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. 2021 Sep 2;134(18):2175–2185. doi: 10.1097/CM9.0000000000001605

Figure 4.

Figure 4

Macrophage exosomes trigger α-collagen I production and activate TGF-β/Smad2/3 pathway by shifting the RAS toward ACE/Ang II/AT1R axis in lung fibroblasts. (A) IHC to analyze AT1R proteins expression in control, BLM, and BLM + GW4869 groups. Original magnification, ×200. Enzyme linked immunosorbent assay (ELISA) kit was used to determine Ang II level of serum (B) and BALF (C) in control, BLM and BLM + GW4869 groups. n = 10 mice per group; ∗P < 0.001 vs. control, †P < 0.001 vs. BLM, for Ang II in serum; ∗P < 0.001 vs. control, †P < 0.001 vs. BLM, for Ang II in BALF. (D) Primary lung fibroblasts were stimulated with exosomes isolated from Ang II treated-macrophages in a dose of 30 μg/mL. As a positive control, fibroblasts were treated with Ang II. After 24 h, the fibroblasts were subjected to western blot analysis of α-collagen I protein, AT1R, TGF-β, p-Smad2/3, and Smad2/3. n = 3 independent experiments; ∗P < 0.05 vs. control for α-collagen I, AT1R, TGF-β, p-Smad2/3, respectively. (E) Fibroblasts were pretreated with IR for 1 h before stimulation with macrophage exosomes for 24 h. The protein levels of α-collagen I, TGF-β, p-Smad2/3, and Smad2/3 were analyzed by western blot. n = 3 independent experiments; ∗P < 0.05 vs. control; †P < 0.05 vs. exoAng II-Mϕ for collagen I, TGF-β, and p-Smad2/3, respectively. ACE: Angiotensin-converting enzyme; Ang II: Angiotensin II; AT1R: Angiotensin II type 1 receptor; BALF: Bronchoalveolar lavage fluid; BLM: Bleomycin; exo: Exosomes; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GW4869: An exosome inhibitor; IHC: Immunohistochemistry; IR: Irbesartan; p-Smad: Phosphorylated-Smad; RAS: Renin-angiotensin system; TGF-β: Transforming growth factor-β.