Myocd regulates airway smooth muscle cell remodeling in response to chronic asthmatic injury

Abstract

Abnormal growth of airway smooth muscle cells is one of the key features in asthmatic airway remodeling, which is associated with asthma severity. The mechanisms underlying inappropriate airway smooth muscle cell growth in asthma remain largely unknown. Myocd has been reported to act as a key transcriptional coactivator in promoting airway-specific smooth muscle development in fetal lungs. Whether Myocd controls airway smooth muscle remodeling in asthma has not been investigated. Mice with lung mesenchyme-specific deletion of Myocd after lung development were generated, and a chronic asthma model was established by sensitizing and challenging the mice with ovalbumin for a prolonged period. Comparison of the asthmatic pathology between the Myocd knockout mice and the wild-type controls revealed that abrogation of Myocd mitigated airway smooth muscle cell hypertrophy and hyperplasia, accompanied by reduced peri-airway inflammation, decreased fibrillar collagen deposition on airway walls, and attenuation of abnormal mucin production in airway epithelial cells. Our study indicates that Myocd is a key transcriptional coactivator involved in asthma airway remodeling. Inhibition of Myocd in asthmatic airways may be an effective approach to breaking the vicious cycle of asthmatic progression, providing a novel strategy in treating severe and persistent asthma. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Keywords: Myocd; airway fibrillar collagen; airway inflammation; airway mesenchymal epithelial interaction; airway remodeling; airway smooth muscle cells; asthma.

Figure 1

Postdevelopmental deletion of Myocd in lung mesenchyme attenuates allergen‐induced airway hyperresponsiveness. (A) CKO of Myocd was induced by Dox administration from 5 to 8 weeks of age in mice with the indicated genotypes. (B) Dox‐induced Cre expression was validated by mGFP expression in the mTmG reporter mouse lung. (C) Lung tissue morphology was compared between WT and Myocd CKO mice. (D) Procedures to establish a chronic asthmatic mouse model. D, day; i.p., intraperitoneal injection; it, intratracheal installation. (E) Comparison of Myocd expression at mRNA level between different mouse lung tissues; ***p < 0.001. (F) Airway hyperresponsiveness to methacholine was compared among different groups as indicated (mean ± SEM, n > 4, *p < 0.05).

Figure 2

Comparison of airway smooth muscle cells (ASMCs) in OVA‐induced asthmatic mouse lungs between Myocd CKO and WT groups. (A) ASMCs were identified by Acta2 immunostaining (red) and proliferating ASMCs were recognized by Ki67 coimmunostaining (magenta). Airway epithelial cells were marked by Cdh1 costaining (green), and all nuclei were counterstained by DAPI (blue). The ASMC layer at greater magnification is shown in the inserts, highlighted by dotted lines. (B–D) Comparison of normalized average area of ASMC layers, normalized ASMC hypertrophy index, and normalized ASMC proliferation index among different groups. There were five mice in the WT‐OVA and CKO‐OVA groups, and four mice in the WT‐PBS and CKO‐PBS groups. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Alteration of inflammatory cells in bronchoalveolar lavage fluid of Myocd CKO lungs. Bronchoalveolar lavage fluid was collected from the mouse 24 h after the last challenge. (A) With the same OVA challenge, the total cell numbers in the BALF recovered from Myocd CKO mouse lungs was significantly decreased compared to those from WT mice. However, the cell composition (B, C, and D) of BALF, measured as percentages of eosinophils, lymphocytes, and macrophages, was not significantly different between Myocd CKO and WT mice. n = 5, ***p < 0.001.

Figure 4

Lung mesenchymal Myocd deletion reduced OVA‐induced lung inflammation. (A) Inflammation assessment of lung tissue sections with H&E staining: massive infiltration of inflammatory cells was seen in peri‐airway (black arrow), perivascular (red arrow), and alveolar structures (blue arrow) of OVA‐challenged WT mouse lungs compared to those in PBS‐treated WT controls. In contrast, the severity of inflammatory cell infiltration in Myocd CKO lungs, particularly in the peri‐airway area, was reduced. (B–D) Inflammatory infiltration in peri‐airway, vascular, and alveolar regions was separately evaluated using a semiquantitative scoring method. Significant difference was detected for peri‐airway inflammation (2.382 ± 0.236 versus 3.480 ± 0.182, *p < 0.05), but not for perivascular and alveolar inflammation between OVA‐treated WT and Myocd CKO groups. Five mice were included in each group, and 10 randomly selected fields were evaluated for each mouse. Ns, no significance. *p < 0.05, **p < 0.01, ***p < 0.001. (E–H) Expression of asthma‐related cytokine/growth factor mRNAs Il4, Il13, Tnf, and Tgfb1 was determined by RT‐qPCR and presented relative to WT‐PBS samples (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001). Actb was used as a reference transcript for normalization.

Figure 5

Lung mesenchyme‐specific knockout of Myocd reduced prolonged OVA treatment‐induced fibrillar collagen deposition in airway walls. (A) Picro‐Sirius Red staining of lung tissue sections of indicated genotypes and allergen challenges viewed under bright field and polarized light microscopy, respectively. The captured birefringence signal under polarizing microscopy was converted into grayscale and quantified using ImageJ. The PBS group was used as normal control. (B) Semiquantitative measurement of the area of fibrillar collagen deposition in airways was performed for more than five fields per sample, five or six samples per condition, and normalized by the internal perimeter of airway epithelium. *p < 0.05, **p < 0.01.

Figure 6

Deletion of mesenchymal Myocd inhibited mucin expression in airway epithelial cells under prolonged allergen exposure. (A) PAS‐staining of lung tissue sections with different genotypes and allergen exposures. Increased mucin expression was obvious in OVA‐challenged WT lung airway epithelial cells, but subtle in OVA‐challenged Myocd CKO lung airway epithelia. (B) The percentage of cells that were PAS‐positive was calculated per bronchiole and compared. ***p < 0.001 (n > 5 per group).