multi-wire myograph system (DMT, Aarhus, Denmark) following the established protocol (24). Following euthanasia, second-order branches of the pulmonary artery were isolated, cleared of surrounding connective tissue and parenchyma, and cut into rings of equal length (~2 mm). The rings were mounted between two tungsten wires in a chamber filled with Krebs-Henseleit (KH) buffer. The buffer was maintained at 37 °C and continuously aerated with 95% O₂-5% CO₂. Rings were stretched to an optimal baseline resting tension (1.5 mN) and allowed to equilibrate for 30 min with buffer exchanges every 10 min. Viability was confirmed via 60 mM KCl contraction. For vasoconstriction studies, rings were exposed to cumulative log-concentrations (10⁻⁹ to 10⁻⁴ M) of serotonin (5-HT). To assess endothelium-dependent vasorelaxation, rings were pre-contracted with a submaximal dose of 5-HT (10⁻⁷ M), followed by the cumulative addition of acetylcholine (ACh; 10⁻⁹ to 10⁻⁴ M). To assess endothelium-independent vasorelaxation, rings were pre-contracted with a submaximal dose of 5-HT, followed by the cumulative addition of sodium nitroprusside (SNP; 10⁻⁹ to 10⁻⁴ M). Isometric tension was continuously recorded using LabChart software (ADInstruments). All contractile responses were normalized as a percentage of the maximum contraction induced by 60 mM KCl.

2.9. Statistical analysis

All experiments were conducted with a minimum of six biological replicates per group, and results are presented as mean ± SD. Statistical analyses were carried out using GraphPad Prism version 10.5.0 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were performed using an unpaired Welch’s t-test, whereas myography dose response data were analyzed via two-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. A p-value < 0.05 was considered statistically significant.

3. Results

3.1. Chronic MA challenge induces airway hyperresponsiveness and right ventricular hypertrophy

To establish the baseline cardiopulmonary phenotype, mice were subjected to a 4-week MA sensitization protocol prior to functional assessment (Figure 1A). Lung function parameters, including Rrs, Ers, and Crs, were measured using the forced oscillation technique on the flexiVent (SCIREQ). In response to increasing concentrations of nebulized methacholine (MCh, 0–50 mg/mL), MA-exposed mice exhibited increased Rrs and Ers, along with concomitant reduction in Crs compared with PBS-challenged controls (Figure 1B–D). This physiological impairment was further confirmed by analyses of maximum response and AUC. MA mice demonstrated significantly increased maximum Rrs (Max Rrs, p<0.001) and Ers (Max Ers, p<0.001), along with reduced maximum Crs (Max Crs, p=0.01) compared with control mice (Figure 1E–G). Consistent with these findings, AUC analysis revealed significantly elevated AUC Rrs (p=0.003) and AUC Ers (p=0.007), whereas AUC Crs (p=0.009) was notably decreased in MA mice (Figure 1H–J). Furthermore, to determine if MA-induced vascular remodeling was associated with right ventricular hypertrophy, we performed Fulton’s Index and visualized RV free wall thickness. MA mice demonstrated significant right ventricular hypertrophy, evidenced by an elevated Fulton Index (Figure 1K, p=0.01) and qualitative thickening of the right ventricular free wall on transverse heart section (Figure 1L).

3.2. Chronic MA challenge induces pulmonary arterial remodeling

Pulmonary artery thickening was assessed using paraffin-embedded lung sections stained with H&E. Compared with control mice, MA mice demonstrated significant increases in medial wall thickness of small, intra-acinar pulmonary arteries (<100 μm diameter; Figure 2A and B). This remodeling was characterized by luminal narrowing associated with significant medial hypertrophy, which was quantitatively confirmed by a significant (p<0.001) increase in the percentage of medial wall thickness (Figure 2A and B). Furthermore, immunohistochemical staining for α-SMA revealed prominent smooth muscle localization throughout the vascular lesions in MA mice (Figure 2C).

Figure 2. Chronic MA challenge induces pulmonary arterial remodeling.

(A) Representative H&E-stained lung sections demonstrating peribronchial pulmonary arteries (<100 μm diameter). (B) Morphometric quantification of the pulmonary artery medial wall thickness, expressed as the percentage of the medial wall area relative to the total vessel area. (C) Representative immunohistochemical staining for α-SMA. Data were analyzed using an unpaired Welch’s t-test. Data are presented as mean ± SD (n = 6 mice per group; biological replicates). In bar graphs, male mice are denoted by circular symbols and female mice by square symbols. ***P < 0.001. PA: Pulmonary artery, Br: Bronchiole, Arrowhead indicates pulmonary arteries.

3.3. Chronic MA challenge promotes hyperreactivity and endothelial dysfunction in isolated pulmonary arteries

To evaluate the effect of chronic inflammation on airway reactivity and endothelial function, second-order branches of the pulmonary artery were isolated (Refer figure 3A) and mounted on a wire myograph. Cumulative concentration-response curves to serotonin (5-HT) demonstrated enhanced vasoconstriction in arteries from MA mice compared to PBS controls (Figure 3B), which was quantitatively confirmed by a significantly elevated maximum contractile response (Figure 3C, p=0.001). To assess endothelium-dependent vasodilation, vessels were pre-contracted with 5-HT and exposed to cumulative doses of acetylcholine (ACh). Arteries from MA mice exhibited significantly blunted endothelium-dependent vasorelaxation (Figure 3D), resulting in a significant decline in maximum percent relaxation (Figure 3E, p<0.001). Importantly, endothelium-independent relaxation in response to cumulative doses of the nitric oxide donor sodium nitroprusside (SNP) remained fully intact and comparable between MA and PBS mice (Figure 3F and G). This confirms that the declined relaxation is primarily due to endothelial dysfunction rather than an inability of the smooth muscle to respond to nitric oxide.

