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Exploring Indirect Effects of MicroRNA-21 in Cardiac Fibrosis: A Proteomics Approach in Platelets

Student thesis: Doctoral ThesisDoctor of Philosophy

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 and inhibitors of miR-21 are currently undergoing clinical trials. In this project, we used a proteomics approach to explore mechanisms of how miR-21 inhibition may attenuate fibrosis. First, primary murine cardiac fibroblasts were transfected with miR-21 mimics and inhibitors. Unexpectedly, proteomic analysis of their secretome showed limited changes in extracellular matrix (ECM) protein secretion despite a pro-proliferative effect of miR-21. Similarly, no significant differences were seen in the ECM of miR-21 null mouse hearts. In light of its ubiquitous expression, we searched for additional explanations as to how miR-21 might regulate fibrosis. In the year 2000 follow-up of the community-based Bruneck study (n=660), we compared circulating miR-21 with a proteomic panel of 229 proteins associated with cardiovascular disease. Several platelet-derived pro-fibrotic factors significantly correlated with plasma miR-21 levels, including the latency-associated peptide of TGF-β1. This correlation was confirmed by ELISA measurements of active TGF-β1 in platelet-poor plasma collected during the Bruneck 2015 follow-up (n=332). Immunohistological staining of murine bone marrow sections revealed marked co-localisation of TGF-β1 with megakaryocytes, whilst immune-mediated thrombocytopaenia in mice indicated platelet dependency of plasma TGF-β1 levels. When mice were treated with antagomiR-21, no differences were observed in the platelet count or their aggregation response. However, a proteomics analysis of the platelet releasate revealed TGF-β1 as differentially regulated. An attenuated TGF-β1 release after antagomiR-21 treatment was validated in platelets activated with collagen and a thrombin receptor-activating peptide. Mechanistically, Wiskott-Aldrich Syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. In conclusion, this study reports a previously unrecognised effect of pharmacological miR-21 inhibition on platelet TGF-β1 release. Furthermore, we observed significantly lower platelet counts in blood from miR-21 null mice. This novel effect of miR-21 on platelets highlights the bone marrow as a potential key contributor to the anti-fibrotic effects of miR-21 inhibition.
Original languageEnglish
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Award date2019

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