Targeting Engrailed‐1 Fibroblasts in Idiopathic Subglottic Stenosis
Niloufar Saeedi, Samuel L. Collins, Diana C. Lopez‐Fay, Yee Choi Chan Li, Ibrahim Abd‐Elazem, Alexander T. Hillel, Kevin M. MotzABSTRACT
Objective
Idiopathic subglottic stenosis (iSGS) is characterized by pathologic deposition of extracellular matrix (ECM) secondary to dysregulated fibroblasts. Engrailed‐1 expressing fibroblasts have been shown to promote ECM deposition in fibrosis. Verteporfin has been shown to reduce EN1 expression in fibroblasts through YAP/TAZ signaling. However, EN1 expression, YAP/TAZ signaling, and verteporfin treatment have not been investigated in iSGS pathogenesis.
Methods
Biopsies of diseased subglottis and distal healthy trachea of iSGS patients ( n = 6) were assessed. qRT‐PCR, analysis of iSGS single‐cell RNA sequencing data, flow cytometry and immunochemistry were used to compare the expression of EN1. Fibroblasts from diseased iSGS tissue were treated with verteporfin (5 μM) or control and gene and protein expression analysis was performed for EN1 , YAP/TAZ pathway and fibrotic genes.
Results
The iSGS subglottis demonstrated increased expression of EN1 (fold change = 58.86, SD = 65.12, p : 0.031) compared to controls. Immunohistochemistry analysis of the diseased iSGS subglottis showed that EN1 was densely expressed. Verteporfin treatment of iSGS fibroblasts in vitro led to an 81.75% (SD = 9.28, p : 0.031) reduction in COL1A1 and 22.77% (SD = 17.11, p : 0.031) reduction in COL3A1 gene expression. This was associated with a 26.32% (SD = 28.25, p : 0.031) reduction in EN1 expression and reductions in the YAP/TAZ associated genes AMOTL2 , CCN1, CCN2, YAP1 , and WWTR1.
Conclusion
EN1 and the associated YAP/TAZ signaling pathway are increased in iSGS. Verteporfin treatment inhibited YAP/TAZ expression and attenuated EN1 and COL1A1 expression in vitro . Targeting EN1 expressing fibroblasts through inhibition of YAP/TAZ signaling represents a potential therapeutic target to reduce fibrosis in iSGS.
Level of Evidence
N/A.