Inhibition of profibrotic microRNA-21 affects platelets and their releasate
Temo Barwari, Seda Eminaga, Ursula Mayr, Ruifang Lu, Paul C. Armstrong, Melissa V. Chan, Mahnaz Sahraei, Marta Fernández-Fuertes, Thomas Moreau, Javier Barallobre-Barreiro, Marc Lynch, Xiaoke Yin, Christian Schulte, Ferheen Baig, Raimund Pechlaner, Sarah R. Langley, Anna Zampetaki, Peter Santer, Martin Weger, Roberto Plasenzotti, Markus Schosserer, Johannes Grillari, Stefan Kiechl, Johann Willeit, Ajay M. Shah, Cedric Ghevaert, Timothy D. Warner, Carlos Fernández-Hernando, Yajaira Suárez, Manuel Mayr
Temo Barwari, Seda Eminaga, Ursula Mayr, Ruifang Lu, Paul C. Armstrong, Melissa V. Chan, Mahnaz Sahraei, Marta Fernández-Fuertes, Thomas Moreau, Javier Barallobre-Barreiro, Marc Lynch, Xiaoke Yin, Christian Schulte, Ferheen Baig, Raimund Pechlaner, Sarah R. Langley, Anna Zampetaki, Peter Santer, Martin Weger, Roberto Plasenzotti, Markus Schosserer, Johannes Grillari, Stefan Kiechl, Johann Willeit, Ajay M. Shah, Cedric Ghevaert, Timothy D. Warner, Carlos Fernández-Hernando, Yajaira Suárez, Manuel Mayr
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Research Article Cardiology Cell biology

Inhibition of profibrotic microRNA-21 affects platelets and their releasate

Abstract

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.

Authors

Temo Barwari, Seda Eminaga, Ursula Mayr, Ruifang Lu, Paul C. Armstrong, Melissa V. Chan, Mahnaz Sahraei, Marta Fernández-Fuertes, Thomas Moreau, Javier Barallobre-Barreiro, Marc Lynch, Xiaoke Yin, Christian Schulte, Ferheen Baig, Raimund Pechlaner, Sarah R. Langley, Anna Zampetaki, Peter Santer, Martin Weger, Roberto Plasenzotti, Markus Schosserer, Johannes Grillari, Stefan Kiechl, Johann Willeit, Ajay M. Shah, Cedric Ghevaert, Timothy D. Warner, Carlos Fernández-Hernando, Yajaira Suárez, Manuel Mayr

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Figure 5

Pharmacological inhibition of miR-21 decreases platelet TGF-β1 release.

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Pharmacological inhibition of miR-21 decreases platelet TGF-β1 release. ...
(A) Platelet counts and their aggregation response were assessed in whole blood and platelet-rich plasma (PRP), respectively, isolated from mice treated with antagomiR-21 or control antagomiR. ELISAs were subsequently performed in the supernatant of aggregated platelets. (B) Platelet counts were assessed by flow cytometry using a CD41-allophycocyanin antibody on gated platelets. Statistical analysis was performed with Mann-Whitney test; n = 5 antagomiR-21–treated mice vs. n = 5 control antagomiR-treated mice. Lines and error bars represent median (IQR).(C) Platelet aggregation in response to arachidonic acid (AA), protease activated receptor 4 (PAR4) amide, or collagen did not show a significant difference after antagomiR-21 treatment (2-way ANOVA with post hoc Šidák’s test; n = 5 for each treatment type). (D) TGF-β1 release from platelets in response to collagen and PAR4 amide was reduced after treatment with antagomiR-21. PF4 was less affected. TGF-β1 levels in platelet-poor plasma (PPP) were unchanged. Statistical analysis was performed with 2-way ANOVA for agonists and Mann-Whitney test for PPP; n = 5 antagomiR-21–treated mice vs. n = 5 control antagomiR-treated mice for each condition; *P < 0.05; ***P < 0.001.