Equine asthma is characterized by lower airway inflammation, bronchial hyperresponsiveness, and remodeling of the bronchial walls.1 In its severe form, increased airway smooth muscle (ASM) mass and deposition of collagen and elastic fibers correlate with the degree of respiratory obstruction.2 These changes are only partially reversible with conventional therapies, including antigen avoidance strategies, inhaled corticosteroids, and bronchodilators,3–5 suggesting that airway remodeling contributes to the incurability and persistence of this condition. While mild and moderate equine asthma (MEA) is highly prevalent and may be transient, inflammation-induced structural alterations of the airway walls are also present, which might hypothetically lead to the progression of the severe form of the disease in some cases. The remodeling of the airway wall in milder forms of equine asthma includes epithelial hyperplasia, thickening of the submucosa, ASM fibrosis, and an increased expression of the fast contracting (+) smooth muscle myosin heavy chain (SMMHC) isoform.6,7 Bronchial angiogenesis, defined as new blood vessel formation originating from the preexisting vasculature, occurs in human asthmatics and horses with severe equine asthma (SEA).8–10 Angiogenesis results from cross-talks between vascular endothelial cells and stromal and inflammatory cells in the organ microenvironments. Chronic inflammation and mediators secreted by structural cells, including ASM cells, may trigger angiogenesis in asthma.11–13 In turn, the new blood vessels facilitate the influx of immune cells and mediators that can amplify inflammation and stimulate the remodeling of the airways. Importantly, the increased vascularity in the submucosa of the bronchial wall has been shown to correlate with airway hyperresponsiveness and airway caliber in human asthma.14
The objective of the current study was to evaluate the presence of angiogenesis in the bronchial tissues of horses with MEA. We hypothesized that increased bronchial vascularity would be observed in MEA and contribute to ASM remodeling.
Methods
Study design
A blinded, retrospective case-control study was undertaken as part of a larger project investigating airway remodeling in MEA and its functional consequences.
Case selection
Endobronchial biopsies from 9 client-owned horses diagnosed with MEA and 7 age-matched healthy controls (6 client owned and 1 mare from a research herd) collected from August 14, 2014, to May 31, 2019, were extracted from the Equine Respiratory Tissue Biobank (Faculty of Veterinary Medicine, Université de Montréal;
Immunohistochemistry
Immunohistochemistry was performed in paraffin-embedded endobronchial biopsy samples, as previously reported.10 Briefly, 2 to 5 tissue sections of a 5-μm thickness were obtained from each biopsy and prepared for the immunohistochemistry analysis. Enzymatic antigen retrieval was performed using pepsin (Ready-to-Use; No. AR-6543-0; ImmunoBioScience). A primary antibody against collagen IV (mouse anti-human, IgG1 monoclonal antibody; Dako No. M0785; Agilent Technologies; dilution 1:50 and overnight incubation at 4 °C) was used as a vascular basement membrane marker. The secondary antibody (No. 715-065-150; Jackson ImmunoResearch Laboratory; dilution 1:1,000) and 3,3′-diaminobenzidine peroxidase (substrate kit; No. SK4100; Vector Laboratories Inc) were then used to outline the vascular basement membrane in brown. The specificity was verified with an isotype control antibody (mouse IgG; No. M5409; Sigma-Aldrich; dilution 1:50). Histologic slides were counterstained with Harris hematoxylin.
Histomorphometry of angiogenesis
Histomorphometric analysis was performed in all tissue samples using a microscope (DM4000-B Life Science Research; Leica), a FLIR camera (FLIR Integrated Imaging Solutions Inc), and an image acquisition computer software (Panoptiq 5.0; ViewsIQ Inc). As previously described, the basement membrane length, number of vessels, total vascular area, and mean vessel size were measured by a blinded operator using a software analysis system (ImageJ, version 1.52p; NIH).10 In brief, a region of interest (ROI) was randomly selected and defined as the area between the epithelial basement membrane to a depth of 150 μm into the submucosa.17,18 The number of vessels in each ROI was manually counted and corrected by the total epithelial basement membrane length. An automated value of the inner area of each bronchial vessel was computed after manual tracing with a freehand tool to calculate the total vascular area of each ROI. Mean vessel size was estimated by dividing the total vascular area value by the number of vessels of each ROI. The ASM remodeling data from a separate study7 on endobronchial biopsies from the same individuals were available for review. Briefly, the expression of the (+) insert SMMHC was assessed by real-time quantitative PCR, and the myocyte proliferation rate was evaluated by colocalization of the proliferating cell nuclear antigen (PCNA) expression and smooth muscle α-actin by immunohistochemistry.
Statistical analyses
Statistical analyses were performed using computer software (GraphPad Prism, version 10.1.2). The normality of the data was analyzed with Shapiro-Wilk tests and QQ plot visualization. Differences between groups were compared with unpaired t tests or with Mann-Whitney tests if the data were not normally distributed. The correlations of the angiogenic parameters, inflammation, and ASM remodeling data (data available from a previous study)7 were studied with Pearson or Spearman correlation tests. Statistical significance was set at P ≤ .05.
