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82 Microrna-21 regulates transforming growth factor beta-1 release from platelets
  1. Temo Barwari1,
  2. Seda Eminaga2,
  3. Paul Armstrong2,
  4. Melissa Chan2,
  5. Ruifang Lu2,
  6. Javier Barallobre-Barreiro2,
  7. Xiaoke Yin2,
  8. Raimund Pechlaner2,
  9. Sarah Langley2,
  10. Anna Zampetaki2,
  11. Peter Santer2,
  12. Martin Weger2,
  13. Timothy Warner2,
  14. Stefan Kiechl2,
  15. Johann Willeit2,
  16. Ajay Shah2,
  17. Manuel Mayr2
  1. 1King’s British Heart Foundation Centre, King’s College London, James Black Centre, London, UK
  2. 2King’s British Heart Foundation Centre, King’s College London


Introduction MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis. Inhibitors of miR-21 are currently undergoing clinical trials. Our aim was to explore mechanisms of how miR-21 inhibition attenuates fibrosis using a proteomics approach.

Methods and results Primary murine cardiac fibroblasts (CF) were transfected with miR-21 mimics and inhibitors. In line with previous reports, miR-21 overexpression increased CF proliferation (n=4 vs. 4; p<0.0001 for 2-way ANOVA). Unexpectedly, proteomic analysis revealed limited effects on extracellular matrix (ECM) secretion. Similarly, the ECM protein composition of hearts from miR-21 null mice was similar to wild-type controls. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In the community-based Bruneck study (n=671), 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 (Pearson r 0.68; q<0.0001). Argonaute2 immunoprecipitation demonstrated enrichment of miR-21 in a human megakaryoblastic cell line. Furthermore, TGF-β1 was abundant in megakaryocytes as demonstrated by immunofluorescence staining of murine bone marrow. Platelet immunodepletion in mice (n=5 vs. 7 for control and anti-CD42b antibody injection, respectively) revealed a strong platelet dependency of plasma TGF-β1 levels (fold change: 2.37; p=0.003). Correlation of miR-21 with total TGF-β1 was confirmed in humans by ELISA measurements in platelet-poor plasma collected during the Bruneck 2015 follow-up (n=332; Pearson r: 0.63; q<0.0001). When mice were treated with antagomiR-21, no differences were observed in the platelet count or in their aggregation response to different agonists. However, a proteomics analysis of the platelet releasate revealed TGF-β1 as differentially regulated. An attenuated TGF-β1 release after antagomiR-21 treatment (n=5 vs. 5 for control and antagomiR-21) was validated in platelets activated with collagen and a thrombin receptor-activating peptide (p=0.004 and 0.002, respectively, for 2-way ANOVA). Mechanistically, Wiskott-Aldrich Syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21.

Conclusions This study reports a previously unrecognised effect of miR-21 inhibition on platelet TGF-β1 release. This novel systemic effect of miR-21 on platelets may contribute to the anti-fibrotic effects of miR-21 inhibitors.

  • microRNA
  • fibrosis
  • platelets

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