Abstract 17714: Downregulation of Tgf-ß By CRISPR/Cas9 Attenuates High Glucose-induced Myofibroblast Transformation: A Novel Therapeutic Approach for Treating Cardiac Fibrosis
Introduction: Cardiac fibrosis is the leading cause of myocardial dysfunction in patients with diabetes. Myofibroblast transformation is a key factor involved in fibrosis, which is induced by activation of TGF-ß signaling pathway. We tested whether high glucose (HG) - induced myofibroblast transformation could be prevented by downregulation of TGF-ß.
Methods and Results: Fibroblasts were treated with either normal levels of glucose (NG, 5 mM) or high glucose (25 mM) for 48 hours. Myofibroblast transformation was examined by the expression of collagen I and α-SMC. HG treatment led to increased mRNA expression of collagen I (2.7 fold) and α-SMC (2.3 fold), compared to fibroblasts treated with NG (p<0.05). The secreted collagen I protein levels were also increased in HG treated cells as determined by ELISA. In contrast, fibroblast proliferation (BrdU assay) and migration (wound scratch migratory assay) were not altered by HG. We used mannitol (25 mM) as osmotic control, which showed no effect on collagen I and α-SMC expression. To determine whether myofibroblast transformation can be prevented by TGF-ß knockdown, we constructed a lentivirus expressing CRISPR-Cas9 and a CRISPR guide RNA targeting TGF-ß. The lentivirus particles were transfected into fibroblast cells. The CRISPR/Cas9 based knockdown approach attenuated HG-induced myofibroblast transformation (reduced collagen I and α-SMC), accompanied by reduced expression of TGF-ß and pSmad2/3 (the downstream of TGF-ß pathway) assessed by Western blot and immunostaining. Furthermore, the CRISPR/Cas9 approach did not alter cell proliferation (BrdU assay), cell survival (TUNEL assay) and migration (wound scratch migratory assay).
Conclusion: This study demonstrates that short term exposure of fibroblast to HG induces myofibroblast formation which is mediated by TGF-ß signaling pathway. Importantly, we showed that downregulation of TGF-ß via CRISPR-Cas9 approach may be a novel therapeutic strategy to prevent HG-induced fibrosis.
Author Disclosures: Y. Li: None. B. Lan: None. L. Shao: None. J. Wang: None. Y. Zhang: None. L. Zhang: None. P. Xiao: None. B. Liu: None. C. Liang: None. X. Pan: None. Y. Geng: None. Z. Shen: None. X. Yu: None.
- © 2016 by American Heart Association, Inc.