Introduction: Fibrosis is a major contributor to cardiac disease. No specific anti-fibrotic therapy is currently available. MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis with inhibitors of miR-21 currently undergoing clinical trials. We aimed to explore how miR-21 inhibition attenuates fibrosis using a proteomics approach.
Methods and Results: Primary murine cardiac fibroblasts (CFs) were transfected with miR-21 mimics and inhibitors. Unexpectedly, proteomic analysis of their secretome showed limited changes in extracellular matrix (ECM) protein secretion. Thus, we searched for additional explanations how miR-21 might regulate fibrosis. In the 2000 follow-up of the community-based Bruneck study (n=669), we compared circulating miR-21 levels with a proteomic panel of 155 proteins associated with cardiovascular disease. Several platelet-derived pro-fibrotic factors significantly correlated with plasma miR-21 levels, including latency-associated peptide (LAP) transforming growth factor beta 1 (TGF-β1). This correlation was confirmed by ELISA measurements of active TGF-β1, a master regulator of fibrosis, in plasma collected during the Bruneck 2015 follow-up (n=332). In subsequent experiments, mice were treated with antagomiR-21. While no differences were observed in the platelet aggregation response, proteomics analysis of the platelet releasate identified TGF-β1 as differentially regulated after antagomiR-21 treatment. This was validated in isolated platelets activated with collagen and a specific thrombin receptor-activating peptide. Mechanistically, Wiskott-Aldrich Syndrome protein (WASp), a regulator of platelet TGF-β1 secretion, was increased in platelets and in bone marrow samples of mice treated with antagomiR-21.
Conclusions: This study reports a previously unrecognized effect of miR-21 inhibition on platelet TGF-β1 release. MiR-21 inhibition was found to reduce TGF-β1 release from platelets through de-repressing WASp. The effect of miR-21 on platelets may contribute to the anti-fibrotic effects of miR-21 inhibitors.