Stimulation of AT2 Receptor Decreases Collagen Expression and Inhibits Myofibroblast Differentiation to Protect Against Bleomycin‐induced Pulmonary Fibrosis
Authors: Kumar Ashok , Rathinasabapathy Anandharajan , Horowitz Alana , Horton Kelsey , Martinez Diana , Raizada Mohan , Steckelings Ulrike Muscha , Unger Thomas , Sumners Colin , Shenoy Vinayak , Katovich Michael 
Published: November 2023

We have observed that activation of the AT2 receptor by a non‐peptide agonist, Compound 21 (C21), protects against Bleomycin (Bleo)‐induced Pulmonary Fibrosis (PF) in rats. This study was designed to provide mechanistic insights for the anti‐fibrotic effects of C21, using in vitro experiments on human lung fibroblasts (MRC5) and alveolar epithelial cells (A549). MRC5 cells at sub‐confluence were incubated with different doses of Bleo (1, 2 and 5 μg/ml) for 48 hours. At 5 μg/ml, Bleo significantly upregulated the expression of type1 and III collagen (COL1A1; 5 fold and COL3; 4 fold), though a dose‐dependent increase in gene expression was observed. Furthermore, Bleo triggered the differentiation of fibroblast to myofibroblast as evidenced by significant increase in the mRNA levels of alpha‐smooth muscle actin (ACTA2; 4 fold) and fibronectin (FN1; 8 fold). In addition, components of the renin angiotensin system (RAS) were considerably upregulated with Bleo incubation (ACE; 7, ACE2; 44, and AT1R; 10 folds). However, pre‐treatment of Bleo‐insulted MRC5 cells with C21 (10 μg/ml) drastically decreased collagen expression and prevented fibroblast‐to‐myofibroblast differentiation. This was supported by complete inhibition of COL1A1, COL3, ACTA2 and FN1 expression, along with downregulation of various RAS components. In a subsequent experiment using A549 cells, co‐incubation with Bleo (5 μg/ml for 48 hours) significantly elevated the levels of ACE2, AT1R and AT2R (ACE2; 3, AT1R; 3 and AT2R; 4 folds), while ACE levels were unaltered. Interestingly, treatment of A549 cells with C21 (10 μg/ml) prior to Bleo insult further augmented the expression of AT2R (12 fold), suggesting that AT2R stimulation may have a positive feedback on its own expression. However, the levels of other RAS components remained unchanged. To further probe whether these in vitro findings are valid in vivo, we also analyzed the gene expression of collagen and tissue remodeling markers in the lungs of Bleo‐challenged animals that were either treated or untreated with C21 (0.03 mg/kg). Bleo administration induced elevation in the gene expression of collagen (COL1A1; 4, COL3; 2, CTGF; 6 and IL‐13; 4 fold) and lung tissue remodeling markers (TIMP1; 3 and MMP‐12; 15 fold). These increases were completely abrogated by C21‐treatment. Collectively, the present study demonstrates that the AT2R agonist, C21, attenuates PF by decreasing collagen expression and inhibiting myofibroblast differentiation, providing new insights on its anti‐fibrotic mechanism of action; however, further studies are warranted to understand the cross talk between AT2R signaling and PF.Support or Funding InformationThis work is supported by NIH Grants HL102033, HL056921 and American Heart Association Scientist Development Grant SDG12080302