Orchestrated response from heterogenous fibroblast subsets contributes to repair from surgery-induced stress after airway reconstruction
Jazmin Calyeca, Zakarie Hussein, Zheng Hong Tan, Lumei Liu, Sayali Dharmadhikari, Kimberly M. Shontz, Tatyana A. Vetter, Christopher K. Breuer, Susan D. Reynolds, Tendy Chiang
Jazmin Calyeca, Zakarie Hussein, Zheng Hong Tan, Lumei Liu, Sayali Dharmadhikari, Kimberly M. Shontz, Tatyana A. Vetter, Christopher K. Breuer, Susan D. Reynolds, Tendy Chiang
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Research Article Cell biology Transplantation

Orchestrated response from heterogenous fibroblast subsets contributes to repair from surgery-induced stress after airway reconstruction

Abstract

Surgery of the tracheobronchial tree carries high morbidity, with over half of the complications occurring at the anastomosis. Although fibroblasts are crucial in airway wound healing, the underlying cellular and molecular mechanisms in airway reconstruction remain unknown. We hypothesized that airway reconstruction initiates a surgery-induced stress (SIS) response, altering fibroblast communication within airway tissues. Using single-cell RNA-Seq, we analyzed native and reconstructed airways and identified 5 fibroblast subpopulations, each with distinct spatial distributions across anastomotic, submucosal, perichondrial, and paratracheal areas. During homeostasis, adventitial and airway fibroblasts (Adventitial-Fb and Airway-Fb, respectively) maintained tissue structure and created cellular niches by regulating ECM turnover. Under SIS, perichondrial fibroblasts (PC-Fb) exhibited chondroprogenitor-like gene signatures, and immune-recruiting fibroblasts (IR-Fb) facilitated cell infiltration. Cthrc1-activated fibroblasts (Cthrc1+ Fb), mainly derived from Adventitial-Fb, primarily contributed to fibrotic scar formation and collagen production, mediated by TGF-β. Furthermore, repeated SIS created an imbalance in fibroblast states favoring emergence of CTHRC1+ Fb and leading to impaired fibroblasts–basal cell crosstalk. Collectively, these data identify PC, IR, and Cthrc1+ Fb as a signaling hub, with SIS emerging as a mechanism initiating airway remodeling after reconstruction that, if not controlled, may lead to complications such as stenosis or anastomotic breakdown.

Authors

Jazmin Calyeca, Zakarie Hussein, Zheng Hong Tan, Lumei Liu, Sayali Dharmadhikari, Kimberly M. Shontz, Tatyana A. Vetter, Christopher K. Breuer, Susan D. Reynolds, Tendy Chiang

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Figure 1

Surgery-induced stress results in fibroblast accumulation in the reconstructed airway.

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Surgery-induced stress results in fibroblast accumulation in the reconst...
(A) Graphical representation of the microsurgical model of airway reconstruction with a corresponding gross image showing the site of surgery. (B) Representative full and section view of H&E images of homeostatic and reconstructed airways at D14–D28. (C) Quantification of the number of cells per mm2 square in the submucosa (Control, n = 4; D14, n = 4; D28, n = 5). (D) Masson’s trichrome staining shows the luminal surface, epithelium, submucosa area, and cartilage (Control, n = 4; D14, n = 4; D28, n = 5). (E) Quantification of submucosa thickness at different time points following reconstruction. Dots represent the mean, and colored area represents upper and lower limits (Control, n = 4; D14, n = 4; D28, n = 5). (F) UMAP and annotation of all scRNA-Seq cells from the normal airway, and D14 and D28 after surgery (n = 63,561 cells). (G) The proportion of cell lineages in the normal airway and D14 and D28 after reconstruction. (H) Representative immunofluorescence staining showing the presence of fibroblasts (Vimentin+, red). (I) Quantification of Fb per mm2 (Vimentin+, red) per condition (Control n = 4, D14 n = 4, D28 n = 5). For all staining, “C” indicates cartilage, and the dotted line indicates the epithelium. Data are shown as mean ± SD. Statistical analysis was performed using 1-way ANOVA with multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bars: 50 µm, 100 µm, 200 µm, and 500 µm as indicated.