Figure 3. Chronic MA challenge promotes hyperreactivity and endothelial dysfunction in isolated pulmonary arteries.

(A) Representative image of dissected mouse pulmonary artery. The heart is in the upper left, right lung lobes are in the upper right, and the left lung lobes are in the lower left (number indicates branching order). (B) Dose-response curves to cumulative concentrations of serotonin (5-HT). (C) Maximum contractile force generated in response to 5-HT. (D) Endothelium-dependent vasorelaxation curves in response to cumulative concentrations of acetylcholine (ACh) following submaximal 5-HT pre-contraction. (E) Maximum percentage of relaxation achieved with ACh. (F) Endothelium-independent vasorelaxation curves in response to cumulative doses of sodium nitroprusside (SNP). (G) Maximum percentage of relaxation achieved with SNP. Dose-response curves were analyzed using two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test for multiple comparisons. All other data were analyzed using an unpaired Welch’s t-test. Data are presented as mean ± SD (n = 6 mice per group; biological replicates). In bar graphs, male mice are denoted by circular symbols and female mice by square symbols. **P < 0.01, ***P < 0.001.

Discussion

In this study, we provide comprehensive in vivo and ex vivo evidence that chronic MA challenge induces airway hyperresponsiveness, pulmonary arterial remodeling, and right ventricular hypertrophy. By integrating lung mechanics, multi-wire myography functional assays, and structural morphometry, our findings establish a novel, robust model of asthma-associated pulmonary vascular dysfunction.

Chronic MA exposure produced significant airway hyperresponsiveness characterized by increased Rrs and Ers with reduced Crs, similar to major functional abnormalities observed in human asthma (25, 26). These findings are consistent with previous studies using MA models that better recapitulate the heterogeneity and severity of clinical asthma compared to single-allergen models (27). Importantly, this chronic airway dysfunction was accompanied by vascular remodeling, impaired relaxation, and right ventricular hypertrophy. These observations align with clinical data showing that severe, chronic asthma can lead to elevated pulmonary vascular resistance and Cor Pulmonale (28, 29).

Significant vascular remodeling occurs before clinical symptoms of PH appear, presenting an opportunity for prevention of advanced, irreversible disease. Our model directly links chronic MA exposure to distinct structural alterations and corresponding functional changes in vasoreactivity within the bronchovascular niche. Here, MA-challenged mice exhibited features of pulmonary vascular remodeling in small intra-acinar arteries, including severe luminal narrowing and increased medial wall thickness. Furthermore, we speculate that the qualitatively increased α-SMA expression in the pulmonary arteries is due to smooth muscle hyperplasia. These structural changes are consistent with growing evidence that the local inflammatory milieu within the bronchovascular bundle can affect adjacent pulmonary vessels and induce pathological vascular remodeling (9, 10, 12, 30).

A strength of this study is the addition of isolated multi-wire myography functional assays, which provide direct insight into intrinsic vascular contractility independent of systemic inflammatory influences or in vivo mechanical stretch (31). While Witzenrath et al. demonstrated that allergic lung inflammation drives pulmonary vascular hyperresponsiveness in isolated perfused lungs of allergen-challenged mice (17), our ex vivo multi-wire myography findings build upon these observations by demonstrating vascular smooth muscle hypercontractility and endothelial impairment in isolated vessel rings. Specifically, MA-derived pulmonary arteries showed exaggerated 5-HT-induced vasoconstriction, supporting our hypothesis that MA exposure induces vascular smooth muscle hypercontractility. Moreover, we observed blunted endothelium-dependent relaxation to ACh in MA-exposed vessels. Contrarily, endothelium-independent relaxation to SNP was not affected by MA exposure, suggesting endothelial dysfunction is a component of the vascular dysfunction in this model.

Some limitations of the present study should be acknowledged. First, while we successfully established the comprehensive structural and functional phenotype of asthma-induced pulmonary arterial remodeling, the molecular mechanisms and explicit paracrine secretome connecting airway inflammation to vascular remodeling were not elucidated. Future studies using spatial transcriptomics and molecular approaches will be important to unravel these specific signaling pathways in detail. Second, in the model utilized, it is difficult to dissect the direct effects of perivascular inflammation and the potential secondary effects of intermittent hypoxia caused by airway obstruction on vascular remodeling. However, the strong structural changes observed specifically in the peribronchial arteries support a direct inflammatory driver. Additionally, while lungs were immersion-fixed in Carnoy’s solution under standardized conditions across all experimental groups, intratracheal inflation-fixation at constant hydrostatic pressure was not performed. Although identical, standardized processing ensures valid relative structural comparisons between groups, future studies employing constant-pressure inflation fixation will further refine precise morphometric assessments of vascular architecture.

Overall, these findings indicate that chronic allergic airway inflammation from MA exposure drives a pulmonary vascular phenotype characterized by structural remodeling, endothelial dysfunction, and smooth muscle hyperreactivity. Further analysis of the pulmonary vascular effects of MA exposure could enable the identification of localized molecular drivers within the bronchovascular bundle and development of targeted therapeutics for vascular remodeling in asthma.