Bronchial angiogenesis in horses with severe asthma and its response to corticosteroids

Abstract

Background: Severe asthma in horses is characterized by structural changes that thicken the lower airway wall, a change that is only partially reversible by current treatments. Increased vascularization contributes to the thickening of the bronchial wall in humans with asthma and is considered a potential new therapeutic target.

Objective: To determine the presence of angiogenesis in the bronchi of severely asthmatic horses, and if present, to evaluate its reversibility by treatment with corticosteroids.

Animals: Study 1: Bronchial samples from asthmatic horses in exacerbation (7), in remission (7), and aged-matched healthy horses. Study 2: Endobronchial biopsy samples from asthmatic horses in exacerbation (6) and healthy horses (6) before and after treatment with dexamethasone.

Methods: Blinded, randomized controlled study. Immunohistochemistry was performed using collagen IV as a marker for vascular basement membranes. Number of vessels, vascular area, and mean vessel size in the bronchial lamina propria were measured by histomorphometry. Reversibility of vascular changes in Study 2 was assessed after 2 weeks of treatment with dexamethasone.

Results: The number of vessels and vascular area were increased in the airway walls of asthmatic horses in exacerbation (P = .01 and P = .02, respectively) and in remission (P = .02 and P = .04, respectively) when compared to controls. In Study 2, the differences observed between groups disappeared after 2 weeks of treatment with corticosteroids because of the increased number of vessels in healthy horses.

Conclusions and clinical importance: Angiogenesis contributes to thickening of the airway wall in asthmatic horses and was not reversed by a 2-week treatment with corticosteroids.

Keywords: angiogenesis; asthma; collagen IV; horses; immunohistochemistry.

FIGURE 1

Representative image of a bronchus from an asthmatic horse (A) and a control horse (B) stained with collagen IV highlighting in brown the basement membrane of blood vessels in the airway lamina propria. The smooth muscle cells are also diffusely stained by collagen IV. BM, basement membrane; ECM, extracellular matrix; EP, epithelium; SM, smooth muscle. Scale bar 100 μm

FIGURE 2

Example of the tracings for the calculation of the vascular area and the number of vessels in the airway lamina propria. A white line was firstly used to calculate basement membrane length (black arrows), and secondly, it was used to outline the extracellular matrix area (red arrows). Luminal circumference of each vessel included in a region of interest is outlined in red (vascular area). Each vessel was manually assigned a number for vessel count. Scale bar 100 μm

FIGURE 3

(A) Pulmonary resistance (R_L) and (B) pulmonary elastance (E_L) measured in control (n = 7) and asthmatic horses in exacerbation (n = 7) and remission (n = 7). The error bars indicate the standard deviation of the mean. Significantly higher R_L and E_L values were observed in horses in exacerbation when compared with control (P < .0001, P = .0008, respectively) and remission (P < .0001, P = .0012, respectively)

FIGURE 4

Histomorphometric results of Study 1. A, Total number of bronchial vessels per μm² of extracellular matrix (ECM) in control and asthmatic horses in exacerbation and remission. An increased number of vessels in horses in exacerbation (P = .007) and remission (P = .02) when compared to control horses was observed. B, Vascular area of control and asthmatic horses in exacerbation and remission. An increased vascular area was observed in horses in exacerbation when compared to the control (P = .02) and remission horses (P = .04). C, Total epithelial basement membrane length values of control and asthmatic horses in exacerbation and remission. No differences were observed between the 3 groups. D, Mean bronchial vessel size of control and asthmatic horses in exacerbation and remission. No differences were observed between the 3 groups. The error bars indicate the standard deviation of the mean

FIGURE 5

Histomorphometric results of Study 2. A, Total number of bronchial vessels per μm of epithelial basement membrane in control and asthmatic horses in exacerbation. A statistically significant increase in the number of vessels was observed in horses in exacerbation (P = .002) when compared to control horses at baseline. No significant differences were observed between control and exacerbation groups (P = .35) after 2 weeks of treatment because of an increase in the number of vessels in control horses (P = .01) after 2 weeks of treatment with dexamethasone. The number of vessels of horses in exacerbation remained unchanged. B, Mean bronchial vessel size of control and asthmatic horses in exacerbation. No significant differences were observed between groups at baseline (P = .1) or at week 2 (P = .24). C, Total epithelial basement membrane length values of control and asthmatic horses in exacerbation. No significant differences were observed between groups at baseline (P = .3) or at week 2 (P = .12). The error bars indicate the standard deviation of the mean