Figure 3: Fibrotic lungs contain α1-adrenoreceptor-expressing myofibroblasts.

(A-D) Representative immunofluorescence imaging of mouse lung tissues at day 14 post-bleomycin treatment (A, B) showed α-SMA (red), ADRA1D (green), and DAPI (blue) cells. A marked increase in ADRA1D-expressing myofibroblasts (white arrows) was observed in bleomycin-treated mice. (C, D) Flow cytometric analysis of wild-type mice post-orotracheal bleomycin administration, with ADRA1D expression in α-SMA-positive cells peaking on day 14 (D, P = 0.0026). (E-L) Immunofluorescence imaging revealed ADRA1D expression (red), α-SMA (green), and nuclear staining with DAPI (blue) in lung explant tissues from IPF, SSc-ILD, and normal lung tissues. (E-G) Normal lung tissues exhibited ADRA1D expression in luminal structures such as airways and blood vessels (E, white arrows), as well as in scattered cells throughout the alveoli (E, white asterisks). Similar patterns were observed in IPF and SSc-ILD tissues, with additional ADRA1D-positive cells in fibrotic areas, resembling cells of inflammatory or stromal lineage (white asterisks and white hashtags, respectively, F, G). The prevalence of ADRA1D-expressing cells was significantly higher in IPF and SSc-ILD tissues than in normal lungs (H, P = 0.0306 and P = 0.0398, respectively). (I-L) Compared to normal lung tissues, a higher accumulation of cells co-expressing ADRA1D and α-SMA (white arrows) was observed in IPF and SSc-ILD tissues (L, P = 0.0003 and P = 0.0296, respectively). Images were captured at 20x magnification. Data are presented as mean ± SEM, and statistical analyses were conducted using Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. ADRA1D, α1-adrenoreceptor subtype D; α-SMA, alpha-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole; HPF, high-power field; IPF, idiopathic pulmonary fibrosis; SSc-ILD, systemic sclerosis-related interstitial lung disease.