(A–C) In vivo studies (n = 3 biological replicates). (A) Schematic of the experimental design (created with BioRender.com. (B) Representative lung sections at day 21. α-SMA, chromogenic IHC. (C) Semiquantification of bronchial α-SMA area (n = 39–45 airways), pulmonary arterial α-SMA (n = 48–52 pulmonary arteries), and ECM deposition (n = 39–40 bronchovascular bundles). (D–F) Human lung tissue. (D and E) Representative 3D immunofluorescence images and semiquantification of α-SMA, type I collagen, and LTBP2 in PCLSs from (D) healthy donors cultured under control (Ctrl) (30–40 mmHg CO₂, pH 7.4) or buffered hypercapnia (50–60 mmHg CO₂, pH 7.4) for 7 days, and (E) COPD donors (n = 8–9 bronchioles from 3 donors). (F) Representative images of lung sections from end-stage COPD (n = 2) and non-COPD (n = 3) donors stained with MTC, α-SMA, or LTBP2. Br, bronchiole; PA, pulmonary artery. Data are presented as a superplot, with individual data points and mean ± SEM values from 3 biological replicates shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA with Tukey’s post hoc test (C) and 2-tailed Welch’s t test (D). Scale bars: 2.5 mm, 250 μm inset (B), 100 μm (C), 50 μm (D and E), and 500 μm (F). Exp., experiment; HC, hypercapnia; MTC, Masson’s trichrome; NC, normocapnia; Re, reversibility.