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Stimulation of AT 2 Receptor Decreases Collagen Expression and Inhibits Myofibroblast Differentiation to Protect Against Bleomycin‐induced Pulmonary Fibrosis
Author(s) -
Kumar Ashok,
Rathinasabapathy Anandharajan,
Horowitz Alana,
Horton Kelsey,
Martinez Diana,
Raizada Mohan,
Steckelings Ulrike Muscha,
Unger Thomas,
Sumners Colin,
Shenoy Vinayak,
Katovich Michael
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb586
Subject(s) - downregulation and upregulation , myofibroblast , fibroblast , receptor , fibronectin , chemistry , pulmonary fibrosis , fibrosis , bleomycin , a549 cell , agonist , microbiology and biotechnology , endocrinology , in vitro , medicine , biology , cell , biochemistry , gene , chemotherapy
We have observed that activation of the AT 2 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 AT 1 R; 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, AT 1 R and AT 2 R (ACE2; 3, AT 1 R; 3 and AT 2 R; 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 AT 2 R (12 fold), suggesting that AT 2 R 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 AT 2 R 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 AT 2 R signaling and PF. Support or Funding Information This work is supported by NIH Grants HL102033, HL056921 and American Heart Association Scientist Development Grant SDG12080302