TY - JOUR
T1 - Dysfunctional Epithelial Barrier Is Characterized by Reduced E-Cadherin in Idiopathic Subglottic Stenosis
AU - Berges, Alexandra J.
AU - Ospino, Rafael
AU - Mafla, Laura
AU - Collins, Samuel
AU - Chan-Li, Yee
AU - Ghosh, Baishakhi
AU - Sidhaye, Venkataramana
AU - Lina, Ioan
AU - Hillel, Alexander T.
N1 - Publisher Copyright:
© 2023 The American Laryngological, Rhinological and Otological Society, Inc.
PY - 2024/1
Y1 - 2024/1
N2 - Objectives: To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. Methods: Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). Results: scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68–890.83 Ω × cm2, p < 0.05), an increase in permeability (paired 95% CI, −6116.00 to −1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814–1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, −2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367–0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748–59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. Conclusion: iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. Level of Evidence: NA Laryngoscope, 134:374–381, 2024.
AB - Objectives: To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium. Methods: Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6). Results: scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68–890.83 Ω × cm2, p < 0.05), an increase in permeability (paired 95% CI, −6116.00 to −1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814–1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, −2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367–0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748–59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium. Conclusion: iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS. Level of Evidence: NA Laryngoscope, 134:374–381, 2024.
KW - E-cadherin
KW - epithelial barrier dysfunction
KW - epithelium
KW - idiopathic subglottic stenosis
KW - occludin
KW - single-cell analysis
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U2 - 10.1002/lary.30951
DO - 10.1002/lary.30951
M3 - Article
C2 - 37565709
AN - SCOPUS:85167572548
SN - 0023-852X
VL - 134
SP - 374
EP - 381
JO - Laryngoscope
JF - Laryngoscope
IS - 1
ER -