Results
Animals
The control group included 2 Quarter Horses, 1 Standardbred, 1 Belgian, 1 Morgan, 1 Paint Horse, and 1 warmblood, with 3 mares and 4 geldings. The MEA group included 4 Quarter Horses, 3 warmblood-type horses, 1 Shire, and 1 Paint Horse, with 5 mares and 4 geldings. The mean and SD of the age of the horses was 9.0 ± 2.9 years in the control group and 7.8 ± 2.4 years in the MEA group (P = .4). Airway neutrophilia was higher in horses with MEA (14.5 ± 4.3%, P < .0001, all horses ≥ 5% neutrophils) compared to controls (2.6 ± 1.1%, all horses < 5% neutrophils). There were no statistical differences for the percentage of mast cells (only 1 MEA horse with mast cells ≥ 2%), eosinophils (2 MEA horses with ≥ 1% eosinophils), macrophages, and lymphocytes between groups. Three horses in the MEA group had received corticosteroids in the 2 months before the collection of samples (1 horse received 0.05 mg/kg of dexamethasone once a week for a month and 0.02 mg/kg of IM triamcinolone 3 weeks before the presentation, 1 horse received 0.08 mg/kg of IM triamcinolone 1.5 months before the presentation, and 1 horse received a decreasing regimen of oral prednisolone starting at 1.4 mg/kg daily for a total of 20 days that ended 2 weeks before the presentation).
Histomorphometric analysis
The number of bronchial vessels (P = .1), vascular area (P = .3), and mean vessel size (P = .1) did not differ significantly between control and asthmatic horses (Figure 1). The vascular area was larger in the 3 MEA horses that had received corticosteroids in the 2 months before the collection of samples compared to those that did not (1,917 ± 728 µm2 vs 1,038 ± 206 µm2, P = .04), while the other parameters did not vary.
Histomorphometric analysis of angiogenic parameters evaluated from endobronchial biopsies collected between 2014 and 2019 from 9 horses with mild and moderate equine asthma (MEA) and 7 healthy controls. A—Number of bronchial vessels per micrometer of epithelial basement membrane (BM). B—Vascular area. C—Mean vessel size. In the dot plot, the bar and the error bars illustrate the mean and the SD, respectively. In the MEA group, square symbols identify horses treated with corticosteroids.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.10.0318
Correlation between angiogenesis, ASM remodeling, and inflammation
The percentage of PCNA-positive ASM cells was positively correlated to the number of bronchial vessels in horses with MEA (Spearman r = 0.73, P = .03) but not in control horses (Pearson r = −0.68, P = .2; data available on 5/7 controls; Figure 2). In control horses, the expression of the (+) insert SMMHC isoform was negatively correlated with the vascular area (Pearson r = −0.83, P = .02) but not in MEA (Spearman r = 0.05, P = .91; Figure 3). Airway neutrophilia correlated negatively with mean vessel size in horses with MEA (Pearson r = −0.83, P = .005) but not in control horses (Pearson r = −.50, P = .26; Figure 4).
Correlation between the airway smooth muscle (ASM) proliferating cell nuclear antigen (PCNA) expression evaluated by immunohistochemistry and the angiogenesis in endobronchial biopsies as described (Figure 1) in control horses (A) and MEA (B).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.10.0318
Correlation between the genic expression of the (+) insert smooth muscle myosin heavy chain (SMMHC) isoform evaluated by real-time quantitative PCR and the vascular area in endobronchial biopsies as described (Figure 1) in control horses (A) and MEA (B).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.10.0318
Correlation between the airway neutrophilia evaluated with bronchoalveolar lavage cytology and the mean vessel size in endobronchial biopsies as described (Figure 1) in control horses (A) and MEA (B).
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.10.0318
Discussion
Angiogenesis is a complex process that involves the growth and development of new blood vessels from preexisting ones.19 As an increased number of bronchial vessels is present in the airways of horses with SEA,10 we postulated that bronchial angiogenesis could also be a feature of milder forms of the disease. However, no significant differences were observed between the MEA and control groups for the number of bronchial vessels, the vascular area, and the vessel sizes. These findings could be attributed to the lesser degree of inflammation and intensity of mechanical strain in mild compared to SEA.20 Similar to our findings, increased bronchial vascularity is observed in humans with severe asthma (patients with high medication requirements) but not in milder forms of the condition.9
Still, the ASM cell proliferation was positively correlated with the number of bronchial vessels in the horses affected by MEA, suggesting that angiogenesis might contribute to ASM remodeling in the disease. The PCNA expression reflects the rate of cellular proliferation and DNA synthesis,21 which are commonly associated with tissue repair and remodeling. As a significant correlation between the percentage of proliferating myocytes and pulmonary neutrophilia was previously reported in this cohort of horses,7 a complex interplay between inflammation, tissue remodeling, and angiogenic processes could occur. Indeed, both the angiogenesis and the increased ASM cell proliferation may have been triggered by growth factors, cytokines, and chemokines released during inflammation.13,22 Furthermore, the proangiogenic hypoxia-inducible factor is upregulated in horses with SEA after an antigen challenge,23 which could lead to both increased vascularity24 and ASM proliferation.25 Whether the hypoxia-inducible factor played a role in the correlation between angiogenesis and ASM remodeling in MEA remains to be determined. Conversely, ASM cells obtained from human asthmatic, but not from controls, may contribute to new blood vessel formation through the release of vascular endothelial growth factor,12 again illustrating the interplay between the ASM remodeling and angiogenesis. To assess this phenomenon, future studies should determine whether ASM remodeling and bronchial angiogenesis occur in anatomical proximity.
The (+) insert SMMHC isoform, also known as SM-B, is a 7-amino acid insert located near the ATP binding site that contributes to airway hyperresponsiveness by increasing the velocity of ASM contraction.26 Its genic expression is increased in the airways of horses affected by MEA and SEA,7,27 a finding that is attenuated by corticosteroid administration and antigen avoidance in the severe form of the disease.5,27 The (+) insert SMMHC isoform is present in ASM and other types of smooth muscle, including vascular smooth muscle. The presence of the insert in equine vascular smooth muscle has not been studied thus far, but in rats, its expression is largely heterogeneous among pulmonary vessels.28 The negative correlation between the expression of the (+) insert SMMHC isoform in the endobronchial biopsies and the vascular area in control horses in the current study illustrates that as the total area of vessels increased, the expression of this isoform decreased. Both vascular and ASM could have contributed to the result as the total (+) insert SMMHC isoform gene expression was measured in the entirety of the endobronchial biopsies. The negative correlation could indicate that the SM-B phenotype expression is lower in vascular than in ASM, but this would need confirmation. This could have been masked in the MEA group where the expression of the (+) insert SMMHC isoform is increased7 or in which ASM could hypothetically have occupied a larger proportion of the biopsy, although this was not measured. Of note, endobronchial biopsies only incompletely sample the smooth muscle layer in horses,15 a limitation that could be overcome in future studies by using endobronchial ultrasound for ASM mass quantification.29
In contrast to the relatively uniform clinical presentation, inflammatory cell populations, and remodeling attributes in SEA, significant variability exists in these traits within the milder forms of the condition. For instance, while sustained airway neutrophilia is characteristic of SEA, mild increases in neutrophils, metachromatic cells, or eosinophils were found in BALF of asthmatic horses in the present study. Although the contribution of neutrophils in angiogenesis is likely complex, these leukocytes may suppress endothelial cell proliferation and angiogenic sprouting by the release of endostatin and thrombospondin-1 or by angiostatin following the cleavage of plasminogen by neutrophil elastase.30,31 Whether the release of these antiangiogenic factors contributed to the negative correlation between airway neutrophilia and the mean vessel sizes in MEA in the present study would require additional investigation.
The main limitation of this study is the low number of horses included. A post hoc power calculation (G*Power 3.1.9.6) indicated that 22 horses per group would have been required to detect a significant increase in the number of vessels in the asthmatic group (α of 0.05 and power of 80%). As 9 horses with SEA and 9 controls were sufficient to demonstrate the presence of angiogenesis in the severe form of the disease,10 it is possible that the lack of difference in the vascularization in the present study was due to greater variability of this phenomenon in MEA. Furthermore, the corticosteroid administration in 3/9 MEA horses might have influenced the results, as illustrated by the increased vascular area in this subgroup. This might have been a direct consequence of corticosteroid treatment as dexamethasone increased the number of bronchial vessels in control horses but not in SEA in a previous study,10 illustrating that the angiogenic response in MEA might be closer to that of healthy horses than SEA. However, only 3 horses received corticosteroids, with varying medication, dose, and length of treatment; therefore, this finding should be confirmed in a larger cohort receiving a standardized treatment regimen.
In conclusion, angiogenesis parameters did not differ in endobronchial biopsies from control animals and horses with MEA. The correlation between the number of vessels and ASM proliferation is a novel finding that could contribute to the development of ASM thickening, a lesion that leads to bronchospasm and lumen narrowing in asthma. The possible causal relationship between ASM remodeling and angiogenesis should be explored in future studies.
Acknowledgments
None reported.
Disclosures
The authors have nothing to disclose. No AI-assisted technologies were used in the generation of this manuscript.
Funding
The collection and storage of samples in the Equine Respiratory Tissue Biobank were possible thanks to the Quebec Respiratory Health Research Network, Equine Research Funds of the Faculty of Veterinary Medicine of the Université de Montréal, and the Canadian Institutes of Health Research (grant No. MOP-102751).
ORCID
S. Mainguy-Seers https://orcid.org/0000-0002-4768-6674
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