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

